Genomic Profiling of BDE-47 Effects on Human Placental ...observed BDE-47 accumulation in CTBs with limited evidence of metabolism. At a functional level, BDE-47 hindered the ability

Genomic Profiling of BDE-47 Effects on Human

Placental CytotrophoblastsJoshua F Robinsondagger1 Mirhan Kapidzicdagger Emily G Hamiltondagger Hao Chendagger

Kenisha W Puckettdagger Yan Zhoudagger Katherine Onadagger Emily ParryDagger YunzhuWangDagger June-Soo ParkDagger Joseph F Costellosect and Susan J Fisherdagger

Department of Obstetrics Gynecology and Reproductive Sciences Center for Reproductive SciencesUniversity of California San Francisco (UCSF) San Francisco California 94143 and

dagger

Department of ObstetricsGynecology and Reproductive Sciences University of California San Francisco (UCSF) San FranciscoCalifornia 94143

Dagger

Environmental Chemistry Laboratory Department of Toxic Substances Control CaliforniaEnvironmental Protection Agency Berkeley California 94710 and sectDepartment of Neurological SurgeryUniversity of California San Francisco (UCSF) San Francisco California 94158To whom correspondence should be addressed at Department of Obstetrics Gynecology and Reproductive Sciences Center for Reproductive Sciences Universityof California San Francisco (UCSF) Box 0665 Bldg 513 Parnassus Ave San Francisco CA 94143-0665 Fax (415) 476-1635 E-mail joshuarobinsonucsfedu

ABSTRACT

Despite gradual legislative efforts to phase out flame retardants (FRs) from the marketplace polybrominated diphenylethers (PBDEs) are still widely detected in human maternal and fetal tissues eg placenta due to their continued globalapplication in consumer goods and inherent biological persistence Recent studies in rodents and human placental celllines suggest that PBDEs directly cause placental toxicity During pregnancy trophoblasts play key roles in uterine invasionvascular remodeling and anchoring of the placenta-fetal unit to the mother Thus to study the potential consequences ofPBDE exposures on human placental development we used an in vitro model primary villous cytotrophoblasts (CTBs)Following exposures the endpoints that were evaluated included cytotoxicity function (migration invasion) thetranscriptome and the methylome In a concentration-dependent manner common PBDE congeners BDE-47 and -99significantly reduced cell viability and increased death Upon exposures to sub-cytotoxic concentrations ( 5mM) weobserved BDE-47 accumulation in CTBs with limited evidence of metabolism At a functional level BDE-47 hindered theability of CTBs to migrate and invade Transcriptomic analyses of BDE-47 effects suggested concentration-dependentchanges in gene expression involving stress pathways eg inflammation and lipidcholesterol metabolism as well asprocesses underlying trophoblast fate eg differentiation migration and vascular morphogenesis In parallel assessmentsBDE-47 induced low-level global increases in methylation of CpG islands including a subset that were proximal to geneswith roles in cell adhesionmigration Thus using a primary human CTB model we showed that PBDEs induced alterationsat cellular and molecular levels which could adversely impact placental development

Key words placenta cytotrophoblast transcriptomics polybrominated diphenyl ether BDE-47 human invasion migrationin vitro methylation

Despite efforts to the contrary the continued use of polybromi-nated diphenyl ethers (PBDEs) as flame retardants (FRs) in con-sumer goods warrants great concern (Jinhui et al 2015) Due totheir bio-persistence these compounds continue to be identified

in human maternal and fetal tissues (Zota et al 2013) In particu-lar of the 209 unique PBDE congeners BDE-47 is found at thehighest levels in the majority of US mothers andor their offspring(Frederiksen et al 2009 Woodruff et al 2011 Zota et al 2018)

VC The Author(s) 2018 Published by Oxford University Press on behalf of the Society of ToxicologyAll rights reserved For permissions please e-mail journalspermissionsoupcom

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Numerous toxicological (Dingemans et al 2011) and epidemiolog-ical (Cowell et al 2015 Herbstman and Mall 2014) studies indicatethat exposures to PBDEs in utero may be harmful to the developinghuman fetus The ramifications of their bioaccumulation remainlargely unknown In human cell lines andor animal models com-mon PBDEs congeners elicit developmental toxicity through sev-eral mechanisms eg alterations in thyroid hormone (TH)signaling inflammation oxidative stress and epigenetic modifi-cations (Costa et al 2014) Further investigations are needed toclarify targeted tissuescell populations and windows of sensitiv-ity during human pregnancy

The placenta is essential for normal fetal development andpregnancy complications linked with abnormal placentation arenegatively associated with neonatal child and adult health(Vinnars et al 2014) Data generated in rodent models and humancell lines (Kalkunte et al 2017 Rajakumar et al 2015 Yang et al2006) suggest that environmental compounds are toxic to the pla-centa In humans the placenta is in direct contact with maternalblood in which these chemicals are routinely detected Thus it isnot surprising that PBDEs are regularly identified in human pla-centas (Leonetti et al 2016) While the consequences associatedwith exposures in utero are unknown recent studies in mamma-lian models suggest that BDE-47 may alter placental developmentcausing oxidative stress and inflammation (Park et al 2014 parkand Loch-Caruso 2014) as well as perturbing hormone signaling(park and Loch-Caruso 2015)

During pregnancy the placenta undergoes numerous mor-phological and molecular transformations to support the grow-ing demands of the developing fetus Specialized placental cellsknown as cytotrophoblasts (CTBs) are critical for (1) initiatingand maintaining the physical anchor between the fetal and ma-ternal units (2) penetration and invasion into the uterine walland (3) remodeling of the uterine vascular architecture toreroute maternal blood flow to the placenta (Red-Horse et al2004 2005) Perturbations in CTB development may underlieseveral pregnancy complications including preeclampsia (PEFisher 2015) intrauterine growth restriction (IUGR) pretermbirth (Romero et al 2014) and over aggressive CTB invasion(Jauniaux and Jurkovic 2012) In vitro isolated cultured primaryhuman villous CTBs (Fisher et al 1989 Hunkapiller and Fisher2008 Kliman et al 1986) have been proposed as a model ofplacentation

CTB invasion is a complicated process with many compo-nents some of which are unique The cells must exit the pla-centa crossing over to the uterus where they attach to itssurface before they deeply invade the parenchyma This processis accompanied by a series of molecular transformations in-volving relevant pathways including cell-cell adhesion migra-tioninvasion and vascular remodeling (Maltepe and Fisher2015) Upon isolation and culture villous CTBs differentiatealong the invasive pathway modulating the same sets of mole-cules that mediate this process in vivo (Robinson et al 2017)

Genomic profiling of isolated CTBs revealed dramatic shiftsat transcriptomic and epigenomic levels across gestation includ-ing many key molecules with known functional roles in placen-tal development and disease (Roadmap Epigenomics et al 2015)In assessing environmental chemical impacts during develop-ment the incorporation of genomic-based approaches in toxico-logical studies ie toxicogenomics provides a global analysis ofmolecular response to environmental exposures (Robinson andPiersma 2013) Given the unusual dynamics of the CTB genomethe potential for environmental chemicals to influence chroma-tin architecturegene expression and the links between placen-tal health and successful birth outcomes (Burton et al 2016

Kovo et al 2012) investigations are needed to address potentialinteractions between these important variables

Here we used a cell culture model to study the effects ofPBDEs on CTB differentiation and invasion Acute exposuresresulted in accumulation of the parent compound and minimalmetabolism Subcytotoxic concentrations of BDE-47 impairedthe ability of CTBs to migrate and invade These functionalalterations were accompanied by alterations at transcriptomicand epigenomic levels Together these data suggest that PBDEexposures could impact process and pathways that are integralto the placentarsquos role in governing pregnancy outcome

MATERIALS AND METHODS

Tissue collection All methods were initially approved by the UCSFInstitutional Review Board Informed consent was obtained fromall donors Second trimester placentas intended for cell isola-tions were collected immediately following elective terminationsand placed in cytowash medium consisting of DMEH-21 (Gibco)125 fetal bovine serum (Hyclone) 1 glutamine plus (AtlantaBiologicals) 1 penicillinstreptomycin (Invitrogen) and 01gentamicin (Gibco) Tissue samples were placed on ice prior todissection

Human primary villous cytotrophoblast isolation CTBs were isolatedfrom second trimester human placentas as described previously(Robinson et al 2017) Single cells were counted using a hemacy-tometer and immediately transferred to a Matrigel (BD bioscien-ces)-coated 12-well plate CTBs were cultured at a density of 500000 CTBswell in 15 ml medium containing DMEH-21 2Nutridoma (Roche) 1 sodium pyruvate (Sigma) 1 Hepes buffer(Invitrogen) 1 glutamate plus (Atlanta Biologicals) and 1 peni-cillinstreptomycin (Invitrogen) Cells were incubated at 37C in5 CO295 air We immunostained with anti-cytokeratin (CKanti-CK rat polyclonal 1100 Damsky et al 1992) a marker regu-larly used to detect for relative trophoblast purity ( 80ndash90CTBs) in our cultures across experiments Cell preparations notmeeting this criteria were not used for downstream analyses

Chemicals PBDE congeners BDE-47 (220440-tetrabromodiphenylether gt99 CAS 5436-43-1 AccuStandard) and BDE-99(2204405-pentabromodiphenyl ether gt99 CAS 60348-60-9AccuStandard) bisphenol A (BPA gt99 80-05-7 Sigma) andperfluorooctanoic acid (PFOA gt96 335-67-1 Sigma) were dis-solved in dimethyl sulfoxide (DMSO Sigma-Aldrich) to makestock solutions and serial dilutions for all experimental studiesChemical exposures were introduced at a 11000 (volvol) mediadilution for all assessments

Cytotoxicity assessments Freshly isolated CTBs were cultured for15 h (t15 h) and supplied with new media containing media only(control) DMSO (vehicle control 01) BDE-47 (01 10 1025 lM) BDE-99 (01 10 10 25 lM) BPA (01 10 10 100 lM) orPFOA (1 10 25 100 250 1000 lM) At t15 h cultured CTBs are ac-tively aggregating one of the initial steps in differentiationin-vasion We evaluated cytotoxicity after a 24-h exposure due toprevious studies suggesting that this time point was optimal todetect significant changes at cellular and molecular levels indeveloping in vitro systems (Costa et al 2015 park and Loch-Caruso 2014) We measured cell viability using the neutral red(NR 40 ngml VWR) lysosomal uptake assay (Borenfreund andPuerner 1985) Briefly after 24 h the media was removed andwells were gently washed with PBS to eliminate residual com-pound New media containing NR was added to each plate and

212 | BDE-47 EFFECTS ON HUMAN CYTOTROPHOBLASTS

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incubated for 2 h at 37C Cells were washed with PBS and a 50ethanol1 acetic acid solution was added to release the NRdye which was measured at an absorbance of 540 nM via aspectrophotometer (Biotek Epoch) In parallel in exposed andcontrol CTB cultures we evaluated lactate dehydrogenase (LDH)activitymdasha marker of cell deathmdashwithin the supernatant usingthe Cytotoxicity Detection Kit (Roche) following the recom-mended manufacturer protocol Absorbance readings for LDHwere acquired at 490 nM For both cytotoxicity endpoints withineach experiment background absorbance readings were sub-tracted from mean absorbance values Adjusted values werenormalized as compared with the control (frac14 100) Average rela-tive percentages and corresponding standard error (SEM) werecomputed across the independent experiments (n 3) Additionalpilot investigations were completed where the initial exposure oc-curred after 3 h (t3 h) post-plating before CTB aggregation occursNo differences in CTB sensitivity to PBDE-induced cytotoxicitywere apparent in regards to when the exposure was initiated (t3

h vs t15 h) We calculated benchmark concentrations (BMCs) for50 cell viability or 200 LDH activity via asymmetrical dose-response modeling (Graphad Prism 70 Giraldo et al 2002)

PBDE accumulation and metabolism in CTBs We tested for PBDEconcentrations in cell and media fractions of CTB cultures ex-posed to 01 DMSO BDE-47 1 or 5 mM after 24 h We evaluatedsamples for BDE-47 related metabolites ie 5-OH-BDE-47 and6-OH-BDE-47 as well as major PBDE congeners eg BDE-17 -28-66 -85 -99 -100 -153 -154 -183 -196 -197 -201 -203 -206-207 -209 PBDE concentrations were measured using gas chro-matographyhigh resolution double-focusing sector mass spec-trometry (GC-HRMS DFS Thermo Fisher Bremen Germany) atthe Department of Toxic Substances Control (DTSC BerkeleyCalifornia) as described previously (Zota et al 2013) Averageconcentrations in cell and media fractions were determinedacross three independent experiments (nfrac14 3) Mass estimates ofBDE-47 in cellmedia fractions were calculated using chemicalconcentrations (mgml) and the molecular weight (48979 gmol)of BDE-47 Percent recovery was estimated to be within 10 ofthe expected total amount (media thorn cell fraction)

Migration of PBDE-exposed CTBs We evaluated the effect(s) ofBDE-47 exposures on CTB migrationaggregation as describedpreviously (Robinson et al 2017) In brief we exposed cells with1 DMSO 1 or 5 mM of BDE-47 after early attachment (t1 h)Controls containing media only were also ran in parallel At 5 or15 h post-exposure the culture media was removed and CTBswere washed once with PBS to remove residual chemical NextCTBs were fixed with 4 paraformaldehyde (PFA 20 min)Cultures were washed again with PBS (2) and stored (in PBS) at4C until further processing Cold methanol was added to per-meabilize the CTBs Cultures were washed with PBS (3) and aPBS-Hoechst 33342 (Life Technologies 12500) solution wasadded After 10 min the liquid was removed and cultures wereplaced in PBS For each well 42 tethered fluorescent imagesconsisting of a 5 mm2 area were captured using a Leica invertedmicroscope with a 10 objective and the tiles can function(Leica Application Suite Advanced Fluorescence) Within eachimage we identified all cells or ldquoobjectsrdquo via detection ofHoescht (nuclear-binding dye) using Volocity software(PerkinElmer version 63) We applied automated erosion anddivision operational functions to enable improved measure-ments of aggregated cell populations Objects that intersectedwith the border of the image or that were initially lt20 mm2 wereeliminated from the analysis to limit potential artifacts Images

containing irregularities eg cell debris high background blurryfeatures were also removed (lt5 of images) We evaluated 20000 cells per well The minimum distance between nuclei (cen-troid to centroid) was evaluated using an automated processwhich measured all possible distances between objects withineach image We identified the average cell number per image ineach independent experiment and removed images outside thenormal range (mean 615 SD 5 of images) to minimize theinfluence of density as a factor of cell-cell proximity Withineach experiment we calculated the difference in the averageminimum distance among cells exposed to DMSO or BDE-47versus the media only control The standard error (SE) to themean was computed across the average of the six independentexperiments We applied ANOVA and pairwise Student pairedt-tests to determine significant differences in migration be-tween controls and BDE-47 exposed CTBs (plt 05 JMP 130)

Invasion of PBDE-exposed CTBs As described previously(Hromatka et al 2013) we evaluated the influence of PBDEexposures on the ability of CTBs to invade We used transwellinserts (8 mm pore size 24-well plate Corning Costar) pre-coatedwith 8 ml of diluted MatrigelTM (31 vv in serum-free medium BDBiosciences) for 20 min at 37C CTBs at a density of 250 000 cellsin 250 ml media were added into the upper compartment ofinserts and placed in 24-well plates containing 800 ml of mediaper well After attachment (t1 h) CTBs were exposed to mediaonly (control) 01 DMSO or BDE-47 (1 5 mM) After an addi-tional 40 h at 37˚C cells were fixed with 3 PFA (30 min) washedin PBS (2) and stored in PBS at 4C until further processing Wequantified CTB projections using immunofluorescence micros-copy and high-content analysis (Volocity) After PBS removalcold methanol was added to permeabilize the CTBs (5 min)Cells were washed with PBS (3) and 5 bovine serum albumin(BSA) (Hyclone)PBS was added to block nonspecific reactivityAfter 1 h the solution was removed and the primary antibodyanti-CK (Fisher_001-clone7D3 RRID AB_2631235 rat monoclo-nal 1100 [Damsky et al 1992]) in 5 BSA was added and incu-bated overnight at 4

C The next day cultures were washed with

PBS (3) inserts were cut using a razor blade probed withVectashield containing Dapi (Vector Bio-Labs) and cover slippedImages were acquired using a Leica inverted microscope (20)per slide Invading CTBs andor significant cell protrusions thatreached the underside of the filter were counted using Volocitysoftware (PerkinElmer version 63) and preset optimized criteria(area gt 4000 mm2 max pixel intensity gt 75 shape factor gt 075)All images and generated counts were double checked manuallyto assure proper identification Within each independent experi-ment (nfrac14 3) we determined the total CTB protrusions per condi-tion across three technical replicates (5 images per slide)Invasion was expressed as a ratio in CTB protrusions per imagebetween each exposure group and the media only control (frac14100) per experiment The standard error (SE) to the mean wascomputed across the average of the experiments Significanteffects were determined via ANOVA and Student t-tests betweenPBDE exposure groups and the vehicle control (plt 05 JMP 130)

RNA isolation from CTBs We isolated RNA from CTBs exposed toBDE-47 and controls for downstream microarray or qRTPCRgene expression assessments CTBs were cultured for 3 h (t3 h)or 15 h (t15 h) and supplied with new media containing mediaonly (control) DMSO (vehicle control) andor BDE-47 (01ndash10lM) Initiated exposure times in culture were selected due toour previous analyses (Robinson et al 2017) indicating that inunexposed cultures single CTBs attach and begin to migrate at

ROBINSON ET AL | 213

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3 h in culture and at 15 h aggregation and cell remodelingactively occur We isolated RNA from CTBs for transcriptomicand qRT-PCR investigations Samples used for transcriptomicstudies consisted of cells (1) exposed to either DMSO or BDE-47(1mM) (2) from an independent placenta (nfrac14 3) and (3) varied inage during the second trimester (gestational week [GW] 166193 or 216) In total 12 samples were used for microarray anal-yses (Supplementary Table 1) Additional samples for qRT-PCRvalidation were also generated from individual placentas andranged from GW 14ndash22 To isolate RNA in brief immediatelyfollowing a 24 h exposure duration the media was removed andRLT Lysis Buffer (Qiagen) was directly added to the culture dishThe lysate was collected and stored at 80C RNA was purifiedusing the RNeasy Micro Kit (Qiagen) The RNA concentrationand quality were estimated (absorbance 260 nm280 nm frac14 19ndash21) by using a Nanodrop spectrometer (Thermo Scientific)Samples destined for microarray analyses were assessed forquality (RIN gt 9) using the Agilent RNA 6000 Nano LabChip Kitand Bioanalyzer 2100 system

Gene expression profiling of BDE-47-exposed CTBs We evaluatedthe effects of BDE-47 exposure on the global transcriptome inCTBs using the Affymetrix Human Gene 20 ST array platformSample processing and hybridization was performed by theUCSF Gladstone Institute as described previously (Winn et al2007) Affymetrix CEL files were processed using the AffymetrixExpression Console and Transcriptome Analysis Console (TAC)software packages Raw values were normalized via the robustmulti-array average (RMA) algorithm Raw and normalized datawere deposited in the Gene Expression Omnibus (GEOGSE104896) We analyzed probes with median intensities gt20of the total distribution and discarded duplicates probes repre-senting the same transcript by using the most variable probewithin the full sample set In total we examined 27 729 uniquegenes using this approach A multivariate ANOVA model wasapplied [y(expression) frac14 B1x(exposure) thorn B2x(time) thornB3x(placenta)] to identify differentially expressed (DE) genes dueto BDE-47 or time while adjusting for differences across geneti-cally unique placental cell preparations ie batch effectsAverage fold change (FC) values were determined by calculat-ing the average ratio of the difference of log 2 intensities be-tween BDE-47 (1 mM) and the respective vehicle control withineach experiment We defined DE genes due to BDE-47 exposure(BDE-47 DE Genes) by applying a cutoff of p 025 (unadjusted)and an absolute average FC 125 between BDE-47 and DMSOfor both of the two exposure scenarios ie initiated either at t3

h or t15 h Approaches used to control for false positives (egBonferonni) were not applied because a limited number ofgenes passed standard criteria thresholds (FDR lt 5) a com-mon observation in toxicological studies using primary humanin vitro systems Thus downstream analyses focused on vali-dated targets and changes in related enriched functional path-ways Hierarchical clustering of FC values was completed byusing average linkage and Euclidean distance (TIGR MEV Saeedet al 2006) We conducted functional enrichment of GeneOntology (GO) Biological Processes (Level 4) of BDE-47 DE Genesdefined using the Official Gene Symbol (OGS) using DAVID(Huang et al 2007) GO terms containing 7 DE Genes and anenrichment significance of p 01 were selected as signifi-cantly overrepresented Corresponding enrichment scores iep-values were also determined for up- and downregulatedgene clusters We grouped terms based on GO classification(Gene Ontology Consortium 2015) and identified parent GOterms to define themes

Transcription factor binding site enrichment analysis of BDE-47 DEgenes We identified potential upstream regulators ie TFs ofgenes found to be DE due to BDE-47 exposure in our transcrip-tomic analyses using OPOSSUM Enriched TFBS motifs were de-fined as (1) -2000 bases upstream of the transcription start site(TSS) (2) an identification score of 04 and (3) an enrichmentscore of Z 7 which indicates the over-occurrence of the TFBSin BDE-47 genessequences as compared with the backgroundtotal genome Within this subset of related TFs we exploredtranscript abundance in CTBs isolated from second trimesterversus term human placentas using an RNA-seq dataset previ-ously generated in our laboratory (Roadmap Epigenomics et al2015) Expression data was processed as described previously(Robinson et al 2016) Abundance and significance of differen-tial expression between second trimester versus term was de-termined using DESeq2 and Waldrsquos test (Chen et al 2011)

Targeted validation of BDE-47 DE genes in CTBs Using CTBs fromindependent placentas (n 3) in addition to samples employedin the microarray analyses we investigated expression levels oftarget genes in control and BDE-47 (1mM initiated at t3 h 001ndash10mM initiated at t15 h) exposed-samples at 24 h to validate micro-array analyses and further interrogate the concentration-response of CTBs to BDE-47 including at more physiologicallyrelevant levels Using purified samples we converted RNA tocDNA using ISCRIPT Universal TaqMan (Bio-Rad) and per-formed qRT-PCR via TaqMan primers for FABP4 FABP7 GPR34GREM1 HMGCS1 IL6 MMP1 NEUROD2 PLAC4 and SCD(Supplementary Table 2) mixed with TaqMan Universal MasterMix II no UNG (Life Technologies) Reactions were carried outfor 40 cycles A minimum of 3 technical replicates were ana-lyzed for all comparisons Differential expression betweenPBDE-exposed and controls was calculated via the DDCTmethod (1) normalized to geometric mean of housekeepinggenes GAPDH and ACTB and (2) adjusted to the vehicle control(DMSO) for each experiment Housekeeping genes were selecteddue to their common application in experiments employing re-productive tissuescells (Arenas-Hernandez and Vega-Sanchez2013) and the fact that our microarray data showed that theirexpression was not significantly altered by BDE-47 exposures(not shown) To determine significant changes across concen-trations BDE-47 we employed ANOVA (JMP) FC values wereexpressed as average log 2 ratios between each exposure groupand the vehicle control

DNA isolation of BDE-47-exposed CTBs Following exposure to BDE-47 (1mM) or vehicle for 24 h (initiated at t3 h or t15 h) CTBs (nfrac14 3independent experiments) were washed with PBS and collectedusing a cell scraper Cells (1 million per sample) were sus-pended in 10 ml of PBS pelleted (800 rpm 5 min) washed 2with PBS and stored at 80 C To extract genomic DNA CTBswere digested with 1 mgml proteinase K in lysis buffer (50 mMTris pH 80 1 mM EDTA pH 80 05 SDS) overnight at 55CFollowing RNase treatment DNA was isolated using the Phenol-Chloroform Isoamyl Alcohol (PCI) method followed by precipi-tation with ethanol and resuspended in TE DNA quality wasevaluated via Nanodrop and the Agilent RNA 6000 NanoLabChip Kit and Bioanalyzer 2100 system

Methylation profiling of BDE-47-exposed CTBs Downstream proc-essing of genomic DNA bisulfite conversion was performed us-ing the EZ DNA Methylation Kit (ZymoResearch) and InfiniumHumanMethylation450 bead arrays (Illumina) following themanufacturerrsquos protocols Methylation data was processed via

arch 2019

Illumina standard background subtraction and control probenormalization and converted to M values using the minfi pack-age and BRB Array Tools (Simon et al 2007) Probes that mappedto regions of sex chromosomes were eliminated from the analy-sis Similar to the model used for transcriptomic analyses weutilized a fixed effect multivariate model (using M-value frac14 ratioof methylated probe vs unmethylated probe intensities) to iden-tify differentially methylated (DM) CpGs due to exposure ortime while controlling for baseline differences across CTBsfrom genetically unique placentas Beta values which reflectthe proportion of methlyated probes (ranging from 0 to 1) weredetermined via logit transformation (log2(M(Mthorn 1)) of M-values(Du et al 2010) The change in methylation was determined bysubtracting b values between BDE-47 (1 mM) and the respectivevehicle control within each experiment Significant DM CpGswere identified using a p 005 (unadjusted) and average abso-lute b 0025 between BDE-47 and DMSO for both exposure win-dows ie initiated either at t3 h or t15 h We evaluatedenrichment of DM CpGs by chromosome location (Fisherrsquos exacttest) We interrogated genes in proximity to DM CpGs for func-tional relevance using DAVID (Biological Level 4) These analy-ses were also conducted for the subset of DM CpGs locatedproximal to promoter regions of genes defined to be locatedlt1500 b upstream of the TSS 50 untranslated region (UTR) orfirst Exon Raw and normalized data were deposited in the NCBIGEO repository GSE115399

We examined correlations between mRNA expression andDM CpGs by aligning the datasets based on associated OGS an-notation For the subset of DM CpGs in proximity of promoterregions we assessed the influence of BDE-47 exposures on ex-pression in comparison with the change in methylation at eachindependent CpG island

RESULTS

Concentration-Dependent PBDE-Induced Cytotoxicity in CTBsAfter 24 h we evaluated the effects of BDE-47 or -99 (01ndash25 mM)exposures on CTB viability and death using the neutral red andLDH activity assays respectively In a concentration-dependentmanner both PBDE congeners significantly reduced cell viabil-ity (ANOVA plt 05 Figure 1A) and increased LDH activity(Figure 1B) In general the two congeners displayed similar po-tencies (p 05) Post-hoc analyses (t-test) comparing cultures ex-posed to PBDE versus vehicle control (1 DMSO) revealedconcentrations 10 mM of BDE-47 or -99 to be significantly cyto-toxic In pilot investigations sensitivity was not dependent on

exposure window (initiated at t3 h vs t15 h not shown)Comparisons of BMCs associated with a 50 loss in cell viabilityor 200 increase in LDH activity suggested that CTBs weremore sensitive to PBDEs versus other common endocrine dis-ruptors (BPA or PFOA) in vitro (Figure 1C) Overall our analysesindicated that PBDEs were relatively potent EDCs that induceconcentration-dependent cytotoxicity in CTBs Due to similari-ties in cytotoxic profiles between BDE-47 and -99 and previousstudies indicating BDE-47 to be the highest detected congenerin human placentas (Zota et al 2018) we focused our efforts onBDE-47 effects at functional and molecular levels in CTBs

Partitioning of BDE-47 in Isolated CTBs and Their Culture MediumUsing gas chromatography-mass spectrometry we evaluated(1) BDE-47 (2) hydroxylated metabolites of the parent speciesand (3) other PBDE co-contaminants in CTBs and their culturemedium which contained subcytotoxic concentrations of BDE-47 or vehicle (01 DMSO) First we quantified BDE-47 in themedium after 24 h of exposure to 1 or 5 mM of this compound Inboth cases the measured amounts were less than the input07 6 03 mM or 12 6 02 mM respectively (Figure 2A) Second weplotted the mass distribution (in mg) of BDE-47 in the mediumand cells versus the expected recovery (Figure 2B) also at 24 hQuantification of BDE-47 in the exposed cultures revealed05 6 02 mg (1 mM) and 09 6 01 mg (5mM) in the medium and03 6 02 mg (1 mM) and 31 6 03 mg (5 mM) in the cells At the con-centrations tested total recovery (medium thorn cells) was within10 of expected estimates (black bars) This suggested an 10increase in CTB accumulation of BDE-47 at the 5 mM versus the1 mM exposure Overall our results suggested bioaccumulationof BDE-47 in the cells and that the uptake rate was nonlinearand concentration dependent

In general hydroxylated metabolites of BDE-47 (5-OH 6-OH)were not detected at appreciable levels The metabolite 6-OH-BDE-47 was identified in one of the three CTB samples thatwere analyzed which was 00003 of the

PBDE-47 recovered

In addition to BDE-47 four of the eighteen PBDE congeners eval-uated ie -17 28 -85 -99 were detected at low levels in the ma-jority of all samples which was lt04 of the

PPBDEs

(Supplementary Table 3) PBDE concentrations were below theminimum detection level (MDL) in all samples exposed to thevehicle control evidence that congeners other than BDE-47which were detected in the exposed CTBs were contaminantsof the chemical stock These results suggested that CTBs had lit-tle to no hydroxylated metabolism of the parent compound dur-ing 24 h of exposure

00 01 10 100

250 01 10 10

Veh BDE-47 BDE-99

00 01 10100

250 01 10100

BMC50Viability

BMC200LDH

BDE-47 181μM 125μM

BDE-99 335μM 246μM

PFOA 3638μM 4329μM

BPA 1165μM 966μM

(μM) (μM)

Figure 1 Concentration-dependent CTB cytotoxicity induced by PBDEs (A) CTB viability or (B) LDH activity at 24 h in cultures that contained PBDEs or 01 DMSO (vehi-

cle control VEH) relative to medium with no additives The standard error (SE black bars) of the mean was computed across experiments (n4) Asterisks () indicate

significant differences between tested concentrations of PBDEs and vehicle control (p 05) (C) Benchmark chemical concentrations (BMCs) corresponding to a 50

loss of viability or a 200 increase in LDH activity

arch 2019

BDE-47 Effects on CTB Migration and InvasionUnder control conditions CTBs plated on MatrigelTM migrate to-ward one another at a rate of 03 mmh forming multicellularaggregates during the first 15 h of culture Significant inhibitionof migrationaggregation was observed with BDE-47 after 5 h ex-posure (ANOVA plt 05) Pairwise comparisons between specificconcentrations of BDE-47 and vehicle control indicated that5 mM significantly reduced migration (D in average minimumdistance frac14 08 mm plt 05) while 1 mM caused only a modest re-duction in inhibition (Figure 3A) At 15 h differences in CTB mi-gration were not observed across groups (not shown)suggesting the ability of CTBs to compensate for the differencesthat were initially observed In addition using a transwell cul-ture system we assessed BDE-47 effects on the cellsrsquo ability toinvade After 40 h exposure with 1 mM BDE-47 there was no

difference between the levels in experimental versus controlcultures In contrast 5 mM significantly impaired invasion (42of media only control plt 05 Figure 3B) Our results suggestedthat BDE-47 perturbs CTB migration and invasion in aconcentration-dependent manner

Global Expression Profiling of BDE-47 in CTBsCTBs were plated for 3 or 15 h before they were exposed for 24 hto BDE-47 (1 mM) or vehicle (01 DMSO) after which global geneexpression profiling was performed We applied a fixed effectslinear model (ANOVA) to identify BDE-47 responsive transcriptsIn total the expression of 276 genes was significantly altered(p 025 absolute FC 125) Hierarchical clustering showedthat 159 genes were upregulated and 117 genes were

MediaCellsExpected(cells + media)

Initial BDE-47 Testing ConcVeh 1μM 5μM

Veh 1μM 5μMInitial BDE-47 Testing Conc

ltMDL ltMDL

Figure 2 Bioaccumulation of BDE-47 in CTBs A CTBs were cultured for 24 h in BDE-47 (1 or 5 mM) or DMSO (01) Gas chromatography-mass spectrometry enabled

measurements of the compound amount in each conditioned medium sample B Distribution of BDE-47 in the medium (light shading) or cells (dark shading) versus

the expected recovery in both compartments (black bars) In general the hydroxylated metabolites (6-OH-BDE-47 and 5-OH-BDE-47) were not detected MDL Minimum

level of detection

Veh BDE-47(1μM)

BDE-47(5μM)

Veh BDE-47(1μM)

BDE-47(5μM)

e of C

ANOVA plt005

Figure 3 Concentration-dependent effects of BDE-47 on CTB migration and invasion A The minimum distance between CTBs exposed to BDE-47 (1 or 5 mM) or vehicle

(01 DMSO) following 5 h exposure durations B The average number of identified projections per image in BDE-47 exposed and control cultures after 40 h Asterisks

signify significant effects between tested concentrations and vehicle control (t-test p 05) The standard error (SE) of the mean was computed across independent

experiments (n4)

arch 2019

downregulated (Figure 4A) On average responses to BDE-47were independent of when exposures were initiated (yfrac14 098xR2 frac14 081 Figure 4B) Thus BDE-47 induced significant transcrip-tomic alterations in cultured CTBs and these changes occurredirrespective of the exposure window

GO Analyzes of BDE-47 Responsive GenesGenes whose expression was modulated by BDE-47 weremapped into GO terms The majority of enriched biological pro-cesses were driven by upregulated transcripts They includedmorphogenesis vasculature development cell differentiationcell migration signal transduction inflammatory response pro-tein metabolism and regulation of biosynthetic process-relatedterms (p 01 of genes changed within each GO term 7Figure 5A) Fewer enriched biological processes were driven bydownregulated genes In general these GO terms were relatedto lipid and steroid metabolism

Next we clustered genes belonging to GO terms related toimportant aspects of CTB biology and placental toxicology mor-phogenesis vasculature development cell migration inflam-matory responses and cellular lipid metabolic processes(Figure 5B) Forty-one genes were included in this subset Thisanalysis highlighted BDE-47-induced perturbations in the ex-pression of genes involved in (1) trophoblast differentiation (egMMP1 MMP8 TEK NODAL BMP2) (2) inflammatory pathways(eg CCL13 IL1A IL6 IL1RN CCL1) and (3) lipidsteroid metabo-lism (eg FABP4 FABP7 FASN INSIG1 HMGCS1) From thisgroup we selected seven genes (in italics) and an additional twotargets (GPR34 PLAC4) to further interrogate the concentration-response relationship of BDE-47 exposures (01ndash10 mM) andmRNA expression We observed significant effects for the 9 tar-gets 8 of which had monotonic relationships with BDE-47 expo-sures (ANOVA p 05) Specifically IL6 MMP1 GREM1 andPLAC4 were significantly upregulated GPR34 SCD HMGCS1and FABP7 were significantly downregulated (Figure 5C)Minimal changes were observed at concentrations 01 mMTrends in response to BDE-47 (1mM) were similar for the two ex-posure windows initiated at 3 or 15 h The qRT-PCR results posi-tively correlated with the microarray data (R2 frac14 074Supplementary Figure 1) Thus our findings suggested specificpathways with known roles in trophoblast development andor

PBDE-induced toxicity were significantly altered by exposure tothis compound

Transcription Factor Binding Site (TFBS) Enrichment Analysis ofBDE-47 Responsive GenesGiven the important role of transcription factors in regulatinggene expression and chemical responses we analyzed theTFBSs of genes that encoded mRNAs whose abundance changeddue to BDE-47 exposure Ten of these motifs for human TFswere enriched in this gene subset (Z 70 Table 1) Based on ourRNA-seq data for second trimester and term CTBs (GEO acces-sion number) nine were among the most highly expressed tran-scripts (gt33 percentile of RNA counts) four (PBX1 RORANFKB TCFCP2L1) were significantly DE between second trimes-ter and term (p 05 highlighted) NFYA (Vaiman et al 2013)NFKB (Vaiman et al 2013) EBF1 (Buckberry et al 2017) RELA(Minekawa et al 2007) and RORA (Qiu et al 2015) are implicatedin placental developmentdisease NFYA (Jin et al 2001) NFKB(Puscheck et al 2015) and RELA (Yamamoto et al 2017) are as-sociated with environmental-induced stressors These analysessuggested that BDE-47 exposure may impact specific transcrip-tional responses of CTBs

Global CpG Methylation Analysis of BDE-47-Exposed CTBsIn parallel with the transcriptomic analyses we profiled globalCpG methylation in BDE-47 (1 mM) and vehicle-exposed CTBs fol-lowing a 24 h duration (initiated at 3 h or 15 h post-plating)Using a fixed effects linear model we identified 758 CpGs as DMCpGs associated with BDE-47 exposure (p 005 absolute aver-age Db 0025) Overall exposure significantly increased globalCpG methylation (08 6 02 Figure 6A) A further increase wasobserved when the analysis was constrained to BDE-47 DMCpGs (34 6 03 Figure 6A) This was consistent with data fromindividual CpGs 93 had increased methylation (708758 total)and 7 had decreased methylation (50758 total Figure 6B) Onaverage methylation changes due to BDE-47 were similarwhether exposures were initiated at 3 h or 15 h (yfrac14 090x R2 frac14069 Figs 6AndashC)

Furthermore within the BDE-47 DM CpG subset we askedwhether there was enrichment based on gene featuresIntergenic sequences were overrepresented and those proximal

(GW)166 193 216y = 098xRsup2 = 081

-15 -1 -05 0 05 1 15Avg FC (BDE-47Veh) t3h+24h

t3h+24h t15h+24h

PSG7CCL1

166 193 216

Figure 4 Gene expression profiling of BDE-47-exposed CTBs A Cultures were exposed to 1 mM BDE-47 for 24 h starting either 3 or 15 h after they were plated Global

transcriptional profiling showed that 276 genes were DE as compared to CTBs that were cultured in 01 DMSO (p 025 absolute FC 125) Hierarchical clustering

demonstrated similar responses among the CTB cultures and between the two exposure windows FC ratios are displayed as the log 2 difference between BDE-47 (1 mM)

and the vehicle control for each experiment B Correlations between the average gene expression FC responses for the different exposure windows Red (upregulated

genes) green (downregulated genes) GW gestational week

arch 2019

to promoters (eg TSS1500 TS200 first Exon) were underrepre-sented (Figure 6D) A relatively large number were also associ-ated with gene bodies although this feature did not achievestatistical significance because the identified regions did notexceed expectations GO analyses of genes near BDE-47 DMCpGs revealed enrichment of processes and pathways that areinvolved in hormone response cellular projection signalingresponse to oxygen-containing compounds morphogenesisand vesicle transportlocalization (Figure 6E) Promoter proxi-mal BDE-47 DM CpGs (178 total) were significantly associated

with vesicle transportlocalization and secretion-relatedterms

Next we examined the chromosomal location of BDE-47 DMCpGs which on the whole showed a larger proportion of in-creased versus decreased methylated CpGs on each chromo-some (Figure 7A) The results revealed enrichment onchromosomes 10 and 11 and a relative absence on chromosome18 Figure 7B shows the chromosome 11 data which had thehighest significance of enrichment by genomic location Thisenabled identification of a BDE-47 DM CpG cluster on Ch 11p155

anatomical struc formationmorphogenesis (14 1)organ morphogenesis (11 2)

reg of cell morphogenesis (8 2)blood vessel morphogenesis (8 1)

circulatory system dev (11 1) cardiovascular system dev (11 1)

vasculature dev (9 1)angiogenesis (7 1)

reg of vasculature dev (7 1)reg of angiogenesis (7 1)cell differentiation (26 15)

(+) reg of cell differentiation (10 3)

(+) reg of cell communication (19 5)signal transduction (36 17)

cell surface receptor signaling pathway (24 8)reg of signal transduction (23 8)

intracellular signal transduction (23 7)reg of intracellular signal transduction (18 6)

(+) reg of signal transduction (18 5)(+) reg of intracellular signal transduction (14 5)

MAPK cascade (13 3)signal transduction by protein phosphorylation (13 3)

protein secretion (8 0)resp to cytokine (11 2)

leukocyte migration (11 2)inflammatory resp (12 0)reg of defense resp (9 2)

reg of inflammatory resp (7 2)

cell motility (16 7)cell migration (15 7)

reg of cell migration (9 1)cell chemotaxis (7 1)

cellular lipid metabolic process (5 9)lipid biosynthetic process (4 6)

reg of lipid metabolic process (5 3)steroid metabolic process (2 5)

reg of protein metabolic process (21 7)reg of cellular protein metabolic process (21 7)

(+) reg of protein metabolic process (18 5)(+) reg of cellular protein metabolic process (18 4)

cellular resp to organic substance (20 7)resp to organic cyclic compound (10 7)

(-) reg of biosynthetic process (13 9)(-) reg of cellular biosynthetic process (13 9)

(-) reg of macromolecule biosynthetic process (12 8)sulfur compound biosynthetic process (3 4)

chemical homeostasis (9 8)(+) reg of cellular component organization (13 4)

reg of transferase activity (12 3)(+) reg of programmed cell death (8 1)

oxoacid metabolic process (2 7)

phosphate-cont compound metabolic process (24 7)reg of phosphorus metabolic process (19 5)

(+) reg of phosphorus metabolic process (16 4)

reg of epithelial cell proliferation (6 1)

ALL uarr darr

p=001p=005 p=0001

Enrichment of GO Biological Process

lative

PLAC4 IL6MMP1

FABP4GREM1

FABP7GPR34

HMGCS1SCD

001μM 01μM1μM5μM10μM

BDE-47

anatomical struc formationmorphogenesis

yvasculature dev

cell migration f

cellular lipid metabolic process

MMP1PDE3BCCL13GREM1MMP8CD48IL1AIL6IL1RNRGCCDCSTAMPHES1FGF1LATTEKPI4K2BSLC3A2CSF1CD36ACP5BMP2ANKRD1NOVCCL1PDK4ERVFRD-1FABP4FABP7FASNCD24GSTM2OPN1LWSCDMYO1GNODALFERMT1SLC37A4OLIG3FGFR4INSIG1HMGCS1

anat morphogenesi

cell m

igration

vasculatu

re dev

lipid metabolism

inflammato

ry res

y ginflammatory resp

t3h+24h t15h+24h

Figure 5 Functional analyses of genes that were DE due to BDE-47 exposures in CTBs A Enriched GO Biological Processes within BDE-47 DE Genes identified using

DAVID (criteria p 01 number of DE genes associated with enriched term 7) GO enrichment scores (-log (p)) are displayed for all BDE-47 DE Genes and upregulated

or downregulated gene subsets The total number of DE Genes due to BDE-47 is located in parentheses (up downregulated) B Clustering of BDE-47 DE Genes associ-

ated with terms anatomical structure formation involved in morphogenesis vasculature development cell migration inflammatory response and cellular lipid meta-

bolic process FC values represent average difference between BDE-47 and vehicle control C Concentration-dependent expression alterations in BDE-47 DE Genes

using qRT-PCR Expression values (DDCT) were normalized to housekeeping genes (GAPDH ACTB) and adjusted by the respective vehicle control Asterisks indicate

significance across all concentrations of BDE-47 and vehicle control (ANOVA plt 05) All targets were observed to be significantly altered with the exception of FABP4

which was significantly altered only with 1 mM in pairwise comparisons with the vehicle control

arch 2019

(see arrow marked with an asterisk genome location 2000000ndash2300000) Interestingly the mRNAs encoded by genes in this re-gion (H19 IGF2 INS-IGF2 and ASCL2) whose products are amongthe most abundant in CTBs play a critical role in gestationaldevelopment (Smith et al 2007) and are responsive to multipleenvironmental exposures (LaRocca et al 2014 Wu et al 2004)

Correlating BDE-47-Induced Changes in DNA Methylation WithAlterations at the mRNA LevelWe identified 178 DM CpGs in gene promoters A subset (156 ofwhich 152 were unique) were correlated with mRNA expressionlevels In total four genes (3 unique mRNAs) whose promoterswere DM had mRNA levels that were also responsive to BDE-47

Table 1 Transcription Factor Binding Site (TFBS) Enrichment Analysis of BDE-47 DE Genes

TF JASPAR TFBSs Z Genes Fisher Count (pct) Age FC (Second Term)

NFYA MA00601 45 166 30 (16 14) 34 73 007AR MA00071 3 108 3 (3 0) 24 33 046PBX1 MA00701 18 107 17 (12 5) 49 85 119EBF1 MA01541 94 106 52 (36 16) 42 35 027RORA (1) MA00711 43 102 26 (17 9) 14 47 168GFI MA00381 126 97 59 (37 22) 34 13 036TBP MA01082 64 90 34 (22 12) 10 56 007NFKB MA00611 48 78 29 (17 12) 10 85 036RELA MA01071 35 75 25 (18 7) 16 88 013TCFCP2L1 MA01451 71 70 40 (28 12) 12 89 164

Overrepresented motifs and associated transcription factors (TFs) in proximity of genes DE by BDE-47 in CTBs Number of DE genes (up vs downregulated) with TF-

binding sites are displayed in combination with corresponding enrichment values for TF binding sites (Z) and genes (Fisher) Abundance of RNA levels (percentile in

top 20 000 genes) of TFs in freshly isolated second trimester and term CTBs as determined via post-hoc analysis of RNA-seq data Significance of differences in expres-

sion between second trimester and term CTBs (plt 05 plt 001)

AllCpGs

BDE-47DMRs

∆β (B

y = 090xRsup2 = 069

-015 -005 005 01501-01

∆β (B

Avg ∆β (BDE-47 vs Veh) t3h + 24h0

(GW)166 193 216

t3h+24h t15h+24h

166 193 216

MRs uarrmethylation

darrmethylation

(darr)

(darr)(darr)

(uarr)

EAll D

MRs (472)

Promoter (17

t of G

ical P

plt001

cellular response to hormone stimulus (23)reg of cell projection organization (22)gland development (18)cellular response to acid chemical (10)system development (112)neuron projection development (30)wound healing (22)cellular response to oxygen-containing compound (32)modulation of synaptic transmission (14)(+) reg of secretion (16)cell-cell signaling (46)(+) reg of secretion by cell (15)(-) reg of cell differentiation (24)cellular component morphogenesis (43)synaptic signaling (24)cellular response to organic substance (67)single-organism transport (95)cell morphogenesis (43)reg of secretion (25)biomineral tissue development (9)neurotransmitter secretion (10)synaptic vesicle cycle (8)synaptic vesicle transport (8)establishment of vesicle localization (11)vesicle localization (11)

t3h+24ht15h+24h∆β Avg

TSS1500

TSS2005rsquoUTR

1st Exon

Body3rsquoU

Intergenic

~PromoterFigure 6 Profiling CpG methylation of BDE-47-exposed CTBs Cultured CTBs were exposed to 1 mM BDE-47 or 01 DMSO for 24 h (initiated at 3 or 15 h post-plating) We

identified 758 CpGs to be DM with BDE-47 versus 01 DMSO (p 005 absolute average Db0025) A Median difference in methylation levels (Db) between BDE-47 ex-

posed and vehicle control cultures in all evaluated CpGs and BDE-47 DM CpGs after 24 h B Hierarchical clustering of the changes in methylation in BDE-47 DM CpGs

(BDE-47 vs concurrent vehicle control) C Cross-scatter plot comparing average Db in BDE-47 response in CTBs exposed at t3 h or t15 h thorn 24 h D Distribution of BDE-47

DM CpGs by regulatory region Asterisks indicate over () or under () representation (Fisherrsquos test () p 05 () p 001)) E Enrichment of GO biological processes

within genes ( in parentheses) in proximity of BDE-47 DM CpGs

arch 2019

(Figure 8A) We plotted the average Db versus the average FC inexpression between BDE-47 and vehicle control for the entiresubset (Figure 8B) We identified three mRNA targets of BDE-47(FERMT1 DPT downregulated CRCT1 upregulated) with in-creased CpG methylation in their promoters due to chemical ex-posure (Solid blue diamonds quadrant IV) Our results suggestthat BDE-47 exposures may alter the expression of specificgenes by modifying CpG methylation within promoter regions

DISCUSSION

Due to widespread identification of PBDEs in human placentaltissues (Leonetti et al 2016 Zota et al 2018) and increasing evi-dence that these compounds cause placental toxicity in rodentand human cell lines (park and Loch-Caruso 2014 2015) weutilized a primary cell model to evaluate the effects of BDEs onhuman CTB differentiation First we surveyed a wide range ofBDE-47 and -99 concentrations (01ndash25 mM) for cytotoxicity(Figure 1) Both congeners induced significant concentration-dependent effects on cell viability and death CTBs displayedsensitivities in the range of other vulnerable human cells egprimary fetal human neural progenitor cells (Schreiber et al2010) Furthermore PBDEs were more potent in inducing

cytotoxicity as compared to other common endocrine disrup-tors BPA or PFOA suggesting PBDEs are relatively potent toxi-cants in the CTB model

In CTBs exposed to subcytotoxic concentrations of BDE-47(Figure 2) we demonstrated acute cellular bioaccumulation ofthe unmetabolized form after 24 h These results agree with pre-vious toxicological studies in rodent in vitro or in vivo modelsthat described cellulartissue accumulation of PBDEs (Mundyet al 2004) Partitioning of BDE-47 between the media and cellu-lar fractions was dependent on the initial testing concentrationwith a greater proportion of BDE-47 distributed in the cells ex-posed to 5 mM versus 1 mM This provided a possible basis forconcentration-dependent nonlinear responses to BDE-47 onthe mRNA level We did not detect a significant amount of themajor hydroxylated metabolites of BDE-47 (5-OH or 6-OH) Thisfinding and the observation that 100 of the parent com-pound was recovered in the cellmedia fractions suggested lim-ited metabolism over 24 h While the placenta may act as abarrier for several xenobiotics (Robins et al 2011) its capacity tometabolize PBDEs remains unknown Other studies from ourlaboratory (Roadmap Epigenomics et al 2015) and other groups(Hakkola et al 1996) indicate that CYP2B6-the primary CYPp450 enzyme involved in BDE-47 metabolism (Feo et al 2013Penell et al 2014)-is expressed at low levels in the placenta

01020304050607080

MRs uarrmethylation

darrmethylation

(uarr)

(darr)

1 2 3 4 5 6 7 8 9 10 111213141516171819202122

-006-004-002

0002004006008

0 25000000 50000000 75000000 100000000 125000000

BDE-47 DMRple005

Avg ∆β

Figure 7 Chromosomal distribution of dysmethylated CpGs in BDE-47-exposed CTBs A Location of BDE-47 DM CpGs by chromosome Asterisks indicate over () or un-

der () representation (Fisherrsquos test p 05 p 001) B Change in methylation (Db) between BDE-47 exposed and vehicle control in CpGs across chromosome 11 Dark

blue and gray dots signify significant BDE-47 DM CpGs and CpGs with p 05 respectively Asterisk indicates cluster of BDE-47 DM CpGs located in Ch 11p155

4(3)152

BDE-47 DMRs(Promoter regions)

ple0005abs ∆βgt0025

ABDE-47 DE Genes

156(152)

Avg ∆β (BDE-47 vs Veh) 24h-01 01-005 0050

DPTFERMT1

BDE-47 DE Genesnot significant

06CRCT1

Figure 8 Correlation between BDE-47 altered methylated promoter regions and gene expression A The overlap of BDE-47 DM CpGs (associated genes located in pro-

moter regions of DM CpGs) and BDE-47 DE Genes B Correlation of average FC in gene expression (y-axis) and Db (x-axis) associated with BDE-47 exposures in gene sub-

set (shaded in Venn) DE Genes are shown as solid blue diamonds

arch 2019

Here we present data suggesting that CTBs are unable to break-down BDE-47 in vitro These mass spectrometry analyzesshowed that other primary PBDE congeners (04) were alsopresent in the mixture Even though these co-contaminantswere a minor component they could contribute to the observeddownstream effects Future studies are needed to addresswhether CTB accumulation of PBDEs depends on the exposureduration (acute vs chronic) and the contribution of other placen-tal cell types eg STBs to the metabolism of these compounds

We tested the ability of BDE-47 to alter CTB migration or in-vasionmdashinherent properties of CTBs that enable successfulpenetration of the decidua and the resident blood vesselsFurthermore perturbations in CTB migrationinvasion under-lie pregnancy complications such as PE PTL and IUGR con-tributing to the pathogenesis of adverse neonatal outcomes(Kaufmann et al 2003) Our results suggest that BDE-47 signifi-cantly disrupts the ability of CTBs to migrate and invade(Figure 3) in line with studies indicating other toxicologicalcompounds eg arsenic (Li and Loch-Caruso 2007) cadmium(Alvarez and Chakraborty 2011) impair trophoblast migrationTogether these results provide a potential link between chemi-cal exposures trophoblast dysfunction and placental-drivenpregnancy complications

Additionally we conducted a comprehensive assessment oftranscriptomic and methylomic changes which correlate withBDE-47 exposure in CTBs We observed that a subcytotoxic con-centration (1 mM) of BDE-47 induces perturbations on transcrip-tomic and methylomic levels in cultured CTBs isolated fromthree independent human placentas We profiled the effects ofBDE-47 following a 24 h exposure duration which was eitherinitiated at t3 h and t15 h corresponding with the (1) initial pro-jection of CTB migration or (2) CTB aggregation respectively(Robinson et al 2017) In general significant genomic responsesto BDE-47 were similar irrespective of when exposures were in-troduced into the media Overall these results suggest a generalconservation of BDE-47 response in our model system Futurestudies which employ larger sample sizes will enable interroga-tion of factors which also may play a role in sensitivity to envi-ronmental exposures in utero such as sex ethnicity andmaternal and gestational age

As for effects on the transcriptome we observed dysregu-lated expression of 276 genes with BDE-47 exposure (Figure 4)Our analyses highlight DE genes associated with (1) proposedmechanisms of PBDE-toxicity eg inflammation D hormone re-sponse and (2) pathways critical for placental and trophoblastdevelopment Below we discuss specific targets of BDE-47 inCTBs that were identified in this study and their proposed rolesin placental development and disease

Interleukins and other inflammatory mediators drive signal-ing aspects of placental development and hyperexpression maysignal or contribute to pregnancy complications (Tjoa et al2003) Here we provide evidence that BDE-47 alters the expres-sion of several genes known to regulate inflammatory response(Figure 5) Genes dysregulated by BDE-47 included IL-6 () IL1A() IL1RN () CCL1 () and CCL13 () Interestingly IL6 may playmajor roles in immune defense (Rose-John et al 2017) and al-tered expression may underlie placental (Prins et al 2012) andneurodevelopmental disease (Shen et al 2008 Sorokin et al2014) Supporting these findings in a human trophoblast cellline in a concentration and time-dependent manner BDE-47co-induced IL-6 IL-8 and oxidative stress mediators (park andLoch-Caruso 2014 2015) Our results support the hypothesisthat PBDE exposures cause inflammation in the placenta whichmay contribute to toxicity

Due to their inherent lipophilic properties and similarities inbiochemistry as endogenous hormones PBDEs and other EDCsmay alter cholesterol and fatty acid metabolism pathwayswhich influence the regulationproduction of critical hormonesfor fetal growth In rodent models PBDE exposure leads to dis-ruption of specific hormones in placenta (Zhu et al 2017a) andLeydig cells (Zhao et al 2011) as well as increased cholesterolserum levels in perinatally exposed juveniles (Tung et al 2017)Here we propose specific mRNA targets such as regulatorybinding proteins (FABP7 [Thumser et al 2014] INSIG1 [Dongand Tang 2010]) and metabolizing enzymes (FASN [Jones andInfante 2015] HMGCS1 [Vock et al 2008] SCD [Zhang et al2005]) involved in lipidcholesterol metabolism to be signifi-cantly downregulated with BDE-47 While these pathways dur-ing fetal development are clearly important the specific role ofthese molecules in the context of chemical toxicity andor tro-phoblast differentiation remains undefined Our results suggestBDE-47 alters the expression of molecules regulating choles-terolfatty acid biosynthesis which may play upstream roles inhormonal regulation in the placenta

We observed several molecules with known critical func-tions in placental development including (1) maintenance oftrophoblast progenitor populations eg BMP2 ( [Golos et al2013]) and NODAL ( [Ma et al 2001]) (2) trophoblast invasioneg MMP1 ( [Cohen et al 2006]) MMP8 ( [Zhu et al 2012]) and(3) villi morphogenesis eg GREM1 ( [OrsquoConnell et al 2013]) tobe significantly altered by BDE-47 exposures (Figure 5B) In addi-tion our analyzes revealed targets with less defined roles in pla-centation with links with placental complications eg PLAC4 ((Tuohey et al 2013]) and candidates yet to be studied in thecontext of placental development For example BDE-47-inducedexpression of GPR34 (Figure 5C) which controls aspects of mi-gration in cancer cells (Jin et al 2015) and may be required forsufficient immune response to pathogens (Liebscher et al2011) Our findings which complement our observations ofBDE-47 functional impairment provide specific pathways withknown roles in trophoblastplacental development to be signifi-cantly altered with exposure

We evaluated for potential upstream regulators of BDE-47DE Genes by conducting enrichment analysis of TF-relatedmotifs in promoter regions of the gene subset (Table 1) In previ-ous studies similar approaches have been applied to proposekey regulatory nodes of developmental toxicity (Robinson et al2011) Based on these analyses we identified TFs involved inPBDE-induced response such as NFKB a highly recognized TFinvolved in regulating inflammation signaling pathways in theplacenta and environmental stress-responsive gene expressionnetworks (Simmons et al 2009) Interestingly other TFs identi-fied through this analysis eg RORA (Qiu et al 2015) RELA(Minekawa et al 2007) were also found to differ in expressionlevels between second trimester and term CTBs and have postu-lated roles in placenta development suggesting that these TFsmay regulate PBDE-response networks in the context of alteredtrophoblast development

While the mechanisms remain poorly understood diverseenvironmental exposures including PBDEs (Byun et al 2015Kappil et al 2016 Sales and Joca 2016 Woods et al 2012) areassociated with epigenetic modifications which may regulatetransient or irreversible genomic changes leading to transge-nerational inheritance of altered phenotypes (Bernal and Jirtle2010 Skinner and Guerrero-Bosagna 2009) Here in parallelwith transcriptomic assessments we provide evidence of BDE-47 exposures to produce subtle but significant global changeson the methylome following a 24 h exposure duration (Figs 6

arch 2019

and 7) Within the subset of BDE-47 DM CpGs the majority ofCpG sites were increased in methylation with BDE-47 (averageDbfrac14 34 93 CpGs were increased bgt 0) and predominatelylocated in nonpromoter regions (ie intergenic positions genebody) Interestingly in general these observations did not cor-relate with a global reduction in transcription (Figure 8) or modi-fied expression of DNA methyltransferases (DNMTs)TET familyenzyme members (not shown) In vitro upon plating proliferat-ing CTBs exit the cell cycle and do not propagate in culture Ateach autosomal CpG site methylation is expected to be 0 50 or100 methylated Thus the subtle changes in methylation ob-served with BDE-47 may represent an active binary shift(methylated vs not methylated) in a subset of cells in cultureWhile increased DNA methylation within promoter regions isa recognized mechanism in silencing mRNA transcription therole of methylation within nonpromoter regions remainsundefined and complex (Szyf 2011) For instance increasedglobal methylation within gene-body regions may actuallypromote gene expression by suppressing cryptic promoterseg antisense targets which compete with RNA Polymerase II-directed transcription (Gagnon-Kugler et al 2009)Furthermore global changes in methylation due to environ-mental exposures are of particular interest in the context ofthe placenta due to its unique DNA methylation and chroma-tin state As compared with adult somatic cells and tissuesthe CTB and placental genome is (1) globally hypomethylated(2) contains a higher proportion of variably methylated CpGsites (b frac14 20ndash80 methylated) and (3) age-dependent in itsspecificity (global methylation higher at term versus secondtrimester [Roadmap Epigenomics et al 2015]) Therefore ourresults which suggest environmental-induced changes on aglobal methylation level could have dramatic long-termimpact(s) on regulatory molecularcellular functions of thedynamically changing placenta

Examining the localized distribution of DM CpGs across thegenome we identified a vulnerable region located on Ch11p155 which included five DM CpGs (all ) due to BDE-47 expo-sure (Figure 7B asterisk) This region of Ch 11 has previouslybeen identified to contain a cluster of imprinted genes impor-tant for fetal growth which have significant associations withseveral gestational diseases (Smith et al 2007) Interestinglygenes within this cluster H19 IGF2 and INS-IGF2 are in the top99 of abundant transcripts in trophoblast cell populationsbased on analyzes of second and term trophoblasts in our labo-ratory (not shown) further suggesting a high level of impor-tance of these molecules in CTB development Recentlyinvestigators have summarized growing evidence linking alter-ations in genomic imprinting of this region and adverse placen-tal ie PE and neurobehavioral disease outcomes (Nomura et al2017)-key outcomes of interest in regards to PBDEs and otherchemical exposures which occur during human pregnancy Ourstudy along with other environmental investigations in rodent(Ouko et al 2009 Susiarjo et al 2013 Wu et al 2004) andhumans (LaRocca et al 2014) suggest associations between en-vironmental exposures and epigenetic alterations within thisparticular gene cluster to have high sensitivity and importancein developmental toxicology

Limited overlap in genes linked with BDE-47 DM CpGs (lo-cated in promoter regions) and BDE-47 DE Genes were identified(4 total 3 unique genes Figure 8) Closer examination of the in-fluence of BDE-47 on methylation within DM CpGs and RNA ex-pression in general suggests a lack of correlation between D inmethylation and expression due to BDE-47 at 24 h Poor

correlation could be due to the lack of temporal data Our com-parative analyses indicate that two candidates may be epige-netically controlled in response to BDE-47 Both targets DPTand FERMT1 were identified to have increased methylation inthe promoter region associated with downregulation of expres-sion with BDE-47 exposure These molecules have relatively un-known functions in placentation but recent evidence impliesroles in adhesion and cell migration (Liu et al 2013) as well aslinks to regulatory pathways known to be important for tropho-blast development (DPT TFG-b signaling [Jones et al 2006Okamoto et al 1999]) FERMT1 epithelial-mesenchymal transi-tionWnt-signaling (Knofler and Pollheimer 2013 Liu et al2017) Future mechanistic studies may explore the relationshipsbetween methylation RNA expression and impaired outcomesin CTBs as related to BDE-47 exposures

We tested 001ndash25mM of BDE-47 which includes concentra-tions proposed as physiologically relevant for human exposuresand which cause deleterious effects in human embryonicfetalcells (Park et al 2014 Schreiber et al 2010) In general toxico-logical responses to BDE-47 were observed at concentrations of 1 mM or higher which are 3 orders of magnitude higher thanthe geometric mean concentrations (6standard deviation) re-cently reported in maternal serum (017 6 197 ngg) cord serum(022 6 175 ngg) or placenta (015 6 196 ngg) (Zota et al 2018)However extrapolating exposure levels between in vitro and inutero is challenging due to the numerous factors that influencesensitivity including (1) absorption metabolism and excretion(2) length of exposure (chronic vs acute) (3) life-style factors (4)nutritional status and (5) life-stage (Doucet et al 2009Grandjean 1992) which are not addressed in cell culture mod-els Accounting for factors that underlie variable exposures aswell as the lipid content of the placenta in vitro concentrationsas high as 8 mM are estimated to be relevant to human expo-sures (Park et al 2014) Furthermore during pregnancy the fe-talplacental unit is exposed to multiple PBDEs and otherenvironmental compounds that may act through similar mech-anisms additively or synergistically (Eriksson et al 2006Tagliaferri et al 2010) Future experiments could use the CTBmodel to evaluate developmental toxicity in the context ofother FRs including emerging alternatives and complex chemi-cal mixtures

In this study we demonstrate the application of a primaryhuman villous CTB model to examine potential environmentalinteractions which occur during placentation using PBDEs as arelevant model chemical toxicant Primary human placentalcells offer multiple advantages as a toxicological model as com-pared with transformed human cell lines (Bilban et al 2010) orrodent (Silva and Serakides 2016) due to suspected differencesin the underlying cellular and molecular components thatregulate placental development Moving forward largersample sizes may be incorporated to improve detection ofenvironmental interactions and control for genetic diversitygestational age and sex Future investigations may also addcomplimentary investigations of other placental cell types egSTBs which may improve resolution of mechanisms proposedto play roles eg D hormone levels (Zhu et al 2017ab) in PBDE-induced placental toxicity in studies examining the completeplacental unit In summary we provide evidence that PBDEs in-duce toxicity in human primary placental cells and alter levelsof expression of specific transcripts and epigenetically con-trolled regions Perturbations may be evaluated as biomarkersin vitro or in vivo to determine correlations between PBDEs andadverse developmentalpregnancy outcomes

arch 2019

SUPPLEMENTARY DATA

Supplementary data are available at Toxicological Sciencesonline

ACKNOWLEDGMENTS

The authors would like to thank Sirirak Buarpung ElaineKwan Jason Farrell and Nicomedes Abello for additionaltechnical support and Michael McMaster and MatthewGormley for their valuable insight in human placental andtrophoblast biology Tissue samples were provided by theNIH Placental Bank at UCSF funded under NICHDNIHEunice Kennedy Shriver National Institute of Child Health ampHuman Development of the National Institutes of Healthunder Award P50HD055764 (to SJF) The authors havenothing to disclose

FUNDING

This work was kindly supported by the United StatesEnvironmental Protection Agency (RD883467801) and theNational Institute of Environmental Health Sciences(P01ES022841 R00ES023846)

REFERENCESAlvarez M M and Chakraborty C (2011) Cadmium inhibits

motility factor-dependent migration of human trophoblastcells Toxicol In Vitro 25(8) 1926ndash1933

Arenas-Hernandez M and Vega-Sanchez R (2013)Housekeeping gene expression stability in reproductive tis-sues after mitogen stimulation BMC Res Notes 6 285

Bernal A J and Jirtle R L (2010) Epigenomic disruption Theeffects of early developmental exposures Birth Defects Res AClin Mol Teratol 88(10) 938ndash944

Bilban M Tauber S Haslinger P Pollheimer J Saleh LPehamberger H Wagner O and Knofler M (2010)Trophoblast invasion Assessment of cellular models usinggene expression signatures Placenta 31 989ndash996

Borenfreund E and Puerner J A (1985) Toxicity determinedin vitro by morphological alterations and neutral red absorp-tion Toxicol Lett 24(2-3) 119ndash124

Buckberry S Bianco-Miotto T Bent S J Clifton V ShoubridgeC Shankar K and Roberts C T (2017) Placental transcrip-tome co-expression analysis reveals conserved regulatory pro-grams across gestation BMC Genomics 18 10

Burton G J Fowden A L and Thornburg K L (2016) Placentalorigins of chronic disease Physiol Rev 96 1509ndash1565

Byun H M Benachour N Zalko D Frisardi M C Colicino ETakser L and Baccarelli A A (2015) Epigenetic effects of lowperinatal doses of flame retardant BDE-47 on mitochondrialand nuclear genes in rat offspring Toxicology 328 152ndash159

Chen Z Liu J Ng H K Nadarajah S Kaufman H L Yang JY and Deng Y (2011) Statistical methods on detecting dif-ferentially expressed genes for RNA-seq data BMC Syst Biol5 S1

Cohen M Meisser A and Bischof P (2006) Metalloproteinasesand human placental invasiveness Placenta 27 783ndash793

Costa L G de Laat R Tagliaferri S and Pellacani C (2014) Amechanistic view of polybrominated diphenyl ether (PBDE)developmental neurotoxicity Toxicol Lett 230 282ndash294

Costa L G Pellacani C Dao K Kavanagh T J and Roque P J(2015) The brominated flame retardant BDE-47 causes oxida-tive stress and apoptotic cell death in vitro and in vivo inmice Neurotoxicology 48 68ndash76

Cowell W J Lederman S A Sjodin A Jones R Wang SPerera F P Wang R Rauh V A and Herbstman J B(2015) Prenatal exposure to polybrominated diphenyl ethersand child attention problems at 3-7 years NeurotoxicolTeratol 52 143ndash150

Damsky C H Fitzgerald M L and Fisher S J (1992)Distribution patterns of extracellular matrix componentsand adhesion receptors are intricately modulated during firsttrimester cytotrophoblast differentiation along the invasivepathway in vivo J Clin Invest 89 210ndash222

Dingemans M M van den Berg M and Westerink R H (2011)Neurotoxicity of brominated flame retardants (in)directeffects of parent and hydroxylated polybrominated diphenylethers on the (developing) nervous system Environ HealthPerspect 119 900ndash907

Dong X Y and Tang S Q (2010) Insulin-induced gene A newregulator in lipid metabolism Peptides 31(11) 2145ndash2150

Doucet J Tague B Arnold D L Cooke G M Hayward S andGoodyer C G (2009) Persistent organic pollutant residues inhuman fetal liver and placenta from Greater MontrealQuebec A longitudinal study from 1998 through 2006Environ Health Perspect 117 605ndash610

Du P Zhang X Huang C C Jafari N Kibbe W A Hou L andLin S M (2010) Comparison of Beta-value and M-valuemethods for quantifying methylation levels by microarrayanalysis BMC Bioinformatics 11 587

Eriksson P Fischer C and Fredriksson A (2006)Polybrominated diphenyl ethers a group of brominatedflame retardants can interact with polychlorinated biphen-yls in enhancing developmental neurobehavioral defectsToxicol Sci 94 302ndash309

Feo M L Gross M S McGarrigle B P Eljarrat E Barcelo DAga D S and Olson J R (2013) Biotransformation of BDE-47to potentially toxic metabolites is predominantly mediatedby human CYP2B6 Environ Health Perspect 121(4) 440ndash446

Fisher S J (2015) Why is placentation abnormal in preeclamp-sia Am J Obstet Gynecol 213 S115ndashS122

Fisher S J Cui T Y Zhang L Hartman L Grahl K Zhang GY Tarpey J and Damsky C H (1989) Adhesive and degra-dative properties of human placental cytotrophoblast cellsin vitro J Cell Biol 109 891ndash902

Frederiksen M Vorkamp K Thomsen M and Knudsen L E(2009) Human internal and external exposure to PBDEs - Areview of levels and sources Int J Hyg Environ Health 212109ndash134

Gagnon-Kugler T Langlois F Stefanovsky V Lessard F andMoss T (2009) Loss of human ribosomal gene CpG methyla-tion enhances cryptic RNA polymerase II transcription anddisrupts ribosomal RNA processing Mol Cell 35 414ndash425

Gene Ontology Consortium (2015) Gene Ontology ConsortiumGoing forward Nucleic Acids Res 43 D1049ndashD1056

Giraldo J Vivas N M Vila E and Badia A (2002) Assessingthe (a)symmetry of concentration-effect curves Empiricalversus mechanistic models Pharmacol Ther 95 21ndash45

Golos T G Giakoumopoulos M and Gerami-Naini B (2013)Review Trophoblast differentiation from human embryonicstem cells Placenta 34 S56ndashS61

Grandjean P (1992) Individual susceptibility to toxicity ToxicolLett 64-65 43ndash51

arch 2019

Hakkola J Pasanen M Hukkanen J Pelkonen O Maenpaa JEdwards R J Boobis A R and Raunio H (1996) Expressionof xenobiotic-metabolizing cytochrome P450 forms in hu-man full-term placenta Biochem Pharmacol 51 403ndash411

Herbstman J B and Mall J K (2014) Developmental exposureto polybrominated diphenyl ethers and neurodevelopmentCurr Environ Health Rep 1(2) 101ndash112

Hromatka B S Drake P M Kapidzic M Stolp H Goldfien GA Shih Ie M and Fisher S J (2013) Polysialic acid enhancesthe migration and invasion of human cytotrophoblastsGlycobiology 23 593ndash602

Huang D W Sherman B T Tan Q Collins J R Alvord W GRoayaei J Stephens R Baseler M W Lane H C andLempicki R A (2007) The DAVID Gene FunctionalClassification Tool A novel biological module-centric algorithmto functionally analyze large gene lists Genome Biol 8 R183

Hunkapiller N M and Fisher S J (2008) Chapter 12 Placentalremodeling of the uterine vasculature Methods Enzymol 445281ndash302

Jauniaux E and Jurkovic D (2012) Placenta accretaPathogenesis of a 20th century iatrogenic uterine diseasePlacenta 33(4) 244ndash251

Jin S Fan F Fan W Zhao H Tong T Blanck P Alomo IRajasekaran B and Zhan Q (2001) Transcription factorsOct-1 and NF-YA regulate the p53-independent induction ofthe GADD45 following DNA damage Oncogene 20 2683ndash2690

Jinhui L Yuan C and Wenjing X (2015) Polybrominateddiphenyl ethers in articles A review of its applications andlegislation Environ Sci Pollut Res Int doi 101007s11356-015-4515-6

Jin Z T Li K Li M Ren Z G Wang F S Zhu J Y Leng X Sand Yu W D (2015) G-protein coupled receptor 34 knock-down impairs the proliferation and migration of HGC-27 gas-tric cancer cells in vitro Chin Med J 128 545ndash549

Jones S F and Infante J R (2015) Molecular pathways Fattyacid synthase Clin Cancer Res 21(24) 5434ndash5438

Jones R L Stoikos C Findlay J K and Salamonsen L A(2006) TGF-beta superfamily expression and actions in theendometrium and placenta Reproduction 132 217ndash232

Kalkunte S Huang Z Lippe E Kumar S Robertson L Wand Sharma S (2017) Polychlorinated biphenyls targetNotchDll and VEGF R2 in the mouse placenta and humantrophoblast cell lines for their anti-angiogenic effects SciRep 7 39885

Kappil M A Li Q Li A Dassanayake P S Xia Y Nanes J ALandrigan P J Stodgell C J Aagaard K M Schadt E Eet al (2016) In utero exposures to environmental organic pol-lutants disrupt epigenetic marks linked to fetoplacental de-velopment Environ Epigenet 2 dvv013

Kaufmann P Black S and Huppertz B (2003) Endovasculartrophoblast invasion Implications for the pathogenesis ofintrauterine growth retardation and preeclampsia BiolReprod 69 1ndash7

Kliman H J Nestler J E Sermasi E Sanger J M and StraussJ F (1986) Purification characterization and in vitro differ-entiation of cytotrophoblasts from human term placentaeEndocrinology 118 1567ndash1582

Knofler M and Pollheimer J (2013) Human placental tropho-blast invasion and differentiation A particular focus on Wntsignaling Front Genet 4 190

Kovo M Schreiber L Ben-Haroush A Gold E Golan A andBar J (2012) The placental component in early-onset andlate-onset preeclampsia in relation to fetal growth restric-tion Prenat Diagn 32 632ndash637

LaRocca J Binder A M McElrath T F and Michels K B(2014) The impact of first trimester phthalate and phenol ex-posure on IGF2H19 genomic imprinting and birth outcomesEnviron Res 133 396ndash406

Leonetti C Butt C M Hoffman K Miranda M L andStapleton H M (2016) Concentrations of polybrominateddiphenyl ethers (PBDEs) and 2 4 6-tribromophenol in hu-man placental tissues Environ Int 88 23ndash29

Li C S and Loch C R (2007) Sodium arsenite inhibits migra-tion of extravillous trophoblast cells in vitro Reprod Toxicol24(3-4) 296ndash302

Liebscher I Muller U Teupser D Engemaier E Engel K MRitscher L Thor D Sangkuhl K Ricken A Wurm A et al(2011) Altered immune response in mice deficient for the Gprotein-coupled receptor GPR34 J Biol Chem 286 2101ndash2110

Liu C C Cai D L Sun F Wu Z H Yue B Zhao S L Wu XS Zhang M Zhu X W Peng Z H et al (2017) FERMT1mediates epithelial-mesenchymal transition to promote co-lon cancer metastasis via modulation of beta-catenin tran-scriptional activity Oncogene 36 1779ndash1792

Liu X Meng L Shi Q Liu S Cui C Hu S and Wei Y (2013)Dermatopontin promotes adhesion spreading and migra-tion of cardiac fibroblasts in vitro Matrix Biol 32 23ndash31

Ma G T Soloveva V Tzeng S J Lowe L A Pfendler K CIannaccone P M Kuehn M R and Linzer D I (2001) Nodalregulates trophoblast differentiation and placental develop-ment Dev Biol 236 124ndash135

Maltepe E and Fisher S J (2015) Placenta The forgotten organAnnu Rev Cell Dev Biol 31 523ndash552

Minekawa R Sakata M Okamoto Y Hayashi M Isobe ATakeda T Yamamoto T Koyama M Ohmichi M TasakaK et al (2007) Involvement of RelA-associated inhibitor inregulation of trophoblast differentiation via interaction withtranscriptional factor specificity protein-1 Endocrinology 1485803ndash5810

Mundy W R Freudenrich T M Crofton K M and DeVito M J(2004) Accumulation of PBDE-47 in primary cultures of ratneocortical cells Toxicol Sci 82 164ndash169

Nomura Y John R M Janssen A B Davey C Finik JButhmann J Glover V and Lambertini L (2017)Neurodevelopmental consequences in offspring of motherswith preeclampsia during pregnancy Underlying biologicalmechanism via imprinting genes Arch Gynecol Obstet 2951319ndash1329

OrsquoConnell B A Moritz K M Walker D W and Dickinson H(2013) Synthetic glucocorticoid dexamethasone inhibitsbranching morphogenesis in the spiny mouse placenta BiolReprod 88 26

Okamoto O Fujiwara S Abe M and Sato Y (1999)Dermatopontin interacts with transforming growth factorbeta and enhances its biological activity Biochem J 337537ndash541

Ouko L A Shantikumar K Knezovich J Haycock P SchnughD J and Ramsay M (2009) Effect of alcohol consumption onCpG methylation in the differentially methylated regions ofH19 and IG-DMR in male gametes Implications for fetal alco-hol spectrum disorders Alcohol Clin Exp Res 33 1615ndash1627

Park H R Kamau P W and Loch-Caruso R (2014)Involvement of reactive oxygen species in brominateddiphenyl ether-47-induced inflammatory cytokine releasefrom human extravillous trophoblasts in vitro Toxicol ApplPharmacol 274 283ndash292

Park H R and Loch-Caruso R (2014) Protective effect of nu-clear factor E2-related factor 2 on inflammatory cytokine

arch 2019

response to brominated diphenyl ether-47 in the HTR-8SVneo human first trimester extravillous trophoblast cellline Toxicol Appl Pharmacol 281(1) 67ndash77

Park H R and Loch-Caruso R (2015) Protective effect of (thorn-)al-pha-tocopherol on brominated diphenyl ether-47-stimulated prostaglandin pathways in human extravilloustrophoblasts in vitro Toxicol In Vitro 29(7) 1309ndash1318

Penell J Lind L Fall T Syvanen A C Axelsson TLundmark P Morris A P Lindgren C Mahajan ASalihovic S et al (2014) Genetic variation in the CYP2B6gene is related to circulating 2 20 4 40-tetrabromodiphenylether (BDE-47) concentrations An observational population-based study Environ Health 13 34

Prins J R Gomez-Lopez N and Robertson S A (2012)Interleukin-6 in pregnancy and gestational disorders JReprod Immunol 95 1ndash14

Puscheck E E Awonuga A O Yang Y Jiang Z and RappoleeD A (2015) Molecular biology of the stress response in theearly embryo and its stem cells Adv Exp Med Biol 84377ndash128

Qiu C Gelaye B Denis M Tadesse M G Luque Fernandez MA Enquobahrie D A Ananth C V Sanchez S E andWilliams M A (2015) Circadian clock-related genetic riskscores and risk of placental abruption Placenta 36 1480ndash1486

Rajakumar C Guan H Langlois D Cernea M and Yang K(2015) Bisphenol A disrupts gene expression in human pla-cental trophoblast cells Reprod Toxicol 53 39ndash44

Red-Horse K Kapidzic M Zhou Y Feng K T Singh H andFisher S J (2005) EPHB4 regulates chemokine-evoked tro-phoblast responses A mechanism for incorporating the hu-man placenta into the maternal circulation Development 1324097ndash4106

Red-Horse K Zhou Y Genbacev O Prakobphol A Foulk RMcMaster M and Fisher S J (2004) Trophoblast differentia-tion during embryo implantation and formation of thematernal-fetal interface J Clin Invest 114 744ndash754

Roadmap Epigenomics C Kundaje A Meuleman W Ernst JBilenky M Yen A Heravi-Moussavi A Kheradpour PZhang Z Wang J et al (2015) Integrative analysis of 111reference human epigenomes Nature 518 7539317ndash7539330

Robins J C Marsit C J Padbury J F and Sharma S S (2011)Endocrine disruptors environmental oxygen epigeneticsand pregnancy Front Biosci E3 690ndash700

Robinson J F Gormley M J and Fisher S J (2016) A genomics-based framework for identifying biomarkers of human neu-rodevelopmental toxicity Reprod Toxicol 60 1ndash10

Robinson J F Kapidzic M Gormley M Ona K Dent TSeifikar H Hamilton E G and Fisher S J (2017)Transcriptional dynamics of cultured human villous cytotro-phoblasts Endocrinology 158 1581ndash1594

Robinson J F and Piersma A H (2013) Toxicogenomicapproaches in developmental toxicology testing MethodsMol Biol 947 451ndash473

Robinson J F Yu X Moreira E G Hong S and Faustman EM (2011) Arsenic- and cadmium-induced toxicogenomic re-sponse in mouse embryos undergoing neurulation ToxicolAppl Pharmacol 250 117ndash129

Romero R Dey S K and Fisher S J (2014) Preterm labor Onesyndrome many causes Science 345 760ndash765

Rose-John S Winthrop K and Calabrese L (2017) The role ofIL-6 in host defence against infections Immunobiology andclinical implications Nat Rev Rheumatol 13 399ndash409

Saeed A I Bhagabati N K Braisted J C Liang W Sharov VHowe E A Li J Thiagarajan M White J A and

Quackenbush J (2006) TM4 microarray software suiteMethods Enzymol 411 134ndash193

Sales A J and Joca S R (2016) Effects of DNA methylationinhibitors and conventional antidepressants on mice behav-iour and brain DNA methylation levels Acta Neuropsychiatr28(1) 11ndash22

Schreiber T Gassmann K Gotz C Hubenthal U Moors MKrause G Merk H F Nguyen N H Scanlan T S Abel Jet al (2010) Polybrominated diphenyl ethers induce develop-mental neurotoxicity in a human in vitro model Evidencefor endocrine disruption Environ Health Perspect 118572ndash578

Shen Q Li Z Q Sun Y Wang T Wan C L Li X W Zhao XZ Feng G Y Li S St Clair D et al (2008) The role of pro-inflammatory factors in mediating the effects on the fetus ofprenatal undernutrition Implications for schizophreniaSchizophr Res 99 48ndash55

Silva J F and Serakides R (2016) Intrauterine trophoblast mi-gration A comparative view of humans and rodents CellAdh Migr 10(1-2) 88ndash110

Simmons S O Fan C Y and Ramabhadran R (2009) Cellularstress response pathway system as a sentinel ensemble intoxicological screening Toxicol Sci 111 202ndash225

Simon R Lam A Li M C Ngan M Menenzes S and Zhao Y(2007) Analysis of gene expression data using BRB-ArrayTools Cancer Inform 3 11ndash17

Skinner M K and Guerrero-Bosagna C (2009) Environmentalsignals and transgenerational epigenetics Epigenomics 1(1)111ndash117

Smith A C Choufani S Ferreira J C and Weksberg R (2007)Growth regulation imprinted genes and chromosome11p155 Pediatr Res 61 43Rndash47R

Sorokin Y Romero R Mele L Iams J D Peaceman A MLeveno K J Harper M Caritis S N Mercer B M Thorp JM et al (2014) Umbilical cord serum interleukin-6 C-reac-tive protein and myeloperoxidase concentrations at birthand association with neonatal morbidities and long-termneurodevelopmental outcomes Am J Perinatol 31 717ndash726

Susiarjo M Sasson I Mesaros C and Bartolomei M S (2013)Bisphenol A exposure disrupts genomic imprinting in themouse PLoS Genet 9 e1003401

Szyf M (2011) The implications of DNA methylation for toxicol-ogy Toward toxicomethylomics the toxicology of DNAmethylation Toxicol Sci 120 235ndash255

Tagliaferri S Caglieri A Goldoni M Pinelli S Alinovi R PoliD Pellacani C Giordano G Mutti A and Costa L G(2010) Low concentrations of the brominated flame retard-ants BDE-47 and BDE-99 induce synergistic oxidative stress-mediated neurotoxicity in human neuroblastoma cellsToxicol In Vitro 24 116ndash122

Thumser A E Moore J B and Plant N J (2014) Fatty acid bind-ing proteins Tissue-specific functions in health and diseaseCurr Opin Clin Nutr Metab Care 17 124ndash129

Tjoa M L van Vugt J M Go A T Blankenstein M AOudejans C B and van Wijk I J (2003) Elevated C-reactiveprotein levels during first trimester of pregnancy are indica-tive of preeclampsia and intrauterine growth restriction JReprod Immunol 59 29ndash37

Tung E W Y Kawata A Rigden M Bowers W J Caldwell DHolloway A C Robaire B Hales B F and Wade M G(2017) Gestational and lactational exposure to anenvironmentally-relevant mixture of brominated flameretardants Effects on neurodevelopment and metabolismBirth Defects Res 109 497ndash512

arch 2019

Tuohey L Macintire K Ye L Palmer K Skubisz M Tong Sand Kaitursquou-Lino T J (2013) PLAC4 is upregulated in severeearly onset preeclampsia and upregulated with syncytialisa-tion but not hypoxia Placenta 34 256ndash260

Vaiman D Calicchio R and Miralles F (2013) Landscape oftranscriptional deregulations in the preeclamptic placentaPLoS One 8 e65498

Vinnars M T Nasiell J Holmstrom G Norman M WestgrenM and Papadogiannakis N (2014) Association between pla-cental pathology and neonatal outcome in preeclampsia Alarge cohort study Hypertens Pregnancy 33 145ndash158

Vock C Doring F and Nitz I (2008) Transcriptional regulationof HMG-CoA synthase and HMG-CoA reductase genes by hu-man ACBP Cell Physiol Biochem 22 515ndash524

Winn V D Haimov-Kochman R Paquet A C Yang Y JMadhusudhan M S Gormley M Feng K T Bernlohr D AMcDonagh S Pereira L et al (2007) Gene expression profil-ing of the human maternal-fetal interface reveals dramaticchanges between midgestation and term Endocrinology 1481059ndash1079

Woodruff T J Zota A R and Schwartz J M (2011)Environmental chemicals in pregnant women in the UnitedStates NHANES 2003ndash2004 Environ Health Perspect 119878ndash885

Woods R Vallero R O Golub M S Suarez J K Ta T AYasui D H Chi L H Kostyniak P J Pessah I N BermanR F et al (2012) Long-lived epigenetic interactions betweenperinatal PBDE exposure and Mecp2308 mutation Hum MolGenet 21 2399ndash2411

Wu Q Ohsako S Ishimura R Suzuki J S and Tohyama C(2004) Exposure of mouse preimplantation embryos to 2 37 8-tetrachlorodibenzo-p-dioxin (TCDD) alters the methyla-tion status of imprinted genes H19 and Igf2 Biol Reprod 701790ndash1797

Yamamoto M Khan N Muniroh M Motomura EYanagisawa R Matsuyama T and Vogel C F (2017)Activation of interleukin-6 and -8 expressions by

methylmercury in human U937 macrophages involves RelAand p50 J Appl Toxicol 37 611ndash620

Yang K Julan L Rubio F Sharma A and Guan H (2006)Cadmium reduces 11 beta-hydroxysteroid dehydrogenasetype 2 activity and expression in human placental tropho-blast cells Am J Physiol Endocrinol Metab 290 E135ndashE142

Zhang S Yang Y and Shi Y (2005) Characterization of humanSCD2 an oligomeric desaturase with improved stability andenzyme activity by cross-linking in intact cells Biochem J388 135ndash142

Zhao Y Ao H Chen L Sottas C M Ge R S and Zhang Y(2011) Effect of brominated flame retardant BDE-47 on an-drogen production of adult rat Leydig cells Toxicol Lett 205209ndash214

Zhu J Y Pang Z J and Yu Y H (2012) Regulation of tropho-blast invasion The role of matrix metalloproteinases RevObstet Gynecol 5 e137ndashe143

Zhu Y Tan Y Q and Leung L K (2017a) Exposure to 2 20 440-tetrabromodiphenyl ether at late gestation modulates pla-cental signaling molecules in the mouse model Chemosphere181 289ndash295

Zhu Y Tan Y Q Wang C C and Leung L K (2017) The flameretardant 2 20 4 40-Tetrabromodiphenyl ether enhances theexpression of corticotropin-releasing hormone in the placen-tal cell model JEG-3 Chemosphere 174 499ndash505

Zota A R Linderholm L Park J S Petreas M Guo TPrivalsky M L Zoeller R T and Woodruff T J (2013)Temporal comparison of PBDEs OH-PBDEs PCBs and OH-PCBs in the serum of second trimester pregnant womenrecruited from San Francisco General Hospital CaliforniaEnviron Sci Technol 47 11776ndash11784

Zota A R Mitro S D Robinson J F Hamilton E G Park J SParry E Zoeller R T and Woodruff T J (2018)Polybrominated diphenyl ethers (PBDEs) and hydroxylatedPBDE metabolites (OH-PBDEs) in maternal and fetal tissuesand associations with fetal cytochrome P450 gene expres-sion Environ Int 112 269ndash278

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R E S E A R C H R E S O U R C E

Transcriptional Dynamics of Cultured HumanVillous Cytotrophoblasts

Joshua F Robinson123 Mirhan Kapidzic123 Matthew Gormley123

Katherine Ona12 Terrence Dent12 Helia Seifikar12 Emily G Hamilton12

and Susan J Fisher123456

1Center for Reproductive Sciences University of California San Francisco California 94143 2Department ofObstetrics Gynecology and Reproductive Sciences University of California San Francisco California 941433Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research University of CaliforniaSan Francisco California 94143 4Division of Maternal Fetal Medicine University of California San FranciscoCalifornia 94143 5Department of Anatomy University of California San Francisco California 94143 and6Human Embryonic Stem Cell Program University of California San Francisco California 94143

During human pregnancy cytotrophoblasts (CTBs) play key roles in uterine invasion vascularremodeling and anchoring of the feto-placental unit Due to the challenges associated withstudying human placentation in utero cultured primary villous CTBs are used as a model of thedifferentiation pathway that leads to invasion of the uterine wall In vitro CTBs emulate in vivo cellbehaviors such as migration aggregation and substrate penetration Although some of themolecular features related to these cell behaviors have been described the underlyingmechanismsat a global level remain undefined at midgestation Thus in this study we characterized second-trimester CTB differentiationinvasion in vitro correlating themajor morphological transitions withthe transcriptional changes that occurred at these steps After plating onMatrigel as individual cellsCTBs migrated toward each other and formed multicellular aggregates In parallel using amicroarray approach we observed differentially expressed (DE) genes across time which wereenriched for numerous functions including cellmigration vascular remodelingmorphogenesis cellcommunication and inflammatory signaling DE genes encoded several molecules that we andothers previously linked to critical CTB function in vivo suggesting that the novel DE molecules wediscovered played important roles Immunolocalization confirmed that CTBs in situ gave a signal fortwo of themost highly expressed genes in vitro In summary we characterized at a global level thetemporal dynamics of primary human CTB gene expression in culture These data will enable futureanalyses of various types of in vitro perturbationsmdashfor example modeling disease processes andenvironmental exposures (Endocrinology 158 1581ndash1594 2017)

W ithin the intervillous space of the human placentanetworks of chorionic villi suspended in circu-

lating maternal blood facilitate the vital exchange ofnutrients wastes and gases between the maternal andembryonicfetal units (Fig 1) The villi are covered in twotrophoblast layers The outer layer is composed of mul-tinucleated syncytiotrophoblasts transport and hormone-producing cells Beneath resides a polarized layer ofmononuclear cytotrophoblast (CTB) progenitors (2)

Depending on location CTBs differentiate into syncytio-trophoblasts or exit the placenta to anchor the embryofetus to the uterus During the latter process the cellsdetach from the trophoblast basement membrane of thevilli and aggregate to form columns of unpolarized cellswhich attach to the uterine wall Invasive CTBs alsoknown as extravillous trophoblasts deeply invade (in-terstitially) into the uterine wall reaching the inner thirdof the myometrium during normal pregnancy During

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in USACopyright copy 2017 Endocrine SocietyReceived 31 August 2016 Accepted 30 January 2017First Published Online 8 March 2017

Abbreviations ANOVA analysis of variance AQP aquaporin BSA bovine serumalbumin CK cytokeratin CTB cytotrophoblast DE differentially expressed ECMextracellular matrix FC fold change FE fold enrichment GO gene ontology IgGimmunoglobulin G MMP matrix metalloproteinase PBS phosphate-buffered saline PEpreeclampsia qRT-PCR quantitative reverse transcription polymerase chain reactionRNA-seq RNA sequencing RPKM reads per kilobase per million mapped reads

doi 101210en2016-1635 Endocrinology June 2017 158(6)1581ndash1594 httpsacademicoupcomendo 1581

icoupcomendoarticle-abstract158615813063792 by C

arch 2019

remodeling of the uterine vessels endovascular CTBspenetrate the walls of the spiral arteries increasing theirdiameter decreasing resistance and diverting maternalblood flow to the placenta (3 4) Due to their diverse andimportant roles in placental development perturbationsin CTB differentiation may be associated with severalpregnancy complications such as preeclampsia (PE) (5)6intrauterine growth restriction preterm labor (6) andthe syndromes associated with excessive invasion (egaccreta percreta and increta) (7)

Influenced by numerous cues (8) CTBs modulate theexpression of molecules that mediate adhesion migra-tion and cellndashcell communication which underlie theirbroad functional capabilities For example as CTBspenetrate the uterine wall they downregulate the ex-pression of adhesionmolecules that inhibit invasion suchas E-cadherin and upregulate others that favor thisprocess (1) To facilitate invasion CTBs release numer-ous degradative molecules including matrix metal-loproteinase (MMP) family members (9) which breakdown basementmembrane components and extracellularmatrix (ECM) molecules they encounter At the sametime the cells upregulate other factors that play a role in

their unusual ability to mimic endothelial and vascularsmooth muscle cells These include other adhesion mol-ecules [eg VE-cadherin (10) neural cell adhesion mol-ecule (11)] as well as Ephephrin and Notch familymembers (12 13) which most likely play crucial rolesin the ability of endovascular CTBs to channel bloodthat flows through the maternal spiral arteries whichthey line Furthermore the cells express a complex net-work of molecules (eg interleukins chemokines andHLA-G) that mediate their interactions with the ma-ternal immune system (14) Determining at a global levelthe mechanisms controlling these behaviors (eg migra-tion adhesion invasion and immune tolerance) which arecritical for CTB functions is key to understanding normalplacental development and disease

Due to the difficulty of studying human CTBs in uteroand known differences between human and rodent pla-centation and pregnancy primary human cell culturemodels are valuable because they enable studies thataddress CTB functions at cellular and molecular levels(15) Over the past 30 years protocols have been de-veloped for isolating and culturingCTBs from placentas ofvarious gestational ages (16 17) Multiple steps have been

Figure 1 Arrangement of cytotrophoblasts (CTBs) at the human maternalndashfetal interface Within the intervillous space of the human placentafloating chorionic villi are suspended in circulating maternal blood These structures facilitate the exchange of nutrients wastes and gasesbetween the maternal and embryonicfetal units Villi are covered in two trophoblast layers as follows (1) an outer layer of multinucleatedsyncytiotrophoblasts and (2) a polarized layer of mononuclear CTBs In anchoring chorionic villi during the first and second trimesters of pregnancyCTBs which are differentiating along the invasive pathway exit the placenta by detaching from the trophoblast basement membrane andaggregating to form columns of unpolarized cells which attach to the uterine wall CTBs invade the decidua (interstitial invasion) or remodel theuterine spiral arteries (endovascular invasion) which increases their elasticity and diverts maternal blood flow to the placenta CTB culturescontain cells isolated from floating and anchoring villi BV fetal blood vessel MF macrophage [modified from Damsky et al (1)]

1582 Robinson et al Transcriptional Dynamics of Cultured Human CTBs Endocrinology June 2017 158(6)1581ndash1594

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introduced to improve the viability and purity of CTBs forexample serial enzymatic digestions Percoll gradientseparations andmagnetic bead immunodepletions (18) Inthis context CTBs isolated fromnormal pregnancies and avariety of pregnancy complications are used to study awide range of placental cell functions in vitro

Transcriptomic-based approaches provide a meansto survey global RNA expression changes at gene andpathway levels Additionally the in vitro and in vivoexpression of the identified molecules can be directlycompared as an independent measure of their potentialrelevance to human pregnancy In placental cells or tissueglobal assessments of RNA expression have been used toinvestigate the molecular changes that occur during preg-nancy and the factors that may influence expression suchas gestational age (19) tissue specification (20) species(21) and normal vs disease states (22) This approach hasalso been used to profile subsets of primary CTBs (23)

Our laboratory routinely isolates and cultures CTBsfrom first- and second-trimester placentas as well as termtissue We use this cell culture model to study at cellularand molecular levels the differentiation pathway thatleads to invasion of the uterine wall (16 24) In this studybuilding on this work we describe CTB morphologicaltransitions and parallel transcriptomic changes over timein vitro as the cells acquire an invasive phenotype Thedetailed characterization of this culture model during thesecond trimester of pregnancy will expand the utility ofthis system for studies of placental development in normalpregnancy and disease and for studies of the effects ofpossible perturbants such as environmental exposures

Materials and Methods

Tissue collectionAll methods in this study were approved by the University of

California San Francisco Institutional Review Board In-formed consent was obtained from all donors Second-trimesterplacentas (gestational age 14 to 22 weeks) were collected im-mediately following elective terminations and placed in cyto-wash medium consisting of DMEH-21 (Gibco) 125 fetalbovine serum (Hyclone) 1glutamine plus (AtlantaBiologicals)1 penicillinstreptomycin (Invitrogen) and 01 gentamicin(Gibco) Tissue samples were placed on ice prior to dissection

Human primary villous CTB isolationCTBs consisting of both extravillous and villous CTBs were

isolated as described (16) with minor modifications fromsecond-trimester human placentas (gestational ages ranged from14 to 22 weeks) In brief the floating and anchoring chorionicvilli were extensively washed in cold phosphate-buffered saline(PBS) dissected into 2- to 4-mm pieces and filtered through a1-mm mesh strainer to remove small pieces of tissue CTBs wereisolated according to the following steps (1) removal of the outersyncytial layer via collagenase (Sigma-Aldrich C-2674) di-gestion (2) release of the CTBs by sequential enzymatic digestion

[trypsin (Sigma-Aldrich T8003 twice) and collagenase] and (3)purification via Percoll density gradient centrifugation Single cellswere counted using a hemacytometer and immediately collected(0-hour samples) or transferred to a Matrigel (BD Biosciences)-coated 12-well plate The substrate consisted of a 11 (volume-to-volume) mixture of Matrigel and culture medium (see later)which was incubated for 15minutes at 37degC CTBs were culturedat a density of 500000 CTBswell in 15 mL medium containingDMEH-21 2 Nutridoma (Roche) 1 sodium pyruvate(Sigma-Aldrich) 1 HEPES buffer (Invitrogen) 1 glutamateplus (Atlanta Biologicals) and 1 penicillinstreptomycin (Invi-trogen) Cells were incubated at 37degC in 5 CO295 air At3 hourspostplatingmediumwas replaced to eliminate unattachedcells As previously reported staining with anti-cytokeratin (CK)showed that cell purity was routinely 80 to 90 (16) Onlycell preparations that met this criterionwere analyzed Because weanalyzed primary CTBs contaminants (eg immune and stromalcells) could contribute to the downstream analyses

Immunolocalization of CTB antigensAt 3 15 or 39 hours of culture medium was removed and

cells were washed once with PBS Next CTBs were fixed with4 paraformaldehyde for 20 minutes washed twice with PBSand stored in PBS at 4degC until further processing PBS wasremoved and cold methanol was added to permeabilize theCTBs After 5 minutes the cells were washed with PBS threetimes and 5 bovine serum albumin (BSA) (Hyclone)PBS wasadded for 1 hour to block nonspecific reactivity The blockingsolution was removed the primary antibody (in 5 BSA) wasadded and the cells were incubated overnight at 4degC Pri-mary antibodies included the following anti-CK [catalogueFisher_001-clone7D3 Research Resource Identifier (RRID)AB_2631235 rat monoclonal 1100] (8) anti-HLA-G (cata-logue Fisher_002-clone4H84 RRIDAB_2631236 mousemonoclonal 1100) (25) and anti-Ki-67 (Thermo Fisher Sci-entific catalogue RM-9106-S1 RRID AB_149792 rabbitmonoclonal 1100) CKs recognized by the rat antibody arehighly expressed by human CTBs vs other placental cell typespossible contaminants (26) HLA-G a major histocompatibilityclass Ib antigen is specific for extravillous CTBs (25 27) Ki-67expression a nuclear antigen is used to identify proliferatingcells (28) The next day the antibody solution was removed andthe CTBs were washed three times in PBS Detection of primaryantibodies was accomplished via incubation (1 hour) withspecies-specific secondary antibodies diluted in PBSgoat anti-rat immunoglobulin G (IgG Alexa Fluor 11000 A11081Life Technologies) donkey anti-mouse (11000 A21202 LifeTechnologies) and goat anti-rabbit IgG (11000 A21206 LifeTechnologies) Cultures were washed with PBS three times andtransferred to 15 mL PBS mixed with Hoechst 33342 (12500Life Technologies) Phase brightfield and fluorescent photomontageswere created by stitching together images (42 covering a5-mm 3 5-mm area) which were captured by using a Leicainvertedmicroscopewith a103objective and the tilescan function(Leica Application Suite Advanced Fluorescence)

We calculated the average fluorescence intensity associatedwith CK HLA-G or Ki-67 on a per cell basis by using Volocitysoftware (PerkinElmer version 63) First we identified all cellsor objects within each image by virtue of Hoechst stainingInitially we excluded objects that intersected with the border ofthe image or were 20 mm2 which eliminated potential arti-facts (eg cell debris) As a result of this process 1 of the

doi 101210en2016-1635 httpsacademicoupcomendo 1583

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objects were discarded from the analysis We applied a sepa-ration of object function which included automated erosionand division operations to enable improved measurements ofaggregated cell populations Next we determined the averageintensity of CKHLA-G or Ki-67 per object For CK orHLA-Gwe used the dilate function (level 4) to measure the intensity ofimmunoreactivity associated with the cytoplasm and plasmamembrane respectively Immunopositive-negative intensitythresholds for CK or HLA-G immunostaining were determinedas the mean intensity of $30 objects with negative signal + 1standard deviation Thresholds (6) for Ki-67 average intensitywere calculated as the mean average intensity of $30 objectswith immunopositive Ki-67 nuclear signals 21 standard de-viation To control for variability in overall fluorescence in-tensity among experiments limits were determined on a per wellbasis for each experiment and time point Percentages of cellsthat expressed each antigen were calculated as the number ofimmunopositive cells per total number of cells per stitchedimage Average values were determined across$3 independentcultures Images captured 20000 cellsper well

Quantification of CTB migrationTo describe migration and aggregation of CTBs over time

we determined the minimum distance between nuclei usingVolocity software Objects were identified as described in theprevious section Then we calculated the minimum distancebetween nuclei (centroid to centroid) This process entailedautomated measurements of all possible distances betweenobjects within each image Three to 12 stitched images wereanalyzed per independent experiment (n = 3) and the averageminimum distance among cells at 3 15 and 39 hours post-plating was calculated The standard error of the mean wascomputed across the average of the three independent experi-ments Representative images at 203 were exported in TIFformat and processed via Photoshop (Adobe)

RNA isolationPlacentas for transcriptional profiling ranged in gestational

age from 14 to 21weeks (mean = 175 weeks) Two experimentsused cells from individual placentas and two experimentsused a combination of cells that were isolated from two pla-centas The contribution of female and male samples to the datawas estimated by evaluating CTB expression (0 hours) of sex-specific genes RPS4Y1 XIST EIF1AY and KDM5D (29)(Supplemental Fig 1) Information about gestational age andsex is included in Supplemental Table 1 Samples for quanti-tative reverse transcription polymerase chain reaction (qRT-PCR) validation ranged in gestational age from 183 to22 weeks (mean = 203 weeks) RNA for these analyses wasisolated from CTBs that were prepared from individual pla-centas Briefly immediately following CTB isolation (0 hours)or after 3 15 19 or 39 hours in culture RLT lysis buffer(Qiagen) was added to either the cell pellet or culture dish wellThe lysate was collected and stored at 280degC We isolated andpurified RNA using the RNeasy Micro Kit (Qiagen) The RNAconcentration and quality were estimated (absorbance 260 nm280 nm = 19 to 21) by using a Nanodrop spectrometer(Thermo Fisher Scientific) Samples destined for microarrayanalyses (0- 3- 15- and 39-hour time points) were assessed forquality (RNA integrity number 9) using the Agilent RNA6000 Nano LabChip Kit and Bioanalyzer 2100 system

Global gene expression profiling of CTBsThe analysis platform was the Affymetrix Human Genome

U133 Plus 20 array Sample processing and hybridization wereperformed by the University of California San FranciscoGladstone Institute as previously described (29) AffymetrixCEL files were processed using the Affymetrix ExpressionConsole and Transcriptome Analysis Console software pack-ages Raw values were normalized via the robust multiarrayaverage algorithm Raw and normalized data were deposited inthe Gene Expression Omnibus (GSE86171) One-way analysisof variance (ANOVA) was applied to identify differentiallyexpressed (DE) genes across time Average fold change (FC)values were determined by calculating the ratio of average log 2intensities between each time point and the 0-hour CTB groupDatasets were annotated using the Affymetrix TranscriptomeAnalysis Console database (10114) In cases ofmultiple probesper gene the one with the lowest P value having the mostsignificant change over time was selected for comparisonpurposes To define DE genes across time and among sampleswe applied a cutoff of P 000005 (one-way ANOVA) anabsolute FC$ 2 between any of the four time points with a falsediscovery rate of1Hierarchical clustering of FC values wascompleted by using average linkage and Euclidean distance(TIGRMEV) (30) A secondary post hoc Student t test was usedto determine the significance of changes between each time pointand time 0 hours (cutoff applied P 0001 absolute FC $ 2)

Functional analyses of DE genesWe used DAVID (31) to identify functional enrichment of gene

ontology (GO) biological processes (level 4) within our DE gene listGenes were defined using the Affymetrix Probe ID GO termscontaining $15 DE genes with a P value of 0005 and a foldenrichment (FE)$15were selected as significantly overrepresentedCorresponding enrichment scores (ie P values and FE were alsodetermined for upregulated and downregulated gene clusters) Aspreviously described (32) relative enrichment scores across GOterms were calculated as2log (p 2 value) 3 FE We grouped GOterms based on GO classification (httpgeneontologyorg) toexpress common themes To evaluate changes within GO bi-ological processes on a temporal level we calculated absoluteaverage FC ratios of DE genes across time within enriched GOswhich were selected based on their relevancy to human placentaldevelopment

Validation of DE genesThese analyses used an independent set of CTBs cultured for

0 3 15 19 and 39 hours First RNA was converted to com-plementary DNA using an ISCRIPT complementary DNA syn-thesis kit (Bio-Rad) Next qRT-PCR was performed usingTaqMan Universal Master Mix II no UNG (Life Technologies)and specific TaqManprimers forCBSCXCL6DHCR7DUSP2F5 FABP7 ITGA2 MMP9 MMP12 PDXK PEG3 andS100A7 (Supplemental Table 2) Selected targets were identifiedbased on observations of robust (absolute FC 2) and significant(P 00005) changes in expression over time and associationwithpathways of interest We also included PEG3 (P = 0005 absoluteFC 2) and MMP12 (P = 04) to estimate the repeatability oftargets with different significance criteria Reactions were carriedout for 40 cycles A minimum of three biological replicates wasanalyzed in all comparisons Differential expression across timewas calculatedbyusing theDD cycle thresholdmethod (normalized

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to glyceraldehyde-3-phosphate dehydrogenase) To determinesignificant changes over time one-way ANOVAwas applied (P005) FC values were expressed as average log 2 ratios betweeneach time point and the 0-hour CTB group

Comparisons between second-trimester andterm samples

We assessed the expression profiles of genes identified to beDE in CTB culture in an RNA sequencing (RNA-seq) datasetgenerated by the Epigenome Roadmap Project (20) which in-cluded CTBs purified in our laboratory from second-trimesterand term placentas (0 hours)We compared the average reads perkilobase per million mapped reads (RPKM) expression values ofsecond-trimester and term CTBs with differences in expressionbetween these time periods The official gene symbol was used toalign the two datasets (merge function) (33)

Immunostaining of tissue sectionsWe used an immunolocalization approach to investigate the

expression at the protein level of transcripts that were abun-dant as well as DE over time in cultured CTBs For this purposewe analyzed tissue sections of the maternalndashfetal interface duringthe second trimester of pregnancy The general method we usedwas published (34) Briefly biopsies were fixed in 3 para-formaldehyde dehydrated in increasing sucrose concentrationsand embedded in OCT (Thermo Fisher Scientific) Immunoloc-alization of proteins was performed using species-specific pri-mary antibodies diluted in blocking buffer (PBS with 3 BSAand 005 Tween 20) for anti-CK (rat polyclonal 1100) (8)anti-NOTUM (Sigma-Aldrich catalogue SAB3500082 RRIDAB_10604118 rabbit polyclonal 1100) and anti-EFEMP1(Abcam catalogue ab14926 RRIDAB_301517 rabbit poly-clonal 1100) for 1 hour at 37degC Detection of primaryantibodieswas done via incubation (1 hour at 37degC)with species-specific secondary antibodies diluted in blocking buffer goatanti-rat IgG (Alexa Fluor 11000 A11081 Life Technologies)and goat anti-rabbit IgG (11000 A21206 Life Technologies)Sectionswerewashedwith PBS three times and coverslippedwithVectashield containing 406-diamidino-2-phenylindole (VectorBio-Laboratories) For these analyses tissue sections from at least12 placentas were evaluated Images were acquired using a Leicainvertedmicroscopewith a 103 or203objectiveRepresentativephotomicrographs were exported in TIF format and processedvia Photoshop (Adobe) No specific immunoreactivity was de-tected staining with the primary or secondary antibody alone orwith an irrelevant isotype-matched antibody

Results

Characterization of cultured CTBsCTBs isolated from floating and anchoring chorionic

villi of second-trimester placentas were plated on Matrigeland cultured for up to 39 hours Within 3 hours CTBsstarted to spread on the matrix and extend cellularprojections [Fig 2(andashc)] By 15 hours CTBs formedmulticellular aggregates Time-lapse imaging indicateda high level of cell movement within the aggregates athigher resolution (data not shown) Furthermore aspreviously described (16) CTBs did not fuse to form

multinucleated syncytiotrophoblasts To complementour visual observations we quantified the distance ofeach CTB to its closest neighbor at 3 15 and 39 hoursWe observed a significant difference in the averageminimum distance among cells at 15 hours (Ddarr45 mm)as compared with 3 hours [P 005 Fig 2(d)] Cell ag-gregation was similar at 15 and 39 hours (P $ 005)Using a semiquantitative approach that combined im-munofluorescence localization and high-throughputcontent image analysis we calculated (on average) thatthe majority of cells in our model expressed CK (861 616) [Fig 2(e)] and HLA-G (7236 44) [Fig 2(f)] Incontrast only 21 6 06 of cells expressed Ki-67 [Fig2(g)] The expression of CK HLA-G and Ki-67 did notchange as a function of time in culture [quantified in Fig2(h)] As a whole these observations suggested that ourtissue culture system models CTB exit from the cell cycleaggregation a proxy for differentiation

We identified 2232 genes as significantly DE acrosstime (0ndash39 hours) as CTBs differentiated in culture(ANOVA P 000005 absolute FC $ 2) [Fig 3(a)]Within this gene set time-dependent changes were highlydynamic Approximately 54 of DE genes followed apositive trend across time (Cluster I) and conversely46 of DE genes displayed a negative trend (Cluster II)As comparedwith the 0-hour data the largest differencesin terms of absolute magnitude and significance wereobserved at 15 hours [Fig 3(b)] These analyses suggestedrobust time-dependent changes in the transcriptome ofCTBs during the initial 39 hours of culture

As to theDE subsetwe observed an enrichment of genesinvolved in a diverse range of GO biological processesincluding anatomical structural morphogenesis vascula-ture development cell motion RNA metabolism trans-lation monosaccharide metabolism energy derivation byoxidation of organic compounds inflammatory responsecoenzyme metabolism regulation of cell proliferation andapoptosis (P 0005 FE $ 15) [Fig 4(a)] We alsoconducted separate GO analyses for Clusters I or II Onaverage genes that were involved in RNAmetabolism andenergy derivation were upregulated over time This wasparticularly true for RNAs encoding proteins that functionin the electron transport chain (30uarr 2darr) Enriched cate-gories of genes that were predominately downregulatedover time in culture included response to lipopolysaccha-ride (6uarr 15darr) and regulation of proliferation (20uarr 47darr)

With regard to selected GO categories expression ofgenes related to inflammatory responses anatomical struc-ture morphogenesis regulation of cell migrationmotionapoptosis RNA metabolism and electron transport chainchanged in a time-dependent manner peaking at 15 hours[absolute FC Fig 4(b)] For example at this time point therewas a fourfold change in expression of genes that are

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involved in inflammatory responses (log 2 =20) In contrastthe expression of genes related to vitamin responses andvascular development peaked at 39 hours with the highestobserved FCs 64 (log 2 = 27) and 33 (log 2 = 17)respectively

Due to our interest in the mechanisms underlyingCTB differentiationinvasion and vascular mimicry wemapped DE genes (additional filter absolute FC 4)associated with three GO biological processes anatom-ical structure morphogenesis (28 genes) cell motion (thefamily term of cell migration 18 genes) and vasculaturedevelopment (23 genes) [Fig 5(a)] This gene subset in-cluded ITGA2 S100A7 ETS1 EDN1 IL6 DHCR7MMP9 and CD44 which were upregulated over timeand DLX6 FOXC1 KISS1R DLX5 FLT1 BMP7NR4A3 and CITED2 which were downregulatedMolecules linked to response to vitamin were also clus-tered due to their strong regulation and the influence ofvitamins on pregnancy outcome This subset includedp450 enzymes (CYP1A1 -27B1) the vitamin D receptorCD44 and aquaporin (AQP)3 which were all upregu-lated in culture [Fig 5(b)] We also listed DE genes withthe largest FCs in our culture system This subsetincluded a variety of molecules that are linked to theaforementioned categories and additional pathwaysincluding chemokines and inflammatory molecules (egCFB SAA1 TNFAIP6 CXCL5 and CXCL6) collagenendopeptidases (MMP3 -10) solute carriers (SCL22A1

-6A11 -1A6) and structural components (eg KRT6AKRT6B ITGA2 and LAMB3) (Supplemental Table 3)

For a subset of DE genes we validated expressionchanges observed bymicroarray via a qRT-PCR approachand RNA samples isolated from a second set of CTBsamples [Fig 5(c)] DHCR7 PDXK CD44 CBS MMP9CXCL6 ITGA2 and S100A7 were upregulated with time

Figure 2 Morphological transitions of cultured primary human CTBs over 39 hours (andashc) CTBs were incubated for 3 15 and 39 hours beforethe nuclei were visualized by Hoechst staining (d) The average minimum distance among CTB nuclei (n = 3) Asterisks signify P 005 (t test)Representative images of immunostaining for (e) CK (f) HLA-G and (g) Ki-67 in CTBs at 3 hours nuclei were stained with Hoechst (h) Averagepercentage of cells that stained for these antigens as a function of time in culture Images are representative of $3 independent experimentsScale bars = 100 mm ns not significant

Figure 3 Characterization of changes in CTB gene expression asa function of time in culture (a) Hierarchical clustering of time-dependent DE genes in CTBs (2232 genes total P 000005absolute FC $ 2) Upregulated or downregulated genes over time inculture were designated as either cluster I or cluster II respectivelyAverage FC values are displayed as the ratio of average intensitiesbetween each time point and the average 0-hour CTB values(log 2 scale) (b) Post hoc t test analysis of DE genes per time point(t test P 0001 absolute FC $ 2)

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in culture (ANOVA P 005) F5 DUSP2 FABP7 andPEG3were downregulated (ANOVA P 005)MMP12mRNA levels which did not change according to themicroarray data were also unchanged when assayed byqRT-PCR Our qRT-PCR and microarray results werehighly correlated in terms of FC in expression (as com-pared with the 0-hour time point) (r = 088 not shown)FC values for genes (PDXK PEG3) which were not sig-nificantly regulated in the microarray data (005 x

00005) correlated between the two analytical methodsWe tested the hypothesis that gene expression changes in

vitro parallel in some cases genes and pathways that areupregulated in second trimester when the placenta is stillremodeling the uterine wall as compared with term whenthis process is completed Thus we interrogated ssRNA-seqprofiles of freshly isolated CTBs from second-trimester andterm placentas (20) to determine transcripts (identified asDE in vitro)whose expressionwas dependent on gestationalage Of the 2232DE genes in vitro 1955 hadRPKMvalues[Fig 6(a)] We plotted the average abundance of this subsetvs the FC difference in expression between second-trimesterand termCTBs [Fig 6(b)] Approximately 95of the geneswe compared were expressed 1 RPKM (log 2 scale = 0)Approximately 16 of genes were DE twofold be-tween the two gestational ages (blue lines log 2 scale = 1)Forty-seven genes that were in the 10th percentile byabundance (585RPKM)were also gestationally regulated

(twofold) This subset included molecules upregulatedin term vs second-trimester CTBs (eg EFEMP1) orupregulated in second-trimester vs term CTBs (egNOTUM) The majority of genes that were modulatedduring gestation (77) were downregulated (green vsred) in culture Furthermore we conducted additionalanalyses using the subset of genes (105 total) that wasassociatedwith pathways underlying CTB differentiationinvasion anatomical structure morphogenesis cell mo-tion and vasculature development [Fig 6(c)] This subsetincludedmolecules upregulated in term vs second-trimesterCTBs (eg BMP7 KLF4 and JAG1) or upregulated insecond-trimester vs term CTBs (eg MMP9 ITGB3 andIL6R) Thus the latter cross comparison identifiedmolecules that are known regulators of CTB inva-sion suggesting that potentially novel candidates thatemerged from this analysis could be interesting tostudy in this context

Next in tissue sections of second-trimester biopsies ofthe maternalndashfetal interface we evaluated protein ex-pression of NOTUM and EFEMP1 using an immunoflu-orescence approach These two molecules stood out in ourtranscriptomic analyses as follows (1) highly abundant (2)significantly DE over time in cultured CTBs and (3) DEbetween second trimester and term In second-trimestertissue sections (n = 12) NOTUM [green Fig 7(a)] wasexpressed in all trophoblast subpopulations that we

Figure 4 Functional GO enrichment analysis of CTB genes that were upregulated or downregulated as a function of time in culture (a) GOterms that were overrepresented (biological processes) among DE genes whose expression changed during CTB culture (P 0005 FE $15total number changed $15 bar denotes significance) (b) Absolute average FC ratios of DE genes within selected enriched GO terms Thenumber of upregulated or downregulated DE genes in each category (1) positive (2) negative

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examined including CTBswithin the floating villi and cellcolumns (upper panels) as well as the placental cells thatinvaded more deeply into the decidua (lower panels) Ex-pressionofNOTUMwasalso apparent in non-CKndashpositivecells within the villous core and decidua As to modulationas a function of invasion the major finding was thatNOTUMexpression tended tomove from the cytoplasm tothe nucleus EFEMP1 was also widely expressed amongthe CTB subpopulations [Fig 7(b)] In general antibodyreactivity which was highest in villous CTBs (upperpanels) decreased as the cells invaded the uterus (lowerpanels) Thus these analyses suggested that NOTUMand EFEMP1 have interesting patterns of modulationas a function of CTB differentiationinvasion

Discussion

Model systems in which TBs isolated from human pla-centas are placed in culture provide important oppor-tunities for studying themechanisms and cellular functionsunderlying normal development and disease We pub-lished protocols for isolating CTBs and culturing themon three-dimensional substrates (16 24) Targeted

analyses showed that under theseconditions the cells differentiate alongthe pathway that leads to migrationaway from the placenta proper andinvasion of the uterine wall (1 8) Inthis work we expanded these reportsby completing a global transcriptomicanalysis that described in detail thechanges in gene expression that paral-leled the CTBmorphological transitionsas the cells acquired an invasive phe-notype in vitro Later we discuss theseresults in the context of CTB differen-tiation in vivo

Quantification of CTB migrationin vitro

We developed a semiautomatedpipeline for doing high throughputimage content analysis and applied thisapproach to our CTB differentiationmodel As described previously themajority of cells expressed CK whichidentifies all TB subpopulations andHLA-G which is upregulated at theprotein level as the cells invade theuterine wall (25 26) Isolation andculture of second-trimester CTBs onMatrigel enabled their attachment (by3 hours) after which they spread on the

substrate becoming highly migratory In the processthey migrated toward each other forming multicellu-lar aggregates while extending elongated filapodia (by15 hours) In agreementwith previous studies we showedthat cultured CTBs that differentiated along the invasivepathway had a very low rate of proliferation (2immunopositive for Ki-67 expression) We quantifiedthese transitions by measuring the average minimumdistance between the cell nuclei at three time points Thisapproach enabled us to quantify migratory activity andaggregation across hundreds of thousands of cells In thisstudy we established baseline conditions that in futureexperiments can be used to evaluate the effects of potentialperturbants including pharmaceuticals environmentalchemicals and disease states (35) on CTB behavior

Transcriptomic changes in cultured CTBsIn parallel with the described morphological transi-

tions we observed robust global RNA expressionchanges over time in cultured CTBs (Fig 3) Using aconservative approach (false discovery rate 1 P

000005 absolute FC $2) 10 of genes were sig-nificantly DE over time Our results suggested changes in

Figure 5 DE genes within enriched categories relevant to CTB invasion (a) Hierarchicalclustering plot of DE genes related to enriched GO terms (anatomical structuremorphogenesis cell motion and vascular development) in CTBs over time in culture DEgenes that passed a secondary filter (absolute FC 4 log 2 = 2) are shown Black boxessignify GO associations (b) DE genes related to the enriched GO term response to vitamin(c) qRT-PCR verification of gene expression changes over time in culture Asterisks indicatesignificant changes (ANOVA P 005 bars denote standard error of the mean)

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specific pathways related to CTB functions in vivo in-cluding morphogenesis vasculature development cellmigration cell communication vitaminnutrition andinflammatory responses These pathways suggested aninterplay between molecules that drive cell behaviors inCTBs (remodeling migration) placental developmentand cellndashcell communication The data highlighted manyinteresting processes with known or potential relevanceto CTB interactions with maternal cells in vivo and novelregulators

Migration and morphogenesis pathwaysAt a transcriptomic level several of the DE molecules

played critical roles in CTB migration andor morpho-genesis [Figs 4 and 5(a)] This subset included MMP9whichwas upregulated (ninefold) in culture In parallelwe observed the increased expression of four otherMMPfamily members (MMP3 -8 -10 and -14) As a classMMPs are critical for CTB invasion and differentiationwithMMP9 playing an especially important role (in vitroand in vivo) (36ndash38) Lower expression levels of MMP9

are associated with PE (39) and the MMP9 (ndash1562CT)variant is linked with PE susceptibility (40) Integrinexpression was also modulated including ITGA2 (uarr)ITGB3 (uarr) (Fig 5) ITGB6 (uarr) and ITGB5 (darr) (data notshown) Regulated expression of integrins and their ECMligands is an integral part of the CTB differentiationpathway that leads to formation of cell columns anduterine invasion (41 42) For example in first-trimesterplacentas column CTBs express high levels of ITGA6B4and low levels of ITGA5B1 whereas invasive extra-villous CTBs have the opposite expression pattern (43)To our knowledge expression of ITGA2 has yet to bedescribed in human CTBs We identified many othergenes that were DE in culture with proposed links to CTBinvasiondifferentiation and placental development Theyincluded the following (1) ETS1 (uarr) a transcriptionfactor that regulates expression of MMPs and othermolecules important for uterine invasion (44 45) (2) thesecreted preproproteinsignaling peptide EDN1 (uarr)which is involved in invasion and vasoconstriction (46)and (3) KISS1R1 (darr) a G proteinndashcoupled receptor that

Figure 6 Mapping CTB genes that were DE in culture (microarray) to CTB gene expression changes from second trimester to term (RNA-seq) (a)The RNA-seq dataset was generated from CTBs that were isolated in our laboratory (20) In total 19552232 DE genes could be compared (b)The average abundance of transcripts of DE genes (y-axis RPKM log 2 scale) vs the FC difference between second-trimester and term CTBs(x-axis log 2 scale) (c) DE genes related to GO terms anatomical morphogenesis cell motion andor vascular development Genes are labeledbased on patterns of regulation over time in culture

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selectively binds to kisspeptins and represses TBmigrationinvasion (47) Our results suggested that CTB differenti-ation in vitro recapitulatedmany of themolecular switchesthat occur as CTBs acquire invasivemigratory propertiesin vivo

Vascular and immune pathwaysCTB endovascular invasion garners a supply of ma-

ternal blood that enables exchange of nutrients wastesand gases at the maternalndashfetal interface In our culturemodel DE genes included molecules that play importantroles in vascular development [Fig 5(a)] such as CD44(uarr) a cell adhesion surface receptor for hyaluronic acidand a major component of the ECM These receptorndashligand complexes regulate intracellular signaling path-ways critical for CTB invasion and remodeling of thedecidua (48) Other DE molecules in this class includedVEGFA and FLT1 which were downregulated Increasedexpression of these molecules underlies maternal vasculardysfunction in PE (49) Additionally several inflammatory

mediators were DE in our model For example CXCL6[Fig 5(c)] and interleukin-6 [Fig 5(a)] were upregulatedIn HTR-8SVneo cells interleukin-6 promotes migrationinvasion and modulation of integrin profiles (50) In thesame cell line (and in primary CTBs) CXCL6 reduces TBmigrationinvasion by reducingMMP2 activity (51) OtherDE inflammatory molecules included numerous chemo-kines and interleukins These results provided a globaltranscriptional context for previous (13) and future in-vestigations using this model system to study inflamma-tory mediators and their mechanistic roles in CTBdifferentiationinvasion vascular remodeling and im-mune interactions with the mother

Vitamin and nutrient regulation pathways in CTBsIn general the importance of vitamins and other nu-

trients for pregnancy health and placental development iswell-recognized (52ndash54) However the underlying mech-anistic links remain unresolved at molecular and cellularlevels In cultured CTBs genes associated with the GO

Figure 7 Immunolocalization of NOTUM and EFEMP1 which were DE in cultured CTBs Representative images of floating and anchoring villi(FV AV respectively upper panels) and invasive (i) CTBs within the decidua (Dec lower panels) Tissue sections of second-trimester samples wereimmunostained for (a) NOTUM or (b) EFEMP1 The samples were costained with anti-CK (trophoblast marker) and 406-diamidino-2-phenylindole(Dapi nuclear dye) Tissue sections from $12 placentas were evaluated Scale bars = 100 mm

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term response to vitamin were among some of the mostdramatically differentially expressed in our dataset [Fig5(b)] This subset included members of the vitamin Dsignaling pathwaymdashthe primary p450-activating enzyme(CYP27B1) and the vitamin D receptor The expression ofboth increased with time in culture suggesting enhancedCTB responsiveness to compounds of this class as theydifferentiate along the invasive pathway This observationis in line with published results regarding the relationshipsbetween vitamin D and the following (1) extravillous TBinvasiveness (55) (2) calciotropic hormone regulation (56)and (3) pregnancy complications [eg PE (57)] In addi-tion we observed increased expression of CBS a memberof the folate metabolismcysteine-synthesis pathway [Fig5(c)] CBS is the critical regulator of homocysteine pro-duction during pregnancy and altered levels may underlieimpaired decidualizationchanges in uterinendashgene expres-sion (58) PE pregnancy loss and congenital birth defects(54) The cytochrome p450 enzymesmdashCYP1A1 -1A2-1B1mdashwhich play roles in xenobioticdrug metabolism aswell as endogenous regulation of fatty acid metabolismangiogenesis and epithelial differentiation (59) were alsoupregulated in culture The functions of these enzymeswith dual roles inmetabolizing exogenous and endogenouscompounds are poorly understood in the context ofplacental development and function Additionally othermolecules in this subset such as AQP3 and AQP9 wereupregulated in culture Thesemembrane proteinswhich actas access points for water molecules may play key rolesduring mammalian pregnancy (60 61) Whether thesemolecules which are highly regulated in our system alsoplay roles in CTB differentiation is an interesting possibility

DE genes in second-trimester vs term CTBsUsing an ssRNA-seq dataset generated as part of the

Epigenome Roadmap Project (20) we defined a subset ofgenes (initially observed to be DE in vitro in CTBs) thatwere DE between second trimester vs term [Fig 6(a)]This analysis revealed several abundant molecules (top10 expression yellow shading) that were associatedwith faulty CTB invasion andor placental disease egFSTL3 (62) FLT1 (63) ADAM12 (64) TFPI2 (65 66)F5 (67) and HSD17B1 (68) (Supplemental Table 4) aswell as new molecules yet to be studied in this context(eg EFEMP1 and NOTUM) [Fig 6(b)] In a follow-upanalysis we identified genes that were up- or down-regulated as a function of CTB differentiationinvasion invitro focusing on a subset of the component processesmdashmorphogenesis cell motion and vascular developmentThen we asked whether they were also regulated as afunction of gestational age [Fig 6(c)] Molecules withknown functions during CTB differentiation such asMMP9 (36ndash38) and ITGB3 (69) were upregulated in

cultured CTBs and the transcripts were expressed athigher levels during second trimester relative to term Ingeneral these findings further supported the concept thatgenes and pathways critical to CTB differentiation andplacental development were actively modulated in ourcell culture model Furthermore these analyses high-lighted new molecules yet to be studied in this contextFor example we identified two genes NOTUM andEFEMP1 as potentially involved in CTB differentiationIn vitro aggregating CTBs downregulated their expres-sion at the RNA level Immunolocalization of NOTUMand EFEMP1 in tissue sections of second-trimesterplacentas confirmed expression of these molecules atthe protein level in villous CTB progenitors as well as inCTBs transiting through the columns and within theuterine wall NOTUM a carboxylesterase was recentlyidentified as a key inhibitor of WNT signaling (70) andcritical for neuralhead induction in Xenopus (71) De-spite the recognized importance of WNT signaling in TBinvasion (72) nothing is known about the potentialrole(s) of NOTUM in placental development In ouranalyses CTB invasion was associated withmovement ofNOTUM from the cytoplasmic to the nuclear compart-ment To our knowledge this phenomenon has not beenpreviously reported

The ECM glycoprotein EFEMP1 acts as a regulatorof MMP expression and cancer metastasis (73) In anestrogen-dependent manner EFEMP1 inhibits WNTB-catenin signaling pathways and thereby the epithelial-to-mesenchymal transition of endometrial carcinoma cells(74) Previously EFEMP1 was shown to be expressed inhuman CTBs that reside within the smooth chorion layerof fetal membranes (75) In this study we add new datasuggesting expression of EFEMP1 in the placenta duringmidgestation and differential patterning of EFEMP1 invillous CTBs (higher expression) as compared with CTBsinvading the uterus (lower expression) In general ourstudy provides evidence of modulation of NOTUM andEFEMP1 expression in CTBs within the maternalndashfetalinterface suggesting possible roles of these twomoleculesin TB differentiation

Conclusion

Primary cultures of human villous CTBs are an importantmodel system for studying their adhesion migration andinvasionmdashbehaviors that are critical determinants ofpregnancy outcomes In this study using a transcriptomic-based approach we profiled CTB gene expression asthe cells differentiated along the invasive pathway invitro The DE genes were involved in key biologicalpathways that are critical to placentation in vivo cellmigration vascular remodeling morphogenesis and

arch 2019

inflammation The rich datasets that were generatedprovide a foundation of gene expression profiles againstwhich the effects of numerous variables including envi-ronmental and pharmacological compounds can beevaluated

Acknowledgments

We thank Cheryl Godwin de Medina for patient recruitmentGabriel Goldfien and Yan Zhou for technical assistance andJason Farrell and Nicomedes Abello for tissue collection

Address all correspondence and requests for reprints toJoshua F Robinson PhD Center for Reproductive SciencesDepartment of Obstetrics Gynecology and ReproductiveSciences University of California San Francisco Box 0665Room RMB-902E 35 Medical Center Way San FranciscoCalifornia 94143-0665 E-mail JoshuaRobinsonucsfedu

This work was supported by the National Institute of En-vironmental Health Sciences (Grants P01ES022841 andK99ES023846) and the Environmental Protection Agency(Grant RD83543301)

Disclosure Summary The authors have nothing to disclose

References

1 Damsky CH Fitzgerald ML Fisher SJ Distribution patterns ofextracellular matrix components and adhesion receptors are in-tricately modulated during first trimester cytotrophoblast differ-entiation along the invasive pathway in vivo J Clin Invest 199289(1)210ndash222

2 Red-Horse K Zhou Y Genbacev O Prakobphol A Foulk RMcMasterM Fisher SJ Trophoblast differentiation during embryoimplantation and formation of the maternal-fetal interface J ClinInvest 2004114(6)744ndash754

3 Zhou Y Fisher SJ JanatpourM Genbacev O Dejana EWheelockM Damsky CH Human cytotrophoblasts adopt a vascular phe-notype as they differentiate a strategy for successful endovascularinvasion J Clin Invest 199799(9)2139ndash2151

4 Zhou Y Damsky CH Fisher SJ Preeclampsia is associated withfailure of human cytotrophoblasts to mimic a vascular adhesionphenotype one cause of defective endovascular invasion in thissyndrome J Clin Invest 199799(9)2152ndash2164

5 Ball E Bulmer JN Ayis S Lyall F Robson SC Late sporadicmiscarriage is associated with abnormalities in spiral artery

transformation and trophoblast invasion J Pathol 2006208(4)535ndash542

6 Romero R Dey SK Fisher SJ Preterm labor one syndrome manycauses Science 2014345(6198)760ndash765

7 Jauniaux E Jurkovic D Placenta accreta pathogenesis of a 20thcentury iatrogenic uterine disease Placenta 201233(4)244ndash251

8 Chen JZ Sheehan PM Brennecke SP Keogh RJ Vessel remodel-ling pregnancy hormones and extravillous trophoblast functionMol Cell Endocrinol 2012349(2)138ndash144

9 Cohen M Bischof P Factors regulating trophoblast invasionGynecol Obstet Invest 200764(3)126ndash130

10 Kokkinos MI Murthi P Wafai R Thompson EW Newgreen DFCadherins in the human placentandashepithelial-mesenchymal transi-tion (EMT) and placental development Placenta 201031(9)747ndash755

11 Blankenship TN King BF Macaque intra-arterial trophoblast andextravillous trophoblast of the cell columns and cytotrophoblasticshell express neural cell adhesion molecule (NCAM) Anat Rec1996245(3)525ndash531

12 Hunkapiller NM Gasperowicz M Kapidzic M Plaks V MaltepeE Kitajewski J Cross JC Fisher SJ A role for Notch signaling introphoblast endovascular invasion and in the pathogenesis of pre-eclampsia Development 2011138(14)2987ndash2998

13 Red-Horse K Kapidzic M Zhou Y Feng KT Singh H Fisher SJEPHB4 regulates chemokine-evoked trophoblast responsesa mechanism for incorporating the human placenta into the ma-ternal circulation Development 2005132(18)4097ndash4106

14 Hemberger M Immune balance at the foeto-maternal interface asthe fulcrum of reproductive success J Reprod Immunol 201397(1)36ndash42

15 Orendi K Kivity V Sammar M Grimpel Y Gonen R Meiri HLubzens E Huppertz B Placental and trophoblastic in vitro modelsto study preventive and therapeutic agents for preeclampsia Pla-centa 201132(Suppl)S49ndashS54

16 Hunkapiller NM Fisher SJ Chapter 12 Placental remodeling ofthe uterine vasculature Methods Enzymol 2008445281ndash302

17 Kliman HJ Nestler JE Sermasi E Sanger JM Strauss JF III Pu-rification characterization and in vitro differentiation of cyto-trophoblasts from human term placentae Endocrinology 1986118(4)1567ndash1582

18 Douglas GC King BF Isolation of pure villous cytotrophoblastfrom term human placenta using immunomagnetic microspheresJ Immunol Methods 1989119(2)259ndash268

19 Mikheev AM Nabekura T Kaddoumi A Bammler TK Govin-darajan R Hebert MF Unadkat JD Profiling gene expression inhuman placentae of different gestational ages an OPRU Networkand UW SCOR Study Reprod Sci 200815(9)866ndash877

20 Kundaje A Meuleman W Ernst J Bilenky M Yen A Heravi-Moussavi A Kheradpour P Zhang Z Wang J Ziller MJ Amin VWhitaker JW Schultz MDWard LD Sarkar A Quon G SandstromRS Eaton ML Wu YC Pfenning AR Wang X Claussnitzer M Liu

Appendix Primary Antibodies

PeptideProteinTarget RRID

Antigen Sequence(if Known)

Name ofAntibody

Manufacturer CatalogNo andor Name ofIndividual Providing

the Antibody

Species Raised inMonoclonal or

PolyclonalDilutionUsed

Cytokeratin AB_2631235 Clone 7D3 Fisher_001-clone7D3 Rat monoclonal 1100HLA-G AB_2631236 Clone 4H84 Fisher_002-clone4H84 Mouse monoclonal 1100Ki-67 AB_149792 Ki-67 Thermo Fisher Scientific

RM-9106-S1Rabbit monoclonal 1100

NOTUM AB_10604118 NOTUM Sigma-AldrichSAB3500082

Rabbit polyclonal 1100

EFEMP1 AB_301517 TYTQCTDGYEWDPVRQQC EFEMP1 Abcam ab14926 Rabbit polyclonal 1100

Abbreviation RRID Research Resource Identifier

arch 2019

Y Coarfa C Harris RA Shoresh N Epstein CB Gjoneska E LeungD Xie W Hawkins RD Lister R Hong C Gascard P Mungall AJMoore R Chuah E Tam A Canfield TK Hansen RS Kaul R SaboPJ Bansal MS Carles A Dixon JR Farh KH Feizi S Karlic R KimAR Kulkarni A Li D Lowdon R Elliott G Mercer TR Neph SJOnuchic V Polak P Rajagopal N Ray P Sallari RC Siebenthall KTSinnott-Armstrong NA Stevens M Thurman RE Wu J Zhang BZhou X Beaudet AE Boyer LA De Jager PL Farnham PJ Fisher SJHaussler D Jones SJ Li W Marra MA McManus MT Sunyaev SThomson JA Tlsty TD Tsai LH Wang W Waterland RA ZhangMQ Chadwick LH Bernstein BE Costello JF Ecker JR Hirst MMeissner A Milosavljevic A Ren B Stamatoyannopoulos JA WangT Kellis M Roadmap Epigenomics Consortium Integrative analysisof 111 reference human epigenomes Nature 2015518(7539)317ndash330

21 Barreto RS Bressan FF Oliveira LJ Pereira FT Perecin FAmbrosio CE Meirelles FV Miglino MA Gene expression inplacentation of farm animals an overview of gene function duringdevelopment Theriogenology 201176(4)589ndash597

22 Zhou Y GormleyMJ Hunkapiller NM Kapidzic M Stolyarov YFeng V NishidaM Drake PM Bianco KWang F McMaster MTFisher SJ Reversal of gene dysregulation in cultured cytotropho-blasts reveals possible causes of preeclampsia J Clin Invest 2013123(7)2862ndash2872

23 Tilburgs T Crespo AC van der Zwan A Rybalov B Raj TStranger B Gardner L Moffett A Strominger JL HumanHLA-G+extravillous trophoblasts immune-activating cells that interactwith decidual leukocytes Proc Natl Acad Sci USA 2015112(23)7219ndash7224

24 Fisher SJ Cui TY Zhang L Hartman L Grahl K Zhang GYTarpey J Damsky CH Adhesive and degradative properties ofhuman placental cytotrophoblast cells in vitro J Cell Biol 1989109(2)891ndash902

25 McMaster MT Librach CL Zhou Y Lim KH Janatpour MJDeMars R Kovats S Damsky C Fisher SJ Human placental HLA-G expression is restricted to differentiated cytotrophoblastsJ Immunol 1995154(8)3771ndash3778

26 Maldonado-Estrada J Menu E Roques P Barre-Sinoussi FChaouat G Evaluation of cytokeratin 7 as an accurate intracellularmarker with which to assess the purity of human placental villoustrophoblast cells by flow cytometry J Immunol Methods 2004286(1-2)21ndash34

27 Kovats S Main EK Librach C Stubblebine M Fisher SJ DeMarsR A class I antigen HLA-G expressed in human trophoblastsScience 1990248(4952)220ndash223

28 Kaya B Nayki U Nayki C Ulug P Oner G Gultekin E YildirimYProliferation of trophoblasts and Ki67 expression in preeclampsiaArch Gynecol Obstet 2015291(5)1041ndash1046

29 Winn VD Haimov-Kochman R Paquet AC Yang YJ Madhu-sudhan MS Gormley M Feng KT Bernlohr DA McDonagh SPereira L Sali A Fisher SJ Gene expression profiling of the humanmaternal-fetal interface reveals dramatic changes between midg-estation and term Endocrinology 2007148(3)1059ndash1079

30 Saeed AI Bhagabati NK Braisted JC Liang W Sharov V HoweEA Li J Thiagarajan M White JA Quackenbush J TM4microarray software suiteMethods Enzymol 2006411134ndash193

31 Huang DW Sherman BT Tan Q Kir J Liu D Bryant D Guo YStephens R Baseler MW Lane HC Lempicki RA DAVID Bio-informatics Resources expanded annotation database and novelalgorithms to better extract biology from large gene lists NucleicAcids Res 200735(Web Server issue)W169ndashW175

32 Robinson JF Verhoef A van Beelen VA Pennings JL Piersma AHDose-response analysis of phthalate effects on gene expression inrat whole embryo culture Toxicol Appl Pharmacol 2012264(1)32ndash41

33 Team RC R A Language and Environment for Statistical Com-puting Vienna R Foundation for Statistical Computing 2014

34 Zhou YMcMasterMWoo K JanatpourM Perry J Karpanen TAlitalo K Damsky C Fisher SJ Vascular endothelial growth factor

ligands and receptors that regulate human cytotrophoblast survivalare dysregulated in severe preeclampsia and hemolysis elevatedliver enzymes and low platelets syndrome Am J Pathol 2002160(4)1405ndash1423

35 Hromatka BS Drake PM Kapidzic M Stolp H Goldfien GA ShihIeM Fisher SJ Polysialic acid enhances the migration and invasionof human cytotrophoblasts Glycobiology 201323(5)593ndash602

36 Luo J Qiao F Yin X Impact of silencing MMP9 gene on thebiological behaviors of trophoblasts J Huazhong Univ Sci Tech-nolog Med Sci 201131(2)241ndash245

37 Librach CL Werb Z Fitzgerald ML Chiu K Corwin NM EstevesRA Grobelny D Galardy R Damsky CH Fisher SJ 92-kD type IVcollagenase mediates invasion of human cytotrophoblasts J CellBiol 1991113(2)437ndash449

38 Plaks V Rinkenberger J Dai J Flannery M Sund M Kanasaki KNi W Kalluri R Werb Z Matrix metalloproteinase-9 deficiencyphenocopies features of preeclampsia and intrauterine growth re-striction Proc Natl Acad Sci USA 2013110(27)11109ndash11114

39 Kolben M Lopens A Blaser J Ulm K Schmitt M Schneider KTTschescheH Proteases and their inhibitors are indicative in gestationaldisease Eur J Obstet Gynecol Reprod Biol 199668(1-2)59ndash65

40 Rahimi Z Rahimi Z Shahsavandi MO Bidoki K Rezaei MMMP-9 (-1562CT) polymorphism as a biomarker of susceptibilityto severe pre-eclampsia Biomarkers Med 20137(1)93ndash98

41 Aplin JD Jones CJ Harris LK Adhesion molecules in humantrophoblast a review I Villous trophoblast Placenta 200930(4)293ndash298

42 Harris LK Jones CJ Aplin JD Adhesion molecules in humantrophoblast a review II Extravillous trophoblast Placenta 200930(4)299ndash304

43 Damsky CH Librach C Lim KH Fitzgerald ML McMaster MTJanatpour M Zhou Y Logan SK Fisher SJ Integrin switchingregulates normal trophoblast invasion Development 1994120(12)3657ndash3666

44 Takai N Ueda T Narahara H Miyakawa I Expression of c-Ets1protein in normal human placenta Gynecol Obstet Invest 200661(1)15ndash20

45 Kessler CA Stanek JW Stringer KF Handwerger S ETS1 induceshuman trophoblast differentiation Endocrinology 2015156(5)1851ndash1859

46 Cervar M Puerstner P Kainer F Desoye G Endothelin-1 stimu-lates the proliferation and invasion of first trimester trophoblasticcells in vitro a possible role in the etiology of pre-eclampsia JInvestig Med 199644(8)447ndash453

47 Hiden U Bilban M Knofler M Desoye G Kisspeptins and theplacenta regulation of trophoblast invasion Rev Endocr MetabDisord 20078(1)31ndash39

48 Takahashi H Takizawa T Matsubara S Ohkuchi A Kuwata TUsui RMatsumotoH Sato Y FujiwaraHOkamotoA SuzukiMTakizawa T Extravillous trophoblast cell invasion is promoted bythe CD44-hyaluronic acid interaction Placenta 201435(3)163ndash170

49 Fan X Rai A Kambham N Sung JF Singh N Petitt M Dhal SAgrawal R Sutton RE DruzinML Gambhir SS Ambati BK CrossJC Nayak NR Endometrial VEGF induces placental sFLT1 andleads to pregnancy complications J Clin Invest 2014124(11)4941ndash4952

50 Jovanovic M Vicovac L Interleukin-6 stimulates cell migrationinvasion and integrin expression in HTR-8SVneo cell line Pla-centa 200930(4)320ndash328

51 Zhang H Hou L Li CM Zhang WY The chemokine CXCL6restricts human trophoblast cell migration and invasion by sup-pressing MMP-2 activity in the first trimesterHum Reprod 201328(9)2350ndash2362

52 De-Regil LM Palacios C Lombardo LK Pe~na-Rosas JP VitaminDsupplementation forwomen during pregnancyCochraneDatabaseSyst Rev 20161(1)CD008873

53 Wei SQ Vitamin D and pregnancy outcomes Curr Opin ObstetGynecol 201426(6)438ndash447

arch 2019

54 Mislanova C Martsenyuk O Huppertz B Obolenskaya M Pla-cental markers of folate-related metabolism in preeclampsia Re-production 2011142(3)467ndash476

55 Chan SY Susarla R Canovas D Vasilopoulou E Ohizua OMcCabe CJ Hewison M Kilby MD Vitamin D promotes humanextravillous trophoblast invasion in vitro Placenta 201536(4)403ndash409

56 OrsquoBrien KO Li S Cao C Kent T Young BV Queenan RAPressman EK Cooper EM Placental CYP27B1 and CYP24A1expression in human placental tissue and their association withmaternal and neonatal calcitropic hormones J Clin EndocrinolMetab 201499(4)1348ndash1356

57 Ma R Gu Y Zhao S Sun J Groome LJ Wang Y Expressions ofvitamin D metabolic components VDBP CYP2R1 CYP27B1CYP24A1 and VDR in placentas from normal and preeclampticpregnancies Am J Physiol Endocrinol Metab 2012303(7)E928ndashE935

58 Nu~no-Ayala M Guillen N Arnal C Lou-Bonafonte JM deMartino A Garcıa-de-Jalon JA Gascon S Osaba L Osada JNavarroMA Cystathionine b-synthase deficiency causes infertilityby impairing decidualization and gene expression networksin uterus implantation sites Physiol Genomics 201244(14)702ndash716

59 Nebert DW Dalton TP The role of cytochrome P450 enzymes inendogenous signalling pathways and environmental carcinogene-sis Nat Rev Cancer 20066(12)947ndash960

60 Liu H Koukoulas I Ross MCWang S Wintour EM Quantitativecomparison of placental expression of three aquaporin genesPlacenta 200425(6)475ndash478

61 Prat C Bouvier D Comptour AMarceau G Belville C ClairefondG Blanc P Gallot D Blanchon L Sapin V All-trans-retinoic acidregulates aquaporin-3 expression and related cellular membranepermeability in the human amniotic environment Placenta 201536(8)881ndash887

62 Guo J Tian T Lu D Xia G Wang H Dong M Alterations ofmaternal serumand placental follistatin-like 3 andmyostatin in pre-eclampsia J Obstet Gynaecol Res 201238(7)988ndash996

63 March MI Geahchan C Wenger J Raghuraman N Berg AHaddow H Mckeon BA Narcisse R David JL Scott J ThadhaniR Karumanchi SA Rana S Circulating angiogenic factors and therisk of adverse outcomes amongHaitianwomenwith preeclampsiaPLoS One 201510(5)e0126815

64 Aghababaei M Beristain AG The Elsevier Trophoblast ResearchAward Lecture Importance of metzincin proteases in trophoblastbiology and placental development a focus onADAM12Placenta201536(Suppl 1)S11ndashS19

65 ZhouQ Xiong Y Chen Y Du Y Zhang J Mu J GuoQWangHMa D Li X Effects of tissue factor pathway inhibitor-2 expressionon biological behavior of BeWo and JEG-3 cell lines Clin ApplThromb Hemost 201218(5)526ndash533

66 Xiong Y Zhou Q Jiang F Zhou S Lou Y Guo Q LiangW KongD Ma D Li X Changes of plasma and placental tissue factorpathway inhibitor-2 in women with preeclampsia and normalpregnancy Thromb Res 2010125(6)e317ndashe322

67 Fong FM Sahemey MK Hamedi G Eyitayo R Yates D Kuan VThangaratinam S Walton RT Maternal genotype and severe pre-eclampsia a HuGE review Am J Epidemiol 2014180(4)335ndash345

68 Ishibashi O Ohkuchi A Ali MM Kurashina R Luo SS IshikawaT Takizawa T Hirashima C Takahashi KMigitaM Ishikawa GYoneyama K Asakura H Izumi A Matsubara S Takeshita TTakizawa T Hydroxysteroid (17-b) dehydrogenase 1 is dysregu-lated by miR-210 and miR-518c that are aberrantly expressed inpreeclamptic placentas a novelmarker for predicting preeclampsiaHypertension 201259(2)265ndash273

69 Chung TW ParkMJ KimHS ChoiHJ HaKT Integrin aVb3 andaVb5 are required for leukemia inhibitory factor-mediated theadhesion of trophoblast cells to the endometrial cells BiochemBiophys Res Commun 2016469(4)936ndash940

70 Kakugawa S Langton PF Zebisch M Howell SA Chang TH LiuY Feizi T Bineva G OrsquoReilly N Snijders AP Jones EY Vincent JPNotum deacylates Wnt proteins to suppress signalling activityNature 2015519(7542)187ndash192

71 Zhang X Cheong SM Amado NG Reis AH MacDonald BTZebisch M Jones EY Abreu JG He X Notum is required forneural and head induction via Wnt deacylation oxidation andinactivation Dev Cell 201532(6)719ndash730

72 Sonderegger SHussleinHLeisserCKnoflerMComplex expressionpattern of Wnt ligands and frizzled receptors in human placenta andits trophoblast subtypes Placenta 200728(Suppl A)S97ndashS102

73 Wang Z Cao CJ Huang LL Ke ZF Luo CJ Lin ZW Wang FZhang YQ Wang LT EFEMP1 promotes the migration and in-vasion of osteosarcoma via MMP-2 with induction by AEG-1 viaNF-kB signaling pathway Oncotarget 20156(16)14191ndash14208

74 Yang T Zhang H Qiu H Li B Wang J Du G Ren C Wan XEFEMP1 is repressed by estrogen and inhibits the epithelial-mesenchymal transition via Wntb-catenin signaling in endome-trial carcinoma Oncotarget 20167(18)25712ndash25725

75 Moore RM Redline RW Kumar D Mercer BM Mansour JMYohannes E Novak JB Chance MR Moore JJ Differential ex-pression of fibulin family proteins in the para-cervical weak zoneand other areas of human fetal membranes Placenta 200930(4)335ndash341

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

Numerous toxicological (Dingemans et al 2011) and epidemiolog-ical (Cowell et al 2015 Herbstman and Mall 2014) studies indicatethat exposures to PBDEs in utero may be harmful to the developinghuman fetus The ramifications of their bioaccumulation remainlargely unknown In human cell lines andor animal models com-mon PBDEs congeners elicit developmental toxicity through sev-eral mechanisms eg alterations in thyroid hormone (TH)signaling inflammation oxidative stress and epigenetic modifi-cations (Costa et al 2014) Further investigations are needed toclarify targeted tissuescell populations and windows of sensitiv-ity during human pregnancy

The placenta is essential for normal fetal development andpregnancy complications linked with abnormal placentation arenegatively associated with neonatal child and adult health(Vinnars et al 2014) Data generated in rodent models and humancell lines (Kalkunte et al 2017 Rajakumar et al 2015 Yang et al2006) suggest that environmental compounds are toxic to the pla-centa In humans the placenta is in direct contact with maternalblood in which these chemicals are routinely detected Thus it isnot surprising that PBDEs are regularly identified in human pla-centas (Leonetti et al 2016) While the consequences associatedwith exposures in utero are unknown recent studies in mamma-lian models suggest that BDE-47 may alter placental developmentcausing oxidative stress and inflammation (Park et al 2014 parkand Loch-Caruso 2014) as well as perturbing hormone signaling(park and Loch-Caruso 2015)

During pregnancy the placenta undergoes numerous mor-phological and molecular transformations to support the grow-ing demands of the developing fetus Specialized placental cellsknown as cytotrophoblasts (CTBs) are critical for (1) initiatingand maintaining the physical anchor between the fetal and ma-ternal units (2) penetration and invasion into the uterine walland (3) remodeling of the uterine vascular architecture toreroute maternal blood flow to the placenta (Red-Horse et al2004 2005) Perturbations in CTB development may underlieseveral pregnancy complications including preeclampsia (PEFisher 2015) intrauterine growth restriction (IUGR) pretermbirth (Romero et al 2014) and over aggressive CTB invasion(Jauniaux and Jurkovic 2012) In vitro isolated cultured primaryhuman villous CTBs (Fisher et al 1989 Hunkapiller and Fisher2008 Kliman et al 1986) have been proposed as a model ofplacentation

CTB invasion is a complicated process with many compo-nents some of which are unique The cells must exit the pla-centa crossing over to the uterus where they attach to itssurface before they deeply invade the parenchyma This processis accompanied by a series of molecular transformations in-volving relevant pathways including cell-cell adhesion migra-tioninvasion and vascular remodeling (Maltepe and Fisher2015) Upon isolation and culture villous CTBs differentiatealong the invasive pathway modulating the same sets of mole-cules that mediate this process in vivo (Robinson et al 2017)

Genomic profiling of isolated CTBs revealed dramatic shiftsat transcriptomic and epigenomic levels across gestation includ-ing many key molecules with known functional roles in placen-tal development and disease (Roadmap Epigenomics et al 2015)In assessing environmental chemical impacts during develop-ment the incorporation of genomic-based approaches in toxico-logical studies ie toxicogenomics provides a global analysis ofmolecular response to environmental exposures (Robinson andPiersma 2013) Given the unusual dynamics of the CTB genomethe potential for environmental chemicals to influence chroma-tin architecturegene expression and the links between placen-tal health and successful birth outcomes (Burton et al 2016

Kovo et al 2012) investigations are needed to address potentialinteractions between these important variables

Here we used a cell culture model to study the effects ofPBDEs on CTB differentiation and invasion Acute exposuresresulted in accumulation of the parent compound and minimalmetabolism Subcytotoxic concentrations of BDE-47 impairedthe ability of CTBs to migrate and invade These functionalalterations were accompanied by alterations at transcriptomicand epigenomic levels Together these data suggest that PBDEexposures could impact process and pathways that are integralto the placentarsquos role in governing pregnancy outcome

MATERIALS AND METHODS

Tissue collection All methods were initially approved by the UCSFInstitutional Review Board Informed consent was obtained fromall donors Second trimester placentas intended for cell isola-tions were collected immediately following elective terminationsand placed in cytowash medium consisting of DMEH-21 (Gibco)125 fetal bovine serum (Hyclone) 1 glutamine plus (AtlantaBiologicals) 1 penicillinstreptomycin (Invitrogen) and 01gentamicin (Gibco) Tissue samples were placed on ice prior todissection

Human primary villous cytotrophoblast isolation CTBs were isolatedfrom second trimester human placentas as described previously(Robinson et al 2017) Single cells were counted using a hemacy-tometer and immediately transferred to a Matrigel (BD bioscien-ces)-coated 12-well plate CTBs were cultured at a density of 500000 CTBswell in 15 ml medium containing DMEH-21 2Nutridoma (Roche) 1 sodium pyruvate (Sigma) 1 Hepes buffer(Invitrogen) 1 glutamate plus (Atlanta Biologicals) and 1 peni-cillinstreptomycin (Invitrogen) Cells were incubated at 37C in5 CO295 air We immunostained with anti-cytokeratin (CKanti-CK rat polyclonal 1100 Damsky et al 1992) a marker regu-larly used to detect for relative trophoblast purity ( 80ndash90CTBs) in our cultures across experiments Cell preparations notmeeting this criteria were not used for downstream analyses

Chemicals PBDE congeners BDE-47 (220440-tetrabromodiphenylether gt99 CAS 5436-43-1 AccuStandard) and BDE-99(2204405-pentabromodiphenyl ether gt99 CAS 60348-60-9AccuStandard) bisphenol A (BPA gt99 80-05-7 Sigma) andperfluorooctanoic acid (PFOA gt96 335-67-1 Sigma) were dis-solved in dimethyl sulfoxide (DMSO Sigma-Aldrich) to makestock solutions and serial dilutions for all experimental studiesChemical exposures were introduced at a 11000 (volvol) mediadilution for all assessments

Cytotoxicity assessments Freshly isolated CTBs were cultured for15 h (t15 h) and supplied with new media containing media only(control) DMSO (vehicle control 01) BDE-47 (01 10 1025 lM) BDE-99 (01 10 10 25 lM) BPA (01 10 10 100 lM) orPFOA (1 10 25 100 250 1000 lM) At t15 h cultured CTBs are ac-tively aggregating one of the initial steps in differentiationin-vasion We evaluated cytotoxicity after a 24-h exposure due toprevious studies suggesting that this time point was optimal todetect significant changes at cellular and molecular levels indeveloping in vitro systems (Costa et al 2015 park and Loch-Caruso 2014) We measured cell viability using the neutral red(NR 40 ngml VWR) lysosomal uptake assay (Borenfreund andPuerner 1985) Briefly after 24 h the media was removed andwells were gently washed with PBS to eliminate residual com-pound New media containing NR was added to each plate and

arch 2019

RESULTS

PBDE-47 recovered

PPBDEs

00 01 10 100

250 01 10 10

Veh BDE-47 BDE-99

00 01 10100

250 01 10100

BMC50Viability

BMC200LDH

BDE-47 181μM 125μM

BDE-99 335μM 246μM

BPA 1165μM 966μM

(μM) (μM)

arch 2019

ltMDL ltMDL

level of detection

Veh BDE-47(1μM)

BDE-47(5μM)

Veh BDE-47(1μM)

BDE-47(5μM)

e of C

ANOVA plt005

experiments (n4)

arch 2019

(GW)166 193 216y = 098xRsup2 = 081

-15 -1 -05 0 05 1 15Avg FC (BDE-47Veh) t3h+24h

t3h+24h t15h+24h

PSG7CCL1

166 193 216

arch 2019

ALL uarr darr

p=001p=005 p=0001

lative

PLAC4 IL6MMP1

FABP4GREM1

FABP7GPR34

HMGCS1SCD

BDE-47

yvasculature dev

cell migration f

anat morphogenesi

cell m

igration

vasculatu

re dev

lipid metabolism

inflammato

ry res

t3h+24h t15h+24h

arch 2019

AllCpGs

BDE-47DMRs

∆β (B

y = 090xRsup2 = 069

-015 -005 005 01501-01

∆β (B

(GW)166 193 216

t3h+24h t15h+24h

166 193 216

MRs uarrmethylation

darrmethylation

(darr)

(darr)(darr)

(uarr)

EAll D

MRs (472)

Promoter (17

t of G

ical P

plt001

TSS1500

TSS2005rsquoUTR

1st Exon

Body3rsquoU

Intergenic

arch 2019

DISCUSSION

01020304050607080

MRs uarrmethylation

darrmethylation

(uarr)

(darr)

1 2 3 4 5 6 7 8 9 10 111213141516171819202122

-006-004-002

0002004006008

0 25000000 50000000 75000000 100000000 125000000

BDE-47 DMRple005

Avg ∆β

4(3)152

ABDE-47 DE Genes

156(152)

Avg ∆β (BDE-47 vs Veh) 24h-01 01-005 0050

DPTFERMT1

06CRCT1

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

RESULTS

PBDE-47 recovered

PPBDEs

00 01 10 100

250 01 10 10

Veh BDE-47 BDE-99

00 01 10100

250 01 10100

BMC50Viability

BMC200LDH

BDE-47 181μM 125μM

BDE-99 335μM 246μM

BPA 1165μM 966μM

(μM) (μM)

arch 2019

ltMDL ltMDL

level of detection

Veh BDE-47(1μM)

BDE-47(5μM)

Veh BDE-47(1μM)

BDE-47(5μM)

e of C

ANOVA plt005

experiments (n4)

arch 2019

(GW)166 193 216y = 098xRsup2 = 081

-15 -1 -05 0 05 1 15Avg FC (BDE-47Veh) t3h+24h

t3h+24h t15h+24h

PSG7CCL1

166 193 216

arch 2019

ALL uarr darr

p=001p=005 p=0001

lative

PLAC4 IL6MMP1

FABP4GREM1

FABP7GPR34

HMGCS1SCD

BDE-47

yvasculature dev

cell migration f

anat morphogenesi

cell m

igration

vasculatu

re dev

lipid metabolism

inflammato

ry res

t3h+24h t15h+24h

arch 2019

AllCpGs

BDE-47DMRs

∆β (B

y = 090xRsup2 = 069

-015 -005 005 01501-01

∆β (B

(GW)166 193 216

t3h+24h t15h+24h

166 193 216

MRs uarrmethylation

darrmethylation

(darr)

(darr)(darr)

(uarr)

EAll D

MRs (472)

Promoter (17

t of G

ical P

plt001

TSS1500

TSS2005rsquoUTR

1st Exon

Body3rsquoU

Intergenic

arch 2019

DISCUSSION

01020304050607080

MRs uarrmethylation

darrmethylation

(uarr)

(darr)

1 2 3 4 5 6 7 8 9 10 111213141516171819202122

-006-004-002

0002004006008

0 25000000 50000000 75000000 100000000 125000000

BDE-47 DMRple005

Avg ∆β

4(3)152

ABDE-47 DE Genes

156(152)

Avg ∆β (BDE-47 vs Veh) 24h-01 01-005 0050

DPTFERMT1

06CRCT1

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

RESULTS

PBDE-47 recovered

PPBDEs

00 01 10 100

250 01 10 10

Veh BDE-47 BDE-99

00 01 10100

250 01 10100

BMC50Viability

BMC200LDH

BDE-47 181μM 125μM

BDE-99 335μM 246μM

BPA 1165μM 966μM

(μM) (μM)

arch 2019

ltMDL ltMDL

level of detection

Veh BDE-47(1μM)

BDE-47(5μM)

Veh BDE-47(1μM)

BDE-47(5μM)

e of C

ANOVA plt005

experiments (n4)

arch 2019

(GW)166 193 216y = 098xRsup2 = 081

-15 -1 -05 0 05 1 15Avg FC (BDE-47Veh) t3h+24h

t3h+24h t15h+24h

PSG7CCL1

166 193 216

arch 2019

ALL uarr darr

p=001p=005 p=0001

lative

PLAC4 IL6MMP1

FABP4GREM1

FABP7GPR34

HMGCS1SCD

BDE-47

yvasculature dev

cell migration f

anat morphogenesi

cell m

igration

vasculatu

re dev

lipid metabolism

inflammato

ry res

t3h+24h t15h+24h

arch 2019

AllCpGs

BDE-47DMRs

∆β (B

y = 090xRsup2 = 069

-015 -005 005 01501-01

∆β (B

(GW)166 193 216

t3h+24h t15h+24h

166 193 216

MRs uarrmethylation

darrmethylation

(darr)

(darr)(darr)

(uarr)

EAll D

MRs (472)

Promoter (17

t of G

ical P

plt001

TSS1500

TSS2005rsquoUTR

1st Exon

Body3rsquoU

Intergenic

arch 2019

DISCUSSION

01020304050607080

MRs uarrmethylation

darrmethylation

(uarr)

(darr)

1 2 3 4 5 6 7 8 9 10 111213141516171819202122

-006-004-002

0002004006008

0 25000000 50000000 75000000 100000000 125000000

BDE-47 DMRple005

Avg ∆β

4(3)152

ABDE-47 DE Genes

156(152)

Avg ∆β (BDE-47 vs Veh) 24h-01 01-005 0050

DPTFERMT1

06CRCT1

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

RESULTS

PBDE-47 recovered

PPBDEs

00 01 10 100

250 01 10 10

Veh BDE-47 BDE-99

00 01 10100

250 01 10100

BMC50Viability

BMC200LDH

BDE-47 181μM 125μM

BDE-99 335μM 246μM

BPA 1165μM 966μM

(μM) (μM)

arch 2019

ltMDL ltMDL

level of detection

Veh BDE-47(1μM)

BDE-47(5μM)

Veh BDE-47(1μM)

BDE-47(5μM)

e of C

ANOVA plt005

experiments (n4)

arch 2019

(GW)166 193 216y = 098xRsup2 = 081

-15 -1 -05 0 05 1 15Avg FC (BDE-47Veh) t3h+24h

t3h+24h t15h+24h

PSG7CCL1

166 193 216

arch 2019

ALL uarr darr

p=001p=005 p=0001

lative

PLAC4 IL6MMP1

FABP4GREM1

FABP7GPR34

HMGCS1SCD

BDE-47

yvasculature dev

cell migration f

anat morphogenesi

cell m

igration

vasculatu

re dev

lipid metabolism

inflammato

ry res

t3h+24h t15h+24h

arch 2019

AllCpGs

BDE-47DMRs

∆β (B

y = 090xRsup2 = 069

-015 -005 005 01501-01

∆β (B

(GW)166 193 216

t3h+24h t15h+24h

166 193 216

MRs uarrmethylation

darrmethylation

(darr)

(darr)(darr)

(uarr)

EAll D

MRs (472)

Promoter (17

t of G

ical P

plt001

TSS1500

TSS2005rsquoUTR

1st Exon

Body3rsquoU

Intergenic

arch 2019

DISCUSSION

01020304050607080

MRs uarrmethylation

darrmethylation

(uarr)

(darr)

1 2 3 4 5 6 7 8 9 10 111213141516171819202122

-006-004-002

0002004006008

0 25000000 50000000 75000000 100000000 125000000

BDE-47 DMRple005

Avg ∆β

4(3)152

ABDE-47 DE Genes

156(152)

Avg ∆β (BDE-47 vs Veh) 24h-01 01-005 0050

DPTFERMT1

06CRCT1

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

ltMDL ltMDL

level of detection

Veh BDE-47(1μM)

BDE-47(5μM)

Veh BDE-47(1μM)

BDE-47(5μM)

e of C

ANOVA plt005

experiments (n4)

arch 2019

(GW)166 193 216y = 098xRsup2 = 081

-15 -1 -05 0 05 1 15Avg FC (BDE-47Veh) t3h+24h

t3h+24h t15h+24h

PSG7CCL1

166 193 216

arch 2019

ALL uarr darr

p=001p=005 p=0001

lative

PLAC4 IL6MMP1

FABP4GREM1

FABP7GPR34

HMGCS1SCD

BDE-47

yvasculature dev

cell migration f

anat morphogenesi

cell m

igration

vasculatu

re dev

lipid metabolism

inflammato

ry res

t3h+24h t15h+24h

arch 2019

AllCpGs

BDE-47DMRs

∆β (B

y = 090xRsup2 = 069

-015 -005 005 01501-01

∆β (B

(GW)166 193 216

t3h+24h t15h+24h

166 193 216

MRs uarrmethylation

darrmethylation

(darr)

(darr)(darr)

(uarr)

EAll D

MRs (472)

Promoter (17

t of G

ical P

plt001

TSS1500

TSS2005rsquoUTR

1st Exon

Body3rsquoU

Intergenic

arch 2019

DISCUSSION

01020304050607080

MRs uarrmethylation

darrmethylation

(uarr)

(darr)

1 2 3 4 5 6 7 8 9 10 111213141516171819202122

-006-004-002

0002004006008

0 25000000 50000000 75000000 100000000 125000000

BDE-47 DMRple005

Avg ∆β

4(3)152

ABDE-47 DE Genes

156(152)

Avg ∆β (BDE-47 vs Veh) 24h-01 01-005 0050

DPTFERMT1

06CRCT1

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

(GW)166 193 216y = 098xRsup2 = 081

-15 -1 -05 0 05 1 15Avg FC (BDE-47Veh) t3h+24h

t3h+24h t15h+24h

PSG7CCL1

166 193 216

arch 2019

ALL uarr darr

p=001p=005 p=0001

lative

PLAC4 IL6MMP1

FABP4GREM1

FABP7GPR34

HMGCS1SCD

BDE-47

yvasculature dev

cell migration f

anat morphogenesi

cell m

igration

vasculatu

re dev

lipid metabolism

inflammato

ry res

t3h+24h t15h+24h

arch 2019

AllCpGs

BDE-47DMRs

∆β (B

y = 090xRsup2 = 069

-015 -005 005 01501-01

∆β (B

(GW)166 193 216

t3h+24h t15h+24h

166 193 216

MRs uarrmethylation

darrmethylation

(darr)

(darr)(darr)

(uarr)

EAll D

MRs (472)

Promoter (17

t of G

ical P

plt001

TSS1500

TSS2005rsquoUTR

1st Exon

Body3rsquoU

Intergenic

arch 2019

DISCUSSION

01020304050607080

MRs uarrmethylation

darrmethylation

(uarr)

(darr)

1 2 3 4 5 6 7 8 9 10 111213141516171819202122

-006-004-002

0002004006008

0 25000000 50000000 75000000 100000000 125000000

BDE-47 DMRple005

Avg ∆β

4(3)152

ABDE-47 DE Genes

156(152)

Avg ∆β (BDE-47 vs Veh) 24h-01 01-005 0050

DPTFERMT1

06CRCT1

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

ALL uarr darr

p=001p=005 p=0001

lative

PLAC4 IL6MMP1

FABP4GREM1

FABP7GPR34

HMGCS1SCD

BDE-47

yvasculature dev

cell migration f

anat morphogenesi

cell m

igration

vasculatu

re dev

lipid metabolism

inflammato

ry res

t3h+24h t15h+24h

arch 2019

AllCpGs

BDE-47DMRs

∆β (B

y = 090xRsup2 = 069

-015 -005 005 01501-01

∆β (B

(GW)166 193 216

t3h+24h t15h+24h

166 193 216

MRs uarrmethylation

darrmethylation

(darr)

(darr)(darr)

(uarr)

EAll D

MRs (472)

Promoter (17

t of G

ical P

plt001

TSS1500

TSS2005rsquoUTR

1st Exon

Body3rsquoU

Intergenic

arch 2019

DISCUSSION

01020304050607080

MRs uarrmethylation

darrmethylation

(uarr)

(darr)

1 2 3 4 5 6 7 8 9 10 111213141516171819202122

-006-004-002

0002004006008

0 25000000 50000000 75000000 100000000 125000000

BDE-47 DMRple005

Avg ∆β

4(3)152

ABDE-47 DE Genes

156(152)

Avg ∆β (BDE-47 vs Veh) 24h-01 01-005 0050

DPTFERMT1

06CRCT1

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

AllCpGs

BDE-47DMRs

∆β (B

y = 090xRsup2 = 069

-015 -005 005 01501-01

∆β (B

(GW)166 193 216

t3h+24h t15h+24h

166 193 216

MRs uarrmethylation

darrmethylation

(darr)

(darr)(darr)

(uarr)

EAll D

MRs (472)

Promoter (17

t of G

ical P

plt001

TSS1500

TSS2005rsquoUTR

1st Exon

Body3rsquoU

Intergenic

arch 2019

DISCUSSION

01020304050607080

MRs uarrmethylation

darrmethylation

(uarr)

(darr)

1 2 3 4 5 6 7 8 9 10 111213141516171819202122

-006-004-002

0002004006008

0 25000000 50000000 75000000 100000000 125000000

BDE-47 DMRple005

Avg ∆β

4(3)152

ABDE-47 DE Genes

156(152)

Avg ∆β (BDE-47 vs Veh) 24h-01 01-005 0050

DPTFERMT1

06CRCT1

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

DISCUSSION

01020304050607080

MRs uarrmethylation

darrmethylation

(uarr)

(darr)

1 2 3 4 5 6 7 8 9 10 111213141516171819202122

-006-004-002

0002004006008

0 25000000 50000000 75000000 100000000 125000000

BDE-47 DMRple005

Avg ∆β

4(3)152

ABDE-47 DE Genes

156(152)

Avg ∆β (BDE-47 vs Veh) 24h-01 01-005 0050

DPTFERMT1

06CRCT1

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

SUPPLEMENTARY DATA

ACKNOWLEDGMENTS

FUNDING

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

Results

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

Discussion

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

Conclusion

arch 2019

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

Acknowledgments

References

Name ofAntibody

the Antibody

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

arch 2019

March 27 2019_A

kfy230-TF1

March 27 2019_B

Documents

Genomic Profiling of BDE-47 Effects on Human Placental ...observed BDE-47 accumulation in CTBs with limited evidence of metabolism. At a functional level, BDE-47 hindered the ability