Placenta: the forgotten organ.

The placenta sits at the interface between the maternal and fetal vascular beds where it mediates nutrient and waste exchange to enable in utero existence. Placental cells (trophoblasts) accomplish this via invading and remodeling the uterine vasculature. Amazingly, despite being of fetal origin, trophoblasts do not trigger a significant maternal immune response. Additionally, they maintain a highly reliable hemostasis in this extremely vascular interface. Decades of research into how the placenta differentiates itself from embryonic tissues to accomplish these and other feats have revealed a previously unappreciated level of complexity with respect to the placenta's cellular composition. Additionally, novel insights with respect to roles played by the placenta in guiding fetal development and metabolism have sparked a renewed interest in understanding the interrelationship between fetal and placental well-being. Here, we present an overview of emerging research in placental biology that highlights these themes and the importance of the placenta to fetal and adult health.

RNA profiling of laser microdissected human trophoblast subtypes at mid-gestation reveals a role for cannabinoid signaling in invasion.

Human placental architecture is complex. Its surface epithelium, specialized for transport, forms by fusion of cytotrophoblast progenitors into multinucleated syncytiotrophoblasts. Near the uterine surface, these progenitors assume a different fate, becoming cancer-like cells that invade its lining and blood vessels. The latter process physically connects the placenta to the mother and shunts uterine blood to the syncytiotrophoblasts. Isolation of trophoblast subtypes is technically challenging. Upon removal, syncytiotrophoblasts disintegrate and invasive cytotrophoblasts are admixed with uterine cells. We used laser capture to circumvent these obstacles. This enabled isolation of syncytiotrophoblasts and two subpopulations of invasive cytotrophoblasts from cell columns and the endovascular compartment of spiral arteries. Transcriptional profiling revealed numerous genes, the placental or trophoblast expression of which was not known, including neurotensin and C4ORF36. Using mass spectrometry, discovery of differentially expressed mRNAs was extended to the protein level. We also found that invasive cytotrophoblasts expressed cannabinoid receptor 1. Unexpectedly, screening agonists and antagonists showed that signals from this receptor promote invasion. Together, these results revealed previously unseen gene expression patterns that translate to the protein level. Our data also suggested that endogenous and exogenous cannabinoids can affect human placental development.

Global proteomic analyses of human cytotrophoblast differentiation/invasion.

During human pregnancy, cytotrophoblasts (CTBs) from the placenta differentiate into specialized subpopulations that play crucial roles in proper fetal growth and development. A subset of these CTBs differentiate along an invasive pathway, penetrating the decidua and anchoring the placenta to the uterus. A crucial hurdle in pregnancy is the ability of these cells to migrate, invade and remodel spiral arteries, ensuring adequate blood flow to nourish the developing fetus. Although advances continue in describing the molecular features regulating the differentiation of these cells, assessment of their global proteomic changes at mid-gestation remain undefined. Here, using sequential window acquisition of all theoretical fragment-ion spectra (SWATH), which is a data-independent acquisition strategy, we characterized the protein repertoire of second trimester human CTBs during their differentiation towards an invasive phenotype. This mass spectrometry-based approach allowed identification of 3026 proteins across four culture time points corresponding to sequential stages of differentiation, confirming the expression dynamics of established molecules and offering new information into other pathways involved. The availability of a SWATH CTB global spectral library serves as a beneficial resource for hypothesis generation and as a foundation for further understanding CTB differentiation dynamics.

High rates of placental inflammation among samples collected by the Multi-Omics for Mothers and Infants consortium.

BACKGROUND: The Multi-Omics for Mothers and Infants consortium aims to improve birth outcomes. Preterm birth is a major obstetrical complication globally and causes significant infant and childhood morbidity and mortality. OBJECTIVE: We analyzed placental samples (basal plate, placenta or chorionic villi, and the chorionic plate) collected by the 5 Multi-Omics for Mothers and Infants sites, namely The Alliance for Maternal and Newborn Health Improvement Bangladesh, The Alliance for Maternal and Newborn Health Improvement Pakistan, The Alliance for Maternal and Newborn Health Improvement Tanzania, The Global Alliance to Prevent Prematurity and Stillbirth Bangladesh, and The Global Alliance to Prevent Prematurity and Stillbirth Zambia. The goal was to analyze the morphology and gene expression of samples collected from preterm and uncomplicated term births. STUDY DESIGN: The teams provided biopsies from 166 singleton preterm (<37 weeks' gestation) and 175 term (≥37 weeks' gestation) deliveries. The samples were fixed in formalin and paraffin embedded. Tissue sections from these samples were stained with hematoxylin and eosin and subjected to morphologic analyses. Other placental biopsies (n=35 preterm, 21 term) were flash frozen, which enabled RNA purification for bulk transcriptomics. RESULTS: The morphologic analyses revealed a surprisingly high rate of inflammation that involved the basal plate, placenta or chorionic villi, and the chorionic plate. The rate of inflammation in chorionic villus samples, likely attributable to chronic villitis, ranged from 25% (Pakistan site) to 60% (Zambia site) of cases. Leukocyte infiltration in this location vs in the basal plate or chorionic plate correlated with preterm birth. Our transcriptomic analyses identified 267 genes that were differentially expressed between placentas from preterm vs those from term births (123 upregulated, 144 downregulated). Mapping the differentially expressed genes onto single-cell RNA sequencing data from human placentas suggested that all the component cell types, either singly or in subsets, contributed to the observed dysregulation. Consistent with the histopathologic findings, gene ontology analyses highlighted the presence of leukocyte infiltration or activation and inflammatory responses in both the fetal and maternal compartments. CONCLUSION: The relationship between placental inflammation and preterm birth is appreciated in developed countries. In this study, we showed that this link also exists in developing geographies. In addition, among the participating sites, we found geographic- and population-based differences in placental inflammation and preterm birth, suggesting the importance of local factors.

Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα.

The placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. The 100,000  g pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to this vesicle type, CD9 and HRS, and the trophoblast proteins placental alkaline phosphatase and HLA-G. Global profiling by mass spectrometry showed that placental EVs were enriched for proteins that function in transport and viral processes. A cytokine array revealed that the CTB 100,000  g pellet contained a significant amount of tumor necrosis factor α (TNFα). CTB EVs increased decidual stromal cell (dESF) transcription and secretion of NF-κB targets, including IL8, as measured by qRT-PCR and cytokine array. A soluble form of the TNFα receptor inhibited the ability of CTB 100,000  g EVs to increase dESF secretion of IL8. Overall, the data suggest that CTB EVs enhance decidual cell release of inflammatory cytokines, which we theorize is an important component of successful pregnancy.

Up-regulated cytotrophoblast DOCK4 contributes to over-invasion in placenta accreta spectrum.

In humans, a subset of placental cytotrophoblasts (CTBs) invades the uterus and its vasculature, anchoring the pregnancy and ensuring adequate blood flow to the fetus. Appropriate depth is critical. Shallow invasion increases the risk of pregnancy complications, e.g., severe preeclampsia. Overly deep invasion, the hallmark of placenta accreta spectrum (PAS), increases the risk of preterm delivery, hemorrhage, and death. Previously a rare condition, the incidence of PAS has increased to 1:731 pregnancies, likely due to the rise in uterine surgeries (e.g., Cesarean sections). CTBs track along scars deep into the myometrium and beyond. Here we compared the global gene expression patterns of CTBs from PAS cases to gestational age-matched control cells that invaded to the normal depth from preterm birth (PTB) deliveries. The messenger RNA (mRNA) encoding the guanine nucleotide exchange factor, DOCK4, mutations of which promote cancer cell invasion and angiogenesis, was the most highly up-regulated molecule in PAS samples. Overexpression of DOCK4 increased CTB invasiveness, consistent with the PAS phenotype. Also, this analysis identified other genes with significantly altered expression in this disorder, potential biomarkers. These data suggest that CTBs from PAS cases up-regulate a cancer-like proinvasion mechanism, suggesting molecular as well as phenotypic similarities in the two pathologies.

Discordant Zika Virus Findings in Twin Pregnancies Complicated by Antenatal Zika Virus Exposure: A Prospective Cohort.

BACKGROUND: There are limited data on the natural history of antenatal Zika virus (ZIKV) exposure in twin pregnancies, especially regarding intertwin concordance of prenatal, placental, and infant outcomes. METHODS: This prospective cohort study included twin pregnancies referred to a single institution from September 2015 to June 2016 with maternal ZIKV. Polymerase chain reaction (PCR) testing of maternal, placental, and neonatal samples was performed. Prenatal ultrasounds were completed for each twin, and histomorphologic analysis was performed for each placenta. Abnormal neonatal outcome was defined as abnormal exam and/or abnormal imaging. Two- to three-year follow-up of infants included physical exams, neuroimaging, and Bayley-III developmental assessment. RESULTS: Among 244 pregnancies, 4 twin gestations without coinfection were identified. Zika virus infection occurred at 16-33 weeks gestation. Zika virus PCR testing revealed discordance between dichorionic twins, between placentas in a dichorionic pair, between portions of a monochorionic placenta, and between a neonate and its associated placenta. Of the 8 infants, 3 (38%) had an abnormal neonatal outcome. Of 6 infants with long-term follow-up, 3 (50%) have demonstrated ZIKV-related abnormalities. CONCLUSIONS: Neonatal PCR testing, placental findings, and infant outcomes can be discordant between co-twins with antenatal ZIKV exposure. These findings demonstrate that each twin should be evaluated independently for vertical transmission.

Severe pre-eclampsia is associated with alterations in cytotrophoblasts of the smooth chorion.

Pre-eclampsia (PE), which affects ∼8% of first pregnancies, is associated with faulty placentation. Extravillous cytotrophoblasts (CTBs) fail to differentiate properly, contributing to shallow uterine invasion and deficient spiral artery remodeling. We studied the effects of severe PE (sPE) on the smooth chorion portion of the fetal membranes. The results showed a significant expansion of the CTB layer. The cells displayed enhanced expression of stage-specific antigens that extravillous CTBs normally upregulate as they exit the placenta. Transcriptomics revealed the dysregulated expression of many genes (e.g. placental proteins, markers of oxidative stress). We confirmed an sPE-related increase in production of PAPPA1, which releases IGF1 from its binding protein. IGF1 enhanced proliferation of smooth chorion CTBs, a possible explanation for expansion of this layer, which may partially compensate for the placental deficits.

The human chorion contains definitive hematopoietic stem cells from the fifteenth week of gestation.

We examined the contribution of the fetal membranes, amnion and chorion, to human embryonic and fetal hematopoiesis. A population of cells displaying a hematopoietic progenitor phenotype (CD34++ CD45low) of fetal origin was present in the chorion at all gestational ages, associated with stromal cells or near blood vessels, but was absent in the amnion. Prior to 15 weeks of gestation, these cells lacked hematopoietic in vivo engraftment potential. Differences in the chemokine receptor and ß1 integrin expression profiles of progenitors between the first and second trimesters suggest that these cells had gestationally regulated responses to homing signals and/or adhesion mechanisms that influenced their ability to colonize the stem cell niche. Definitive hematopoietic stem cells, capable of multilineage and long-term reconstitution when transplanted in immunodeficient mice, were present in the chorion from 15-24 weeks gestation, but were absent at term. The second trimester cells also engrafted secondary recipients in serial transplantation experiments. Thus, the human chorion contains functionally mature hematopoietic stem cells at mid-gestation.

Gravidity-dependent associations between interferon response and birth weight in placental malaria.

BACKGROUND: Maternal malarial infection leads to poor perinatal outcomes, including low birth weight from preterm delivery and/or fetal growth restriction, particularly in primigravidas. In placental malaria, Plasmodium falciparum-infected red blood cells cause an inflammatory response that can interfere with maternal-fetal exchange, leading to poor growth. The type I interferon (IFN-I) pathway plays an immunomodulatory role in viral and bacterial infections, usually by suppressing inflammatory responses. However, its role in placental malaria is unknown. This study examines the cytokine responses in placental tissue from subsets of malaria-infected and uninfected women, and attempts to correlate them with particular birth outcomes. METHODS: 40 whole placental biopsy samples were obtained from pregnant women at least 16 years of age recruited to a larger prospective chemoprevention trial against malaria. These were patients at Tororo District Hospital in Uganda, an area of high malaria endemicity where approximately 40% of women have evidence of malaria infection at delivery. They were regularly followed at a local clinic and monitored for fever, with blood smears performed then and at time of delivery to diagnose malaria infection. Placenta biopsies were taken for histological diagnosis of placental malaria, as well as quantitative PCR analysis of genes in the IFN-I pathway (IFN-ß, IL-10 and MX-1). Parameters such as infant birth weight and gestational age were also recorded. RESULTS: Histological analysis revealed placental malaria in 18 samples, while 22 were found to be uninfected. RT-PCR analysis showed a four-fold increase in IFN-ß and IL-10 expression in multigravidas with placental malaria when compared to gravidity-matched, uninfected controls. This effect was not observed in primigravidas. Interestingly, linear regression analysis showed a positive association between IFN-ß levels and higher birth weights (ß = 101.2 g per log2-fold increase in IFN-ß expression, p = 0.042). This association was strongest in primigravidas with placental malaria (ß = 339.0, p = 0.006). CONCLUSIONS: These results demonstrate differential regulation of the IFN-I pathway in placental malaria according to gravidity, with the greatest anti-inflammatory response seen in multigravidas. The association between IFN-ß levels and higher birth weight also suggests a protective role for IFN-I against fetal growth restriction in placental malaria.

Association of polybrominated diphenyl ether (PBDE) levels with biomarkers of placental development and disease during mid-gestation.

BACKGROUND: Polybrominated diphenyl ether (PBDE) exposures have been associated with adverse pregnancy outcomes. A hypothesized mechanism is via alterations in placental development and function. However, we lack biomarkers that can be used as early indicators of maternal/fetal response to PBDE exposures and/or perturbations in placental development or function. METHODS: To evaluate the relationship between PBDE levels and placental biomarkers during mid-gestation of human pregnancy (n = 62), we immunolocalized three molecules that play key roles in cytotrophoblast (CTB) differentiation and interstitial/endovascular uterine invasion-integrin alpha-1 (ITGA1), vascular endothelial-cadherin (CDH5), and metalloproteinase-1 (MMP1)-and assessed three morphological parameters as potential indicators of pathological alterations using H&E-stained tissues-leukocyte infiltration, fibrinoid deposition, and CTB endovascular invasion. We evaluated associations between placental PBDE levels and of biomarkers of placental development and disease using censored Kendall's tau correlation and linear regression methods. RESULTS: PBDEs were detected in all placental samples. We observed substantial variation in antigen expression and morphological endpoints across placental regions. We observed an association between PBDE concentrations and immunoreactivity of endovascular CTB staining with anti-ITGA1 (inverse) or interstitial CTBs staining with anti-CDH5 (positive). CONCLUSIONS: We found several molecular markers that may be sensitive placental indicators of PBDE exposure. Further, this indicates that placental biomarkers of development and disease could be useful barometers of exposure to PBDEs, a paradigm that could be extended to other environmental chemicals and placental stage-specific antigens.

Defective decidualization during and after severe preeclampsia reveals a possible maternal contribution to the etiology.

In preeclampsia (PE), cytotrophoblast (CTB) invasion of the uterus and spiral arteries is often shallow. Thus, the placenta's role has been a focus. In this study, we tested the hypothesis that decidual defects are an important determinant of the placental phenotype. We isolated human endometrial stromal cells from nonpregnant donors with a previous pregnancy that was complicated by severe PE (sPE). Compared with control cells, they failed to decidualize in vitro as demonstrated by morphological criteria and the analysis of stage-specific antigens (i.e., IGFBP1, PRL). These results were bolstered by global transcriptional profiling data that showed they were transcriptionally inert. Additionally, we used laser microdissection to isolate the decidua from tissue sections of the maternal-fetal interface in sPE. Global transcriptional profiling revealed defects in gene expression. Also, decidual cells from patients with sPE, which dedifferentiated in vitro, failed to redecidualize in culture. Conditioned medium from these cells failed to support CTB invasion. To mimic aspects of the uterine environment in normal pregnancy, we added PRL and IGFBP1, which enhanced invasion. These data suggested that failed decidualization is an important contributor to down-regulated CTB invasion in sPE. Future studies will be aimed at determining whether this discovery has translational potential with regard to assessing a woman's risk of developing this pregnancy complication.

Quantitative proteomic analyses of mammary organoids reveals distinct signatures after exposure to environmental chemicals.

Common environmental contaminants such as bisphenols and phthalates and persistent contaminants such as polychlorinated biphenyls are thought to influence tissue homeostasis and carcinogenesis by acting as disrupters of endocrine function. In this study we investigated the direct effects of exposure to bisphenol A (BPA), mono-n-butyl phthalate (Pht), and polychlorinated biphenyl 153 (PCB153) on the proteome of primary organotypic cultures of the mouse mammary gland. At low-nanomolar doses each of these agents induced distinct effects on the proteomes of these cultures. Although BPA treatment produced effects that were similar to those induced by estradiol, there were some notable differences, including a reduction in the abundance of retinoblastoma-associated protein and increases in the Rho GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein CDC42. Both Pht and PCB153 induced changes that were distinct from those induced by estrogen, including decreased levels of the transcriptional corepressor C-terminal binding protein 1. Interestingly, the three chemicals appeared to alter the abundance of distinct splice forms of many proteins as well as the abundance of several proteins that regulate RNA splicing. Our combined results indicate that the three classes of chemical have distinct effects on the proteome of normal mouse mammary cultures, some estrogen-like but most estrogen independent, that influence diverse biological processes including apoptosis, cell adhesion, and proliferation.

Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification.

Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active ß-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines.

Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions.

Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.

Bacterial Interactomes: Interacting Protein Partners Share Similar Function and Are Validated in Independent Assays More Frequently Than Previously Reported.

Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

Alterations in the Salivary Proteome and N-Glycome of Sjögren's Syndrome Patients.

We used isobaric mass tagging (iTRAQ) and lectin affinity capture mass spectrometry (MS)-based workflows for global analyses of parotid saliva (PS) and whole saliva (WS) samples obtained from patients diagnosed with primary Sjögren's Syndrome (pSS) who were enrolled in the Sjögren's International Collaborative Clinical Alliance (SICCA) as compared with two control groups. The iTRAQ analyses revealed up- and down-regulation of numerous proteins that could be involved in the disease process (e.g., histones) or attempts to mitigate the ensuing damage (e.g., bactericidal/permeability increasing fold containing family (BPIF) members). An immunoblot approach applied to independent sample sets confirmed the pSS associated up-regulation of ß2-microglobulin (in PS) and down-regulation of carbonic anhydrase VI (in WS) and BPIFB2 (in PS). Beyond the proteome, we profiled the N-glycosites of pSS and control samples. They were enriched for glycopeptides using lectins Aleuria aurantia and wheat germ agglutinin, which recognize fucose and sialic acid/N-acetyl glucosamine, respectively. MS analyses showed that pSS is associated with increased N-glycosylation of numerous salivary glycoproteins in PS and WS. The observed alterations of the salivary proteome and N-glycome could be used as pSS biomarkers enabling easier and earlier detection of this syndrome while lending potential new insights into the disease process.

Preeclampsia: novel insights from global RNA profiling of trophoblast subpopulations.

BACKGROUND: The maternal signs of preeclampsia, which include the new onset of high blood pressure, can occur because of faulty placentation. We theorized that transcriptomic analyses of trophoblast subpopulations in situ would lend new insights into the role of these cells in preeclampsia pathogenesis. OBJECTIVE: Our goal was to enrich syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts from the placentas of severe preeclampsia cases. Total RNA was subjected to global transcriptional profiling to identify RNAs that were misexpressed compared with controls. STUDY DESIGN: This was a cross-sectional analysis of placentas from women who had been diagnosed with severe preeclampsia. Gestational age-matched controls were placentas from women who had a preterm birth with no signs of infection. Laser microdissection enabled enrichment of syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts. After RNA isolation, a microarray approach was used for global transcriptional profiling. Immunolocalization identified changes in messenger RNA expression that carried over to the protein level. Differential expression of non-protein-coding RNAs was confirmed by in situ hybridization. A 2-way analysis of variance of non-coding RNA expression identified particular classes that distinguished trophoblasts in cases vs controls. Cajal body foci were visualized by coilin immunolocalization. RESULTS: Comparison of the trophoblast subtype data within each group (severe preeclampsia or noninfected preterm birth) identified many highly differentially expressed genes. They included molecules that are known to be expressed by each subpopulation, which is evidence that the method worked. Genes that were expressed differentially between the 2 groups, in a cell-type-specific manner, encoded a combination of molecules that previous studies associated with severe preeclampsia and those that were not known to be dysregulated in this pregnancy complication. Gene ontology analysis of the syncytiotrophoblast data highlighted the dysregulation of immune functions, morphogenesis, transport, and responses to vascular endothelial growth factor and progesterone. The invasive cytotrophoblast data provided evidence of alterations in cellular movement, which is consistent with the shallow invasion often associated with severe preeclampsia. Other dysregulated pathways included immune, lipid, oxygen, and transforming growth factor-beta responses. The data for endovascular cytotrophoblasts showed disordered metabolism, signaling, and vascular development. Additionally, the transcriptional data revealed the differential expression in severe preeclampsia of 2 classes of non-coding RNAs: long non-coding RNAs and small nucleolar RNAs. The long non-coding RNA, urothelial cancer associated 1, was the most highly up-regulated in this class. In situ hybridization confirmed severe preeclampsia-associated expression in syncytiotrophoblasts. The small nucleolar RNAs, which chemically modify RNA structure, also correlated with severe preeclampsia. Thus, we enumerated Cajal body foci, sites of small nucleolar RNA activity, in primary cytotrophoblasts that were isolated from control and severe preeclampsia placentas. In severe preeclampsia, cytotrophoblasts had approximately double the number of these foci as the control samples. CONCLUSION: A laser microdissection approach enabled the identification of novel messenger RNAs and non-coding RNAs that were misexpressed by various trophoblast subpopulations in severe preeclampsia. The results suggested new avenues of investigation, in particular, the roles of PRG2, Kell blood group determinants, and urothelial cancer associated 1 in syncytiotrophoblast diseases. Additionally, many of the newly identified dysregulated molecules might have clinical utility as biomarkers of severe preeclampsia.

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma.

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.