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.

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.

Establishment of human trophoblast progenitor cell lines from the chorion.

Placental trophoblasts are key determinants of in utero development. Mouse trophoblast (TB) stem cells, which were first derived over a decade ago, are a powerful cell culture model for studying their self-renewal or differentiation. Our attempts to isolate an equivalent population from the trophectoderm of human blastocysts generated colonies that quickly differentiated in vitro. This finding suggested that the human placenta has another progenitor niche. Here, we show that the chorion is one such site. Initially, we immunolocalized pluripotency factors and TB fate determinants in the early gestation placenta, amnion, and chorion. Immunoreactive cells were numerous in the chorion. We isolated these cells and plated them in medium containing fibroblast growth factor which is required for human embryonic stem cell self-renewal, and an inhibitor of activin/nodal signaling. Colonies of polarized cells with a limited lifespan emerged. Trypsin dissociation yielded continuously self-replicating monolayers. Colonies and monolayers formed the two major human TB lineages-multinucleate syncytiotrophoblasts and invasive cytotrophoblasts (CTBs). Transcriptional profiling experiments revealed the factors associated with the self-renewal or differentiation of human chorionic TB progenitor cells (TBPCs). They included imprinted genes, NR2F1/2, HMGA2, and adhesion molecules that were required for TBPC differentiation. Together, the results of these experiments suggested that the chorion is one source of epithelial CTB progenitors. These findings explain why CTBs of fully formed chorionic villi have a modest mitotic index and identify the chorionic mesoderm as a niche for TBPCs that support placental growth.

Human placental cytotrophoblast epigenome dynamics over gestation and alterations in placental disease.

The human placenta and its specialized cytotrophoblasts rapidly develop, have a compressed lifespan, govern pregnancy outcomes, and program the offspring's health. Understanding the molecular underpinnings of these behaviors informs development and disease. Profiling the extraembryonic epigenome and transcriptome during the 2nd and 3rd trimesters revealed H3K9 trimethylation overlapping deeply DNA hypomethylated domains with reduced gene expression and compartment-specific patterns that illuminated their functions. Cytotrophoblast DNA methylation increased, and several key histone modifications decreased across the genome as pregnancy advanced. Cytotrophoblasts from severe preeclampsia had substantially increased H3K27 acetylation globally and at genes that are normally downregulated at term but upregulated in this syndrome. In addition, some cases had an immature pattern of H3K27ac peaks, and others showed evidence of accelerated aging, suggesting subtype-specific alterations in severe preeclampsia. Thus, the cytotrophoblast epigenome dramatically reprograms during pregnancy, placental disease is associated with failures in this process, and H3K27 hyperacetylation is a feature of severe preeclampsia.

A proteomic approach identifies early pregnancy biomarkers for preeclampsia: novel linkages between a predisposition to preeclampsia and cardiovascular disease.

Preeclampsia (PE) is a common, potentially life-threatening pregnancy syndrome triggered by placental factors released into the maternal circulation, resulting in maternal vascular dysfunction along with activated inflammation and coagulation. Currently there is no screening test for PE. We sought to identify differentially expressed plasma proteins in women who subsequently develop PE that may perform as predictive biomarkers. In seven DIGE experiments, we compared the plasma proteome at 20 wk gestation in women who later developed PE with an appropriate birth weight for gestational age baby (n=27) or a small for gestational age baby (n=12) to healthy controls with uncomplicated pregnancies (n=57). Of the 49 differentially expressed spots associated with PE-appropriate for gestational age, PE-small for gestational age or both (p<0.05, false discovery rate corrected), 39 were identified by LC-MS/MS. Two protein clusters that accurately (>90%) classified women at risk of developing PE were identified. Immunoblots confirmed the overexpression of fibrinogen gamma chain and alpha-1-antichymotrypsin in plasma prior to PE. The proteins identified are involved in lipid metabolism, coagulation, complement regulation, extracellular matrix remodeling, protease inhibitor activity and acute-phase responses, indicating novel synergism between pathways involved in the pathogenesis of PE. Our findings are remarkably similar to recently identified proteins complexed to high-density lipoprotein and linked to cardiovascular disease.

Placental Structure in Preterm Birth Among HIV-Positive Versus HIV-Negative Women in Kenya.

BACKGROUND: Preterm birth (PTB) is a major cause of infant morbidity and mortality in developing countries. Recent data suggest that in addition to Human Immunodeficiency Virus (HIV) infection, use of antiretroviral therapy (ART) increases the risk of PTB. As the mechanisms remain unexplored, we conducted this study to determine whether HIV and ART were associated with placental changes that could contribute to PTB. SETTING: We collected and evaluated placentas from 38 HIV-positive women on ART and 43 HIV-negative women who had preterm deliveries in Nairobi, Kenya. METHODS: Anatomical features of the placentas were examined at gross and microscopic levels. Cases were matched for gestational age and compared by the investigators who were blinded to maternal HIV serostatus. RESULTS: Among preterm placentas, HIV infection was significantly associated with thrombosis (P = 0.001), infarction (P = 0.032), anomalies in cord insertion (P = 0.02), gross evidence of membrane infection (P = 0.043), and reduced placental thickness (P = 0.010). Overall, preterm placentas in both groups were associated with immature villi, syncytial knotting, villitis, and deciduitis. Features of HIV-positive versus HIV-negative placentas included significant fibrinoid deposition with villus degeneration, syncytiotrophoblast delamination, red blood cell adhesion, hypervascularity, and reduction in both surface area and perimeter of the terminal villi. CONCLUSIONS: These results imply that HIV infection and/or ART are associated with morphological changes in preterm placentas that contribute to delivery before 37 weeks. Hypervascularity suggests that the observed pathologies may be attributable, in part, to hypoxia. Further research to explore potential mechanisms will help elucidate the pathways that are involved perhaps pointing to interventions for decreasing the risk of prematurity among HIV-positive women.

Expression of HLA-G in malignant mesothelioma and clinically aggressive breast carcinoma.

The aim of the present study was to evaluate HLA-G expression in breast carcinoma and malignant mesothelioma (MM). Malignant breast carcinoma effusions (46) and corresponding solid tumors (39) and 104 MM (26 effusions, 78 solid tumors) were analyzed using immunohistochemistry (IHC). HLA-G protein and mRNA expression were further studied using immunoblotting (IB) and RT-PCR. HLA-ABC expression was analyzed using flow cytometry (FCM). IHC showed predominantly focal HLA-G expression in 12 of 46 (26%) breast carcinoma effusions and 16 of 39 (41%) solid lesions. In MM, 20 of 78 (26%) solid lesions and 14 of 26 (54%) effusions were focally HLA-G positive. Expression in MM was higher in effusions (p=0.008). IB showed more frequent HLA-G expression in MM compared with breast carcinoma effusions, while RT-PCR showed HLA-G mRNA expression in both tumors. FCM showed conserved HLA-ABC expression in 15 of 15 effusions. Breast cancer patients with HLA-G-positive tumor cells had shorter disease-free survival (mean 37 vs 85, median 25 vs 31 months), though not significantly (p=0.14). In conclusion, HLA-G is focally expressed in MM and breast carcinoma, while HLA-ABC expression is conserved. However, the up-regulated expression of HLA-G in MM effusions and its possible association with shorter disease-free survival in advanced stage of breast carcinoma suggest a possible role in immune response evasion in some tumors.

HLA-G is a potential tumor marker in malignant ascites.

PURPOSE: Molecular approaches as supplements to cytological examination of malignant ascites may play an important role in the clinical management of cancer patients. HLA-G is a potential tumor-associated marker and that one of its isoforms, HLA-G5, produces a secretory protein. This study is to assess the clinical utility of secreted HLA-G levels in differential diagnosis of malignant ascites. EXPERIMENTAL DESIGN: We used ELISA to assess whether secretory HLA-G (sHLA-G) could serve as a marker of malignant ascites in ovarian and breast carcinomas, which represent the most common malignant tumors causing ascites in women. RESULTS: On the basis of immunohistochemistry, 45 (61%) of 74 ovarian serous carcinomas and 22 (25%) invasive ductal carcinomas of the breast demonstrated HLA-G immunoreactivity ranging from 2 to 100% of the tumor cells. HLA-G staining was not detected in a wide variety of normal tissues, including ovarian surface epithelium and normal breast tissue. Revese transcription-PCR demonstrated the presence of HLA-G5 isoform in all of the tumor samples expressing HLA-G. ELISA was performed to measure the sHLA-G in 42 malignant and 18 benign ascites supernatants. sHLA-G levels were significantly higher in malignant ascites than in benign controls (P < 0.001). We found that the area under the receiver-operating characteristic curve for sHLA-G was 0.95 for malignant versus benign ascites specimens. At 100% specificity, the highest sensitivity to detect malignant ascites was 78% (95% confidence interval, 68-88%) at a cutoff of 13 ng/ml. CONCLUSIONS: Our findings suggest that measurement of sHLA-G is a useful molecular adjunct to cytology in the differential diagnosis of malignant versus benign ascites.

HLA-G upregulation in pre-malignant and malignant lesions of the gastrointestinal tract.

HLA-G belongs to the nonclassical MHC class Ib group of molecules and has been implicated in mediating immune-responsiveness in various cancerous and non-cancerous cell types. We have examined HLA-G expression in a number of human gastrointestinal malignancies, including pancreatic ductal adenocarcinoma, ampullary cancer, biliary cancer, and colorectal cancer by immunolabeling analysis. We used indices of <5% (negative), 6-25%, 26-50%, 51-75%, and >75% (diffuse) to subclassify lesions based on percentage of positive cell labeling. Across all cancer subtypes, 52-79% of lesions demonstrated expression of HLA-G, with up to 33% of lesions demonstrating diffuse (>75%) expression. In addition, we utilized the neoplastic progression model of colorectal cancer to evaluate HLA-G protein expression in normal colon, tubulovillous adenomas, invasive cancer, and liver metastases arising from colorectal cancer. Focal HLA-G expression was detected in regions of normal colon adjacent to sites of adenomatous and cancerous lesions, as well as in all stages of cancer progression. Overall, the percentage of diffusely (>75%) labeled lesions appeared increased in preneoplastic and neoplastic conditions, as compared to normal colon. Specifically, tubulovillous adnenomas demonstrated pronounced diffuse labeling in 58% of lesions examined. No correlation with HLA-G expression and CD4+ or CD8+ T cells was identified. We propose that HLA-G expression is upregulated in a large percentage of gastrointestinal lesions and may serve to mediate immune-responsiveness in certain instances.

HLA-G immunoreactivity is specific for intermediate trophoblast in gestational trophoblastic disease and can serve as a useful marker in differential diagnosis.

HLA-G is a nonclassical MHC class I antigen that has been shown to be a specific marker for normal intermediate trophoblast (IT). In this study HLA-G immunoreactivity assessed with an HLA-G specific antibody (4H84) was detected in all 14 cases of choriocarcinoma, 14 placental site trophoblastic tumors, 13 epithelioid trophoblastic tumors, 16 placental site nodules, and nine exaggerated placental sites. In contrast, HLA-G immunoreactivity was not detected in 34 nontrophoblastic uterine neoplasms. HLA-G immunoreactivity was present in all the IT cells of exaggerated placental sites and placental site trophoblastic tumors and in 70-100% of IT cells in placental site nodules and epithelioid trophoblastic tumors. The pattern of distribution of HLA-G in different subpopulations of IT confirms the relationship of various trophoblastic lesions to different types of IT (exaggerated placental site and placental site trophoblastic tumor to implantation site IT and placental site nodule and epithelioid trophoblastic tumor to chorionic-type IT) and suggests that choriocarcinoma is related to villous-type IT because the majority of mononucleate cells in this neoplasm were HLA-G immunoreactive. In conclusion, HLA-G immunoreactivity appears to be specific for IT in gestational trophoblastic disease and can serve as a useful marker in the differential diagnosis of these lesions.

Truncated HLA-G isoforms are retained in the endoplasmic reticulum and insufficiently provide HLA-E ligands.

The preferential expression of the non-polymorphic human leukocyte antigen G (HLA-G) on invading extravillous cytotrophoblast cells that are, with the exception of HLA-C and -E, HLA class I negative led to the hypothesis that HLA-G plays a major role in controlling the effector functions of the large granular leukocytes (LGL), a specialized natural killer (NK) cell population present in large numbers in the decidua. Transcription of the HLA-G gene is characterized by extensive alternative splicing producing at least seven potentially membrane bound or secreted isoforms. Except for HLA-G1 and its soluble variant (HLA-G1s), there is still dispute as to whether any of the other isoforms displays a major immunological function. Here we describe that the membrane-bound isoforms HLA-G2, -G3, and G4 as well as the soluble variant of HLA-G2 (HLA-G2s) do not egress the endoplasmic reticulum as determined by Endo H sensitivity assays. Moreover these isoforms seem not to have a major immunological function with respect to NK cell inhibition by providing a ligand for HLA-E, which would allow the interaction of this molecule with the inhibitory CD94/NKG2A NK cell receptor.

Abnormal placentation and the syndrome of preeclampsia.

Preeclampsia, particularly the severe cases that occur early in pregnancy, is associated with defects in the (placental) cytotrophoblast differentiation pathway that leads to uterine invasion. At a morphologic level, interstitial invasion often is shallow. Perhaps more significantly, endovascular invasion, particularly the arterial component, is rudimentary. The latter defect is thought to lead to hypoperfusion of the placenta. At a molecular level, these defects are associated with particular deficits in the differentiation process whereby cytotrophoblasts--epithelial cells of ectodermal origin--assume vascular-like properties. Until recently, the question was how the latter defects could lead to the maternal symptoms of this condition. Now a possible link in the form of preeclampsia-associated changes in placental production of vasculogenic/angiogenic substances and their inhibitors has been discovered. It is likely that this new paradigm will improve both diagnosis and treatment of this life-threatening pregnancy complication.

Disruption of oxygen-regulated responses underlies pathological changes in the placentas of women who smoke or who are passively exposed to smoke during pregnancy.

Previously, we showed that maternal smoking harms human placental development by changing the balance between cytotrophoblast (CTB) proliferation and differentiation. To understand the mechanisms involved, we studied the effects of maternal smoking and in vitro exposure of CTBs to nicotine and on CTB expression of molecules that govern cellular responses to oxygen tension: the von Hippel-Lindau tumor suppressor protein (pVHL), the hypoxia-inducible transcription factors (HIFs), and the vascular endothelial growth factors (VEGFs). We previously reported that hypoxia upregulates CTB pVHL expression (1). Here we show that in vitro exposure of CTBs to nicotine has the same effect. Maternal smoking also dysregulated CTB expression of all three molecules. Remarkably, we found that passive exposure to cigarette smoke had many of the same effects as active smoking, a graphic demonstration of the ill effects of cigarette smoke, even secondhand, on placental development. Together, these findings explain, in part, how smoking damages the placenta by altering expression of key mediators of placental development.

Human trophoblast progenitors: where do they reside?

In humans, very little is known about the factors that regulate trophoblast (TB) specification, expansion of the initial TB population, and formation of the cytotrophoblast (CTB) populations that populate the chorionic villi. The absence of human trophoblast progenitor cell (hTPC) lines that can be propagated in vitro has been a limiting factor. Because attempts to derive TB stem cells from the trophectoderm of the human blastocyst have so far failed, investigators use alternative systems as cell culture models including TBs derived from human embryonic stem cells (hESCs), immortalized CTBs, and cell lines established from TB tumors. Additionally, the characteristics of mature TBs have been extensively studied using primary cultures of CTBs and explants of placental chorionic villi. However, none of these models can be used to study TB progenitor self-renewal and differentiation. Furthermore, the propagation of human TB progenitors from villous CTBs (vCTBs) has not been achieved. The downregulation of key markers of cell cycle progression in vCTBs by the end of the first trimester of pregnancy may indicate that these cells are not a source of human TB progenitors later in pregnancy. In contrast, mesenchymal cells of the villi and chorion continue to proliferate until the end of pregnancy. We recently reported isolation of continuously self-renewing hTPCs from chorionic mesenchyme and showed that they differentiated into the mature TB cell types of the villi, evidence that they can function as TB progenitors. This new cell culture model enables a molecular analysis of the seminal steps in human TB differentiation that have yet to be studied in humans. In turn, this information can be used to trace the origins of pregnancy complications that are associated with faulty TB growth and differentiation.

Reversal of gene dysregulation in cultured cytotrophoblasts reveals possible causes of preeclampsia.

During human pregnancy, a subset of placental cytotrophoblasts (CTBs) differentiates into cells that aggressively invade the uterus and its vasculature, anchoring the progeny and rerouting maternal blood to the placenta. In preeclampsia (PE), CTB invasion is limited, reducing placental perfusion and/or creating intermittent flow. This syndrome, affecting 4%-8% of pregnancies, entails maternal vascular alterations (e.g., high blood pressure, proteinuria, and edema) and, in some patients, fetal growth restriction. The only cure is removal of the faulty placenta, i.e., delivery. Previously, we showed that defective CTB differentiation contributes to the placental component of PE, but the causes were unknown. Here, we cultured CTBs isolated from PE and control placentas for 48 hours, enabling differentiation and invasion. In various severe forms of PE, transcriptomics revealed common aberrations in CTB gene expression immediately after isolation, including upregulation of SEMA3B, which resolved in culture. The addition of SEMA3B to normal CTBs inhibited invasion and recreated aspects of the PE phenotype. Additionally, SEMA3B downregulated VEGF signaling through the PI3K/AKT and GSK3 pathways, effects that were observed in PE CTBs. We propose that, in severe PE, the in vivo environment dysregulates CTB gene expression; the autocrine actions of the upregulated molecules (including SEMA3B) impair CTB differentiation, invasion and signaling; and patient-specific factors determine the signs.

HLA-G expression in effusions is a possible marker of tumor susceptibility to chemotherapy in ovarian carcinoma.

OBJECTIVE: We recently showed that the levels of secreted human leukocyte antigen-G (HLA-G), a nonclassical MHC class I antigen, are significantly elevated in malignant effusions in ovarian carcinoma compared to benign ones. The objective of this study was to evaluate the expression and clinical role of HLA-G in effusions and corresponding solid tumors from patients diagnosed with advanced-stage ovarian carcinoma. METHODS: Effusions (= 148), corresponding primary tumors (= 66), and metastatic lesions (= 122) were analyzed using immunohistochemistry with an anti-HLA-G monoclonal antibody. RESULTS: HLA-G was detected in cancer cells in 49/148 (33%) effusions, 33/66 (50%) primary tumors, and 59/122 (48%) solid metastases. These differences did not reach statistical significance. Expression in effusions and solid metastases significantly correlated (P = 0.029). HLA-G expression in tumor cells was significantly lower in effusions obtained during or following chemotherapy (P = 0.038). The presence of HLA-G-positive tumor cells in effusions obtained prior to the institution of chemotherapy correlated with better overall survival (P = 0.042). HLA-G expression in primary tumors and solid metastases did not correlate with any of the clinicopathologic parameters studied. CONCLUSIONS: HLA-G is expressed in a significant number of ovarian carcinomas at all anatomic sites. The reduced expression of HLA-G in post-chemotherapy effusions and its correlation with improved survival may be related to preferential susceptibility of HLA-G-expressing cells at this site. Our findings suggest a new role for HLA-G as a prognostic indicator in advanced-stage ovarian cancer in effusions.

Longitudinal serum concentrations of placental growth factor: evidence for abnormal placental angiogenesis in pathologic pregnancies.

OBJECTIVE: Complicated pregnancies demonstrate abnormal decidual and placental villous vasculature. We examined maternal concentrations of vascular endothelial growth factor and placental growth factor in normal pregnancies and in pregnancies that were complicated by isolated idiopathic small-for-gestational-age (SGA) newborn infants, preeclampsia alone, or preeclampsia with SGA newborn infants at the time of clinical disease and before the onset of clinical signs. STUDY DESIGN: Serum vascular endothelial growth factor and placental growth factor were measured by enzyme-linked immunosorbent assay in cross-sectional and longitudinal cohorts of pregnant nulliparous women. The results were compared by Wilcoxon tests or a mixed-models method, respectively. RESULTS: In the cross-sectional study, serum placental growth factor was reduced in abnormal pregnancy relative to control subjects (SGA newborn infants, 18 [P =.04]; preeclampsia, 20; or preeclampsia with small- for-gestational-age newborn infants, 11 [P =.0001]) as early as 15 to 19 weeks of gestation in preeclampsia with SGA newborn infants. Vascular endothelial growth factor was <30 pg/mL in all serum specimens from pregnant women. CONCLUSION: We postulate that decreased placental growth factor production results in abnormalities of placental angiogenesis through direct and indirect effects on other vasculotropic growth factors.