Epigenetic changes regulating the epithelial-mesenchymal transition in human trophoblast differentiation.

The phenotype of human placental extravillous trophoblast (EVT) at the end of pregnancy reflects both first trimester differentiation from villous cytotrophoblast (CTB) and later gestational changes, including loss of proliferative and invasive capacity. Invasion abnormalities are central to two major placental pathologies, preeclampsia and placenta accreta spectrum, so characterization of the corresponding normal processes is crucial. In this report, our gene expression analysis, using purified human CTB and EVT cells, highlights an epithelial-mesenchymal transition (EMT) mechanism underlying CTB-EVT differentiation and provides a trophoblast-specific EMT signature. In parallel, DNA methylation profiling shows that CTB cells, already hypomethylated relative to non-trophoblast cell lineages, show further genome-wide hypomethylation in the transition to EVT. However, a small subgroup of genes undergoes gains of methylation (GOM) in their regulatory regions or gene bodies, associated with differential mRNA expression (DE). Prominent in this GOM-DE group are genes involved in the EMT, including multiple canonical EMT markers and the EMT-linked transcription factor RUNX1, for which we demonstrate a functional role in modulating the migratory and invasive capacities of JEG3 trophoblast cells. This analysis of DE associated with locus-specific GOM, together with functional studies of an important GOM-DE gene, highlights epigenetically regulated genes and pathways acting in human EVT differentiation and invasion, with implications for obstetric disorders in which these processes are dysregulated.

Transcriptomics-Based Subphenotyping of the Human Placenta Enabled by Weighted Correlation Network Analysis in Early-Onset Preeclampsia With and Without Fetal Growth Restriction.

BACKGROUND: Placental disorders contribute to pregnancy complications, including preeclampsia and fetal growth restriction (FGR), but debate regarding their specific pathobiology persists. Our objective was to apply transcriptomics with weighted gene correlation network analysis to further clarify the placental dysfunction in these conditions. METHODS: We performed RNA sequencing with weighted gene correlation network analysis using human placental samples (n=30), separated into villous tissue and decidua basalis, and clinically grouped as follows: (1) early-onset preeclampsia (EOPE)+FGR (n=7); (2) normotensive, nonanomalous preterm FGR (n=5); (2) EOPE without FGR (n=8); (4) spontaneous idiopathic preterm birth (n=5) matched for gestational age; and (5) uncomplicated term births (n=5). Our data was compared with RNA sequencing data sets from public databases (GSE114691, GSE148241, and PRJEB30656; n=130 samples). RESULTS: We identified 14 correlated gene modules in our specimens, of which most were significantly correlated with birthweight and maternal blood pressure. Of the 3 network modules consistently predictive of EOPE±FGR across data sets, we prioritized a coexpression gene group enriched for hypoxia-response and metabolic pathways for further investigation. Cluster analysis based on transcripts from this module and the glycolysis/gluconeogenesis metabolic pathway consistently distinguished a subset of EOPE±FGR samples with an expression signature suggesting modified tissue bioenergetics. We demonstrated that the expression ratios of LDHA/LDHB and PDK1/GOT1 could be used as surrogate indices for the larger panels of genes in identifying this subgroup. CONCLUSIONS: We provide novel evidence for a molecular subphenotype consistent with a glycolytic metabolic shift that occurs more frequently but not universally in placental specimens of EOPE±FGR.

Conformation-dependent anti-Aß monoclonal antibody signatures of disease status and severity in urine of women with preeclampsia.

Prior research has shown that urine of women with preeclampsia (PE) contains amyloid-like aggregates that are congophilic (exhibit affinity for the amyloidophilic dye Congo red) and immunoreactive with A11, a polyclonal serum against prefibrillar ß-amyloid oligomers, thereby supporting pathogenic similarity between PE and protein conformational disorders such as Alzheimer's and prion disease. The objective of this study was to interrogate PE urine using monoclonal antibodies with previously characterized A11-like epitopes. Over 100 conformation-dependent monoclonals were screened and three (mA11-09, mA11-89, and mA11-205) selected for further confirmation in 196 urine samples grouped as follows: severe features PE (sPE, n = 114), PE without severe features (mPE, n = 30), chronic hypertension (crHTN, n = 14) and normotensive pregnant control (P-CRL, n = 38). We showed that the selected conformation-specific monoclonals distinguished among patients with varying severities of PE from P-CRL and patients with crHTN. By use of latent class analysis (LCA) we identified three classes of subjects: Class 1 (n = 94) comprised patients whose urine was both congophilic and reactive with the monoclonals. These women were more likely diagnosed with early-onset sPE and had severe hypertension and proteinuria; Class 2 patients (n = 55) were negative for congophilia and against the antibodies. These were predominantly P-CRL and crHTN patients. Lastly, Class 3 patients (n = 48) were positive for urine congophilia, albeit at lower intensity, but negative for monoclonal immunoreactivities. These women were diagnosed primarily as mPE or late-onset sPE. Collectively, our study validates conformation-dependent Aß imunoreactivity of PE urine which in conjunction to urine congophilia may represent an additional indicator of disease severity.

Molecular signatures of labor and nonlabor myometrium with parsimonious classification from 2 calcium transporter genes.

Clinical phenotyping of term and preterm labor is imprecise, and disagreement persists on categorization relative to underlying pathobiology, which remains poorly understood. We performed RNA sequencing (RNA-seq) of 31 specimens of human uterine myometrium from 10 term and 21 preterm cesarean deliveries with rich clinical context information. A molecular signature of 4814 transcripts stratified myometrial samples into quiescent (Q) and nonquiescent (NQ) phenotypes, independent of gestational age and incision site. Similar stratifications were achieved using expressed genes in Ca2+ signaling and TGF-ß pathways. For maximal parsimony, we evaluated the expression of just 2 Ca2+ transporter genes, ATP2B4 (encoding PMCA4) and ATP2A2 (coding for SERCA2), and we found that their ratio reliably distinguished NQ and Q specimens in the current study, and also in 2 publicly available RNA-seq data sets (GSE50599 and GSE80172), with an overall AUC of 0.94. Cross-validation of the ATP2B4/ATP2A2 ratio by quantitative PCR in an expanded cohort (by 11 additional specimens) achieved complete separation (AUC of 1.00) of NQ versus Q specimens. While providing additional insight into the associations between clinical features of term and preterm labor and myometrial gene expression, our study also offers a practical algorithm for unbiased classification of myometrial biopsies by their overall contractile program.

Knockout of MAPK Phosphatase-1 Exaggerates Type I IFN Response during Systemic Escherichia coli Infection.

We have previously shown that Mkp-1-deficient mice produce elevated TNF-α, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coli-infected wild-type and Mkp-1 -/- mice. A large number of IFN-stimulated genes were more robustly expressed in E. coli-infected Mkp-1 -/- mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-ß, IFN-γ, TNF-α, IL-1α and ß, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coli-infected Mkp-1 -/- mice relative to wild-type mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1 -/- mice prior to E. coli infection augmented mortality and disease severity. Mkp-1 -/- bone marrow-derived macrophages (BMDM) produced higher levels of IFN-ß mRNA and protein than did wild-type BMDM upon treatment with LPS, E. coli, polyinosinic:polycytidylic acid, and herring sperm DNA. Augmented IFN-ß induction in Mkp-1 -/- BMDM was blocked by a p38 inhibitor but not by an JNK inhibitor. Enhanced Mkp-1 expression abolished IFN-ß induction by both LPS and E. coli but had little effect on the IFN-ß promoter activity in LPS-stimulated RAW264.7 cells. Mkp-1 deficiency did not have an overt effect on IRF3/7 phosphorylation or IKK activation but modestly enhanced IFN-ß mRNA stability in LPS-stimulated BMDM. Our results suggest that Mkp-1 regulates IFN-ß production primarily through a p38-mediated mechanism and that IFN-ß plays a beneficial role in E. coli-induced sepsis.

Connecting the dots on vertical transmission of SARS-CoV-2 using protein-protein interaction network analysis - Potential roles of placental ACE2 and ENDOU.

We conducted a protein-protein interaction (PPI) network study searching for proteins relevant to pregnancy-associated COVID-19 in pregnancy complicated with severe preeclampsia (sPE) and intra-amniotic infection and/or inflammation (Triple-I). PPI networks from sPE and Triple-I were intersected with the PPI network from coronavirus infection. Common proteins included the SARS-CoV-2 entry receptor ACE2 and ENDOU, a placental endoribonuclease homologous to Nsp15, a protein produced by the virus to escape host immunity. Remarkably, placental ENDOU mRNA expression far exceeded that of ACE2. Immunohistochemistry confirmed ENDOU localization at the hemochorial maternal-fetal interface. Investigation of ENDOU's relevance to vertical transmission of SARS-CoV-2 is further warranted.

Electrophysiological Maturation of Cerebral Organoids Correlates with Dynamic Morphological and Cellular Development.

Cerebral organoids (COs) are rapidly accelerating the rate of translational neuroscience based on their potential to model complex features of the developing human brain. Several studies have examined the electrophysiological and neural network features of COs; however, no study has comprehensively investigated the developmental trajectory of electrophysiological properties in whole-brain COs and correlated these properties with developmentally linked morphological and cellular features. Here, we profiled the neuroelectrical activities of COs over the span of 5 months with a multi-electrode array platform and observed the emergence and maturation of several electrophysiologic properties, including rapid firing rates and network bursting events. To complement these analyses, we characterized the complex molecular and cellular development that gives rise to these mature neuroelectrical properties with immunohistochemical and single-cell transcriptomic analyses. This integrated approach highlights the value of COs as an emerging model system of human brain development and neurological disease.

Unique transcriptomic landscapes identified in idiopathic spontaneous and infection related preterm births compared to normal term births.

Preterm birth (PTB) is leading contributor to infant death in the United States and globally, yet the underlying mechanistic causes are not well understood. Histopathological studies of preterm birth suggest advanced villous maturity may have a role in idiopathic spontaneous preterm birth (isPTB). To better understand pathological and molecular basis of isPTB, we compared placental villous transcriptomes from carefully phenotyped cohorts of PTB due to infection or isPTB between 28-36 weeks gestation and healthy term placentas. Transcriptomic analyses revealed a unique expression signature for isPTB distinct from the age-matched controls that were delivered prematurely due to infection. This signature included the upregulation of three IGF binding proteins (IGFBP1, IGFBP2, and IGFBP6), supporting a role for aberrant IGF signaling in isPTB. However, within the isPTB expression signature, we detected secondary signature of inflammatory markers including TNC, C3, CFH, and C1R, which have been associated with placental maturity. In contrast, the expression signature of the gestational age-matched infected samples included upregulation of proliferative genes along with cell cycling and mitosis pathways. Together, these data suggest an isPTB molecular signature of placental hypermaturity, likely contributing to the premature activation of inflammatory pathways associated with birth and providing a molecular basis for idiopathic spontaneous birth.

Toll-like receptor 9, maternal cell-free DNA and myometrial cell response to CpG oligodeoxynucleotide stimulation.

PROBLEM: Among mechanisms triggering onset of parturition, it has been recently postulated that Toll-Like Receptor (TLR)9 engagement by cell-free DNA (cfDNA) triggers inflammation, myometrial contractions, and labor in absence of infection. The current study evaluated whether direct (myometrial) or indirect (decidual) TLR9 engagement enhances human myometrial contractility. METHOD OF STUDY: Toll-like receptor 9 expression and cellular localization were surveyed by immunohistochemistry of placenta, fetal membranes, and myometrium in term (gestational age [GA]: >37 weeks) labor (TL, n = 7) or term non-labor (TNL, n = 7) tissues. Non-pregnant myometrium (n = 4) served as reference. TLR9 mRNA expression relative to other TLRs was evaluated through the mining of an RNA-seq dataset and confirmed by RT-PCR. Immortalized human myometrial cells (hTERT-HM) were treated with incremental concentrations of TLR9 agonist ODN2395, TNF-α, or LPS. Secreted cytokines were quantified by multiplex immunoassay, and contractility was assessed by an in-gel cell contraction assay (n = 9). Induction of hTERT-HM contractility was also evaluated indirectly following exposure to conditioned media from primary term decidual cells (n = 4) previously stimulated with ODN2395. RESULTS: Toll-like receptor 9 immunostaining in placenta and amniochorion was strongest in decidual cells, but unrelated to labor. TLR9 staining intensity was significantly decreased in TL compared with TNL myometrium (P = 0.002). Although total cfDNA in maternal circulation increased in TL (P = 0.025 vs TNL), difference in cffDNA was non-significant. Myometrial TLR9 mRNA levels were unaffected by contractile status and far less abundant than other pro-inflammatory TLRs. hTERT-HM contractility was enhanced by LPS (P = 0.002) and TNF-α (P = 0.003), but not by ODN2395 (P = 0.345) or supernatant of TLR9-stimulated decidual cells. CONCLUSION: Myometrial and decidual TLR9 are unlikely to directly regulate human parturition.

Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis.

Mitogen-activated protein kinase phosphatase (Mkp)-1 exerts its anti-inflammatory activities during Gram-negative sepsis by deactivating p38 and c-Jun N-terminal kinase (JNK). We have previously shown that Mkp-1+/+ mice, but not Mkp-1-/- mice, exhibit hypertriglyceridemia during severe sepsis. However, the regulation of hepatic lipid stores and the underlying mechanism of lipid dysregulation during sepsis remains an enigma. To understand the molecular mechanism underlying the sepsis-associated metabolic changes and the role of Mkp-1 in the process, we infected Mkp-1+/+ and Mkp-1-/- mice with Escherichia coli i.v., and assessed the effects of Mkp-1 deficiency on tissue lipid contents. We also examined the global gene expression profile in the livers via RNA-seq. We found that in the absence of E. coli infection, Mkp-1 deficiency decreased liver triglyceride levels. Upon E. coli infection, Mkp-1+/+ mice, but not Mkp-1-/- mice, developed hepatocyte ballooning and increased lipid deposition in the livers. E. coli infection caused profound changes in the gene expression profile of a large number of proteins that regulate lipid metabolism in wildtype mice, while these changes were substantially disrupted in Mkp-1-/- mice. Interestingly, in Mkp-1+/+ mice E. coli infection resulted in downregulation of genes that facilitate fatty acid synthesis but upregulation of Cd36 and Dgat2, whose protein products mediate fatty acid uptake and triglyceride synthesis, respectively. Taken together, our studies indicate that sepsis leads to a substantial change in triglyceride metabolic gene expression programs and Mkp-1 plays an important role in this process.

Human Placenta Expresses α2-Adrenergic Receptors and May Be Implicated in Pathogenesis of Preeclampsia and Fetal Growth Restriction.

α2-Adrenergic receptors (α2ARs) are G-protein-coupled receptors involved in catecholamine signaling by extracellular regulated protein kinase 1 and 2 (ERK1/2) pathways. We examined placental expression and function of α2AR subtypes in women with severe preeclampsia (sPE) with and without intrauterine growth restriction (IUGR). Placental biopsies were analyzed from 52 women with i) sPE (n = 8); ii) sPE + IUGR (n = 9); iii) idiopathic IUGR (n = 8); iv) idiopathic preterm birth (n = 16); and v) healthy term controls (n = 11). Expression of α2AR subtypes (α2A, α2B, α2C) and phospho-ERK1/2 (receptor activation marker) was investigated by immunohistochemistry and/or quantitative real-time RT-PCR. The effects of α2CAR knockdown on syncytialization (syncytin-1 and -2) and ß-human chorionic gonadotropin secretion were examined in BeWo cells stimulated with forskolin. The effects of α2AR agonist UK 14,304 and specific α2CAR antagonist were tested, using a trophoblast migration assay. All three α2ARs were expressed and functionally active in human placenta with site-specific localization. Highest α2BAR and α2CAR mRNA expression was identified in sPE + IUGR. α2CAR knockdown increased expression of syncytin-1 and -2 but decreased secretion of ß-human chorionic gonadotropin. UK 14,304 impaired trophoblast migration. The observed α2AR expression pattern suggests different function for each subtype. α2CAR modulates trophoblast syncytialization and migration and may carry pathogenic role in sPE + IUGR.

Skin Microbiota in Obese Women at Risk for Surgical Site Infection After Cesarean Delivery.

The obesity pandemic in the obstetrical population plus increased frequency of Cesarean delivery (CD) has increased vulnerability to surgical site infection (SSI). Here we characterized the microbiome at the site of skin incision before and after CD. Skin and relevant surgical sites were sampled before and after surgical antisepsis from obese (n = 31) and non-obese (n = 27) pregnant women. We quantified bacterial biomass by qPCR, microbial community composition by 16sRNA sequencing, assigned operational taxonomic units, and stained skin biopsies from incision for bacteria and biofilms. In obese women, incision site harbors significantly higher bacterial biomass of lower diversity. Phylum Firmicutes predominated over Actinobacteria, with phylotypes Clostridales and Bacteroidales over commensal Staphylococcus and Propionbacterium spp. Skin dysbiosis increased post-surgical prep and at end of surgery. Biofilms were identified post-prep in the majority (73%) of skin biopsies. At end of surgery, incision had significant gains in bacterial DNA and diversity, and obese women shared more genera with vagina and surgeon's glove in CD. Our findings suggest microbiota at incision differs between obese and non-obese pregnant women, and changes throughout CD. An interaction between vaginal and cutaneous dysbiosis at the incision site may explain the a priori increased risk for SSI among obese pregnant women.

Integrated microRNA and mRNA network analysis of the human myometrial transcriptome in the transition from quiescence to labor.

We conducted integrated transcriptomics network analyses of miRNA and mRNA interactions in human myometrium to identify novel molecular candidates potentially involved in human parturition. Myometrial biopsies were collected from women undergoing primary Cesarean deliveries in well-characterized clinical scenarios: (1) spontaneous term labor (TL, n = 5); (2) term nonlabor (TNL, n = 5); (3) spontaneous preterm birth (PTB) with histologic chorioamnionitis (PTB-HCA, n = 5); and (4) indicated PTB nonlabor (PTB-NL, n = 5). RNAs were profiled using RNA sequencing, and miRNA-target interaction networks were mined for key discriminatory subnetworks. Forty miRNAs differed between TL and TNL myometrium, while seven miRNAs differed between PTB-HCA vs. PTB-NL specimens; six of these were cross-validated using quantitative PCR. Based on the combined sequencing data, unsupervised clustering revealed two nonoverlapping cohorts that differed primarily by absence or presence of uterine quiescence, rather than gestational age or original clinical cohort. The intersection of differentially expressed miRNAs and their targets predicted 22 subnetworks with enriched representation of miR-146b-5p, miR-223-3p, and miR-150-5p among miRNAs, and of myocyte enhancer factor-2C (MEF2C) among mRNAs. Of four known MEF2 transcription factors, decreased MEF2A and MEF2C expression in women with uterine nonquiescence was observed in the sequencing data, and validated in a second cohort by quantitative PCR. Immunohistochemistry localized MEF2A and MEF2C to myometrial smooth muscle cells and confirmed decreased abundance with labor. Collectively, these results suggest altered MEF2 expression may represent a previously unrecognized process through which miRNAs contribute to the phenotypic switch from quiescence to labor in human myometrium.

Cellular Mechanics of Primary Human Cervical Fibroblasts: Influence of Progesterone and a Pro-inflammatory Cytokine.

The leading cause of neonatal mortality, pre-term birth, is often caused by pre-mature ripening/opening of the uterine cervix. Although cervical fibroblasts play an important role in modulating the cervix's extracellular matrix (ECM) and mechanical properties, it is not known how hormones, i.e., progesterone, and pro-inflammatory insults alter fibroblast mechanics, fibroblast-ECM interactions and the resulting changes in tissue mechanics. Here we investigate how progesterone and a pro-inflammatory cytokine, IL-1ß, alter the biomechanical properties of human cervical fibroblasts and the fibroblast-ECM interactions that govern tissue-scale mechanics. Primary human fibroblasts were isolated from non-pregnant cervix and treated with estrogen/progesterone, IL-1ß or both. The resulting changes in ECM gene expression, matrix remodeling, traction force generation, cell-ECM adhesion and tissue contractility were monitored. Results indicate that IL-1ß induces a significant reduction in traction force and ECM adhesion independent of pre-treatment with progesterone. These cell level effects altered tissue-scale mechanics where IL-1ß inhibited the contraction of a collagen gel over 6 days. Interestingly, progesterone treatment alone did not modulate traction forces or gel contraction but did result in a dramatic increase in cell-ECM adhesion. Therefore, the protective effect of progesterone may be due to altered adhesion dynamics as opposed to altered ECM remodeling.

Comprehensive RNA profiling of villous trophoblast and decidua basalis in pregnancies complicated by preterm birth following intra-amniotic infection.

INTRODUCTION: We performed RNA sequencing with the primary goal of discovering key placental villous trophoblast (VT) and decidua basalis (DB) transcripts differentially expressed in intra-amniotic infection (IAI)-induced preterm birth (PTB). METHODS: RNA was extracted from 15 paired VT and DB specimens delivered of women with: 1) spontaneous PTB in the setting of amniocentesis-proven IAI and histological chorioamnionitis (n = 5); 2) spontaneous idiopathic PTB (iPTB, n = 5); and 3) physiologic term pregnancy (n = 5). RNA sequencing was performed using the Illumina HiSeq 2500 platform, and a spectrum of computational tools was used for gene prioritization and pathway analyses. RESULTS: In the VT specimens, 128 unique long transcripts and 7 mature microRNAs differed significantly between pregnancies complicated by IAI relative to iPTB (FDR<0.1). The up-regulated transcripts included many characteristic of myeloblast-derived cells, and bioinformatic analyses revealed enrichment for multiple pathways associated with acute inflammation. In an expanded cohort including additional IAI and iPTB specimens, the expression of three proteins (cathepsin S, lysozyme, and hexokinase 3) and two microRNAs (miR-133a and miR-223) was validated using immunohistochemistry and quantitative PCR, respectively. In the DB specimens, only 11 long transcripts and no microRNAs differed significantly between IAI cases and iPTB controls (FDR<0.1). Comparison of the VT and DB specimens in each clinical scenario revealed signatures distinguishing these placental regions. DISCUSSION: IAI is associated with a transcriptional signature consistent with acute inflammation in the villous trophoblast. The present findings illuminate novel signaling pathways involved in IAI, and suggest putative therapeutic targets and potential biomarkers associated with this condition.

Evidence for participation of neutrophil gelatinase-associated lipocalin/matrix metalloproteinase-9 (NGAL•MMP-9) complex in the inflammatory response to infection in pregnancies complicated by preterm birth.

PROBLEM: Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in neutrophils and involved in innate immunity by sequestering iron. NGAL's ability to complex with matrix metalloproteinase-9 (MMP-9) and extend its gelatinolytic activity led us to investigate its role in pregnancies complicated by preterm birth (PTB) and intra-amniotic infection/inflammation (IAI). METHOD OF STUDY: We assayed the amniotic fluid (AF) levels of NGAL and MMP-9 in 308 women that had a clinically indicated amniocentesis and a normal pregnancy outcome or PTB. qRT-PCR was employed to determine NGAL mRNA expression of placental villous trophoblast and amniochorion. Immunohistochemistry was used for cellular localization. RESULTS: AF NGAL levels were gestational age-regulated. Women with IAI and PTB had significantly higher levels of NGAL, MMP-9 and NGAL•MMP-9 complex. CONCLUSION: The amniochorion is a source of NGAL and similarly to other inflammatory conditions, this protein may augment the collagenolytic effect of MMP-9 and modulate host-microbe interactions in pregnancies complicated by IAI.

Comparing human and macaque placental transcriptomes to disentangle preterm birth pathology from gestational age effects.

INTRODUCTION: A major issue in the transcriptomic study of spontaneous preterm birth (sPTB) in humans is the inability to collect healthy control tissue at the same gestational age (GA) to compare with pathologic preterm tissue. Thus, gene expression differences identified after the standard comparison of sPTB and term tissues necessarily reflect differences in both sPTB pathology and GA. One potential solution is to use GA-matched controls from a closely related species to tease apart genes that are dysregulated during sPTB from genes that are expressed differently as a result of GA effects. METHODS: To disentangle genes whose expression levels are associated with sPTB pathology from those linked to GA, we compared RNA sequencing data from human preterm placentas, human term placentas, and rhesus macaque placentas at 80% completed gestation (serving as healthy non-human primate GA-matched controls). We first compared sPTB and term human placental transcriptomes to identify significantly differentially expressed genes. We then overlaid the results of the comparison between human sPTB and macaque placental transcriptomes to identify sPTB-specific candidates. Finally, we overlaid the results of the comparison between human term and macaque placental transcriptomes to identify GA-specific candidates. RESULTS: Examination of relative expression for all human genes with macaque orthologs identified 267 candidate genes that were significantly differentially expressed between preterm and term human placentas. 29 genes were identified as sPTB-specific candidates and 37 as GA-specific candidates. Altogether, the 267 differentially expressed genes were significantly enriched for a variety of developmental, metabolic, reproductive, immune, and inflammatory functions. Although there were no notable differences between the functions of the 29 sPTB-specific and 37 GA-specific candidate genes, many of these candidates have been previously shown to be dysregulated in diverse pregnancy-associated pathologies. DISCUSSION: By comparing human sPTB and term transcriptomes with GA-matched control transcriptomes from a closely related species, this study disentangled the confounding effects of sPTB pathology and GA, leading to the identification of 29 promising sPTB-specific candidate genes and 37 genes potentially related to GA effects. The apparent similarity in functions of the sPTB and GA candidates may suggest that the effects of sPTB and GA do not correspond to biologically distinct processes. Alternatively, it may reflect the poor state of knowledge of the transcriptional landscape underlying placental development and disease.

Agonist-Dependent Downregulation of Progesterone Receptors in Human Cervical Stromal Fibroblasts.

Progesterone (P(4)) maintains uterine quiescence during the majority of pregnancy, whereas diminished progesterone receptor (PR) expression and/or activity (ie, functional P(4) withdrawal) promotes parturition. To investigate the regulation of PR expression in cervical stroma, fibroblasts from premenopausal hysterectomy specimens were prepared. Greater than 99% of the cultures were vimentin positive (mesenchymal cell marker) with only occasional cytokeratin-8 positivity (epithelial cell marker) and no evidence of CD31-positive (endothelial cell marker) cells. Cells were immunolabeled with antibodies directed against PRs (PR-A and PR-B), estrogen receptor α (ER-α), and glucocorticoid receptor-α/ß (GR-α/ß). All cells were uniformly immunopositive for ER-α and GR-α/ß but did not express PRs. Incubation of cells with 10(-8) mol/L 17ß-estradiol induced a time-dependent increase in PR-A and PR-B messenger RNAs (mRNAs) by quantitative real-time polymerase chain reactions and proteins by immunoblotting and immunofluorescence. Incubation of cervical fibroblasts with PR ligands (medroxyprogesterone acetate or Org-2058) downregulated PR-A and PR-B levels. Coincubation of cells with PR ligands plus RU-486, a PR antagonist, partially abrogated agonist-induced receptor downregulation. Dexamethasone, a pure glucocorticoid, had no inhibitory effect on PR expression. These results indicate that progestins and estrogens regulate PR expression in cervical fibroblasts. We postulate that hormonal regulation of PR expression in the cervical stroma may contribute to functional P(4) withdrawal in preparation for parturition.