Single nucleotide polymorphisms in the human progesterone receptor gene and spontaneous preterm birth.

Progesterone supplementation can prevent preterm birth in some high-risk women. Progesterone binds to progesterone receptor (PR) and modulates the expression of target genes. This study investigates the association between single nucleotide polymorphisms (SNPs) in the PR gene and spontaneous preterm birth. DNA was extracted from consecutive patients with preterm birth (n = 78) and term controls (n = 415), and genotyping was performed for 3 PR SNPs (+331[G>A], + 770[C>T], +660[G>T]) using Sequenom matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Data were analyzed by chi(2) test and logistic regression analysis. Multivariate analysis showed no association between maternal carriage of minor + 331T, +770T, and/or +660T alleles and preterm birth when controlled for maternal age, ethnicity, gravidity, parity, prior preterm birth, route of delivery, or neonatal outcome. Carriage of +770T and +660T (but not +331T) was associated with preterm birth in women with a body mass index <18.5 kg/m(2) (relative risk, 10.8; 95% confidence interval, 1.4-82.6; P = .02). Maternal carriage of minor alleles of +331(G>A), +770(C>T), and +660(G> T) SNPs in the PR gene is not associated with spontaneous preterm birth.

Thrombin activation of endometrial endothelial cells: a possible role in intrauterine growth restriction.

Preeclampsia (PE), intrauterine growth restriction (IUGR) and abruption with or without fetal loss are associated with reduced uteroplacental blood flow, decidual vasculopathy, endothelial cell dysfunction, thrombosis, inflammation and hemorrhage. Our hypothesis is that reduced uteroplacental blood flow causes focal decidual hypoxia that generates vascular endothelial growth factor (VEGF). The latter acts directly on decidual endothelial cells to induce aberrant expression of tissue factor (TF), the primary initiator of coagulation. This in turn generates thrombin that induces: i) further TF expression; and ii) inflammatory cytokines. Both VEGF and TF induce aberrant angiogenesis-vessel maintenance reflected by endothelial cell fenestrations and induction of a prothrombotic surface causing both the decidual hemorrhage (i.e. abruption) and thrombosis (i.e. uteroplacental vascular insufficiency) observed in these adverse pregnancy outcomes. This novel hypothesis is supported by our finding of TF expression in decidual endothelium of pregnancies complicated by IUGR and/or fetal loss. Moreover, treatment of cultured endometrial endothelial cells with VEGF or thrombin induces TF protein and mRNA expression. Quantitative RT-PCR analysis indicates that thrombin enhances (>10-fold) the output of diverse inflammatory cytokines in these cultures. The greatest effect (>2-log) was seen on macrophage inflammatory protein 3alpha (MIP3alpha). In vitro, thrombin results in endometrial endothelial cell aggregations and changes in the apoptotic pathway. Thus, we postulate that reductions in uteroplacental flow initiate a cascade of molecular effects leading to hypoxia, thrombosis, inflammation, and endothelial cell dysfunction resulting in untoward pregnancy outcomes.

Regulation of monocyte chemoattractant protein-1 expression by tumor necrosis factor-alpha and interleukin-1beta in first trimester human decidual cells: implications for preeclampsia.

The current study describes a statistically significant increase in macrophages (CD68-positive cells) in the decidua of preeclamptic patients. To elucidate the regulation of this monocyte infiltration, expression of monocyte chemoattractant protein-1 (MCP-1) was assessed in leukocyte-free first trimester decidual cells. Confluent decidual cells were primed for 7 days in either estradiol or estradiol plus medroxyprogesterone acetate to mimic the decidualizing steroidal milieu of the luteal phase and early pregnancy. The medium was exchanged for a serum-free defined medium containing corresponding steroids +/- tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta. After 24 hours, enzyme-linked immunosorbent assay measurements indicated that the addition of medroxyprogesterone acetate did not affect MCP-1 output, whereas 10 ng/ml of TNF-alpha or IL-1beta increased output by 83.5-fold +/- 20.6 and 103.1-fold +/- 14.7, respectively (mean +/- SEM, n = 8, P < 0.05). Concentration-response comparisons revealed that even 0.01 ng/ml of TNF-alpha or IL-1beta elevated MCP-1 output by more than 15-fold. Western blotting confirmed the enzyme-linked immunosorbent assay results, and quantitative reverse transcriptase-polymerase chain reaction confirmed corresponding effects on MCP-1 mRNA levels. The current study demonstrates that TNF-alpha and IL-1beta enhance MCP-1 in first trimester decidua. This finding suggests a mechanism by which recruitment of excess macrophages to the decidua impairs endovascular trophoblast invasion, the primary placental defect of preeclampsia.

Activation of the protein kinase A pathway in human endometrial stromal cells reveals sequential categorical gene regulation.

Decidualization of endometrial stromal cells is a prerequisite for human implantation and occurs in vivo in response to progesterone and involves activation of the protein kinase A (PKA) pathway. The objective of this study was to determine the molecular signatures and patterns of gene expression during stimulation of this pathway with an analog of cAMP. Endometrial stromal cells from two subjects were treated with or without 8-Br-cAMP (1 mM) for 0, 2, 12, 24, 36, and 48 h and were processed for microarray analysis, screening for 12,686 genes and ESTs. Most abundantly upregulated genes included neuropeptides, immune genes, IGF family members, cell cycle regulators, extracellular matrix proteases, cholesterol trafficking, cell growth and differentiation, hormone signaling, and signal transduction. Most abundantly downregulated genes included activator of NF-kappaB, actin/tropomyosin/calmodulin binding protein, cyclin B, IGFBP-5, alpha1 type XVI collagen, lipocortin III, l-kynurenine hydrolase, frizzle-related protein, and cyclin E2. RT-PCR validated upregulation of IGFBP-1, preprosomatostatin, and IL-11, and Northern analysis validated their kinetic upregulation. RT-PCR confirmed downregulation of IGFBP-5, cyclin B, and TIL-4. K-means analysis revealed four major patterns of up- and downregulated genes, and genes within each ontological group were categorized into these four kinetic patterns. Within each ontological group different patterns of temporal gene expression were observed, indicating that even genes within one functional category are regulated differently during activation of the PKA pathway in human endometrial stromal cells. Overall, the data demonstrate kinetic reprogramming of genes within specific functional groups and changes in genes associated with nucleic acid binding, cell proliferation, decreased G protein signaling, increased STAT pathway signaling, structural proteins, cellular differentiation, and secretory processes. These changes are consistent with cAMP modulating early events (0-6 h) primarily involving cell cycle regulation, subsequent events (12-24 h) involving cellular differentiation (including changes in morphology and secretory phenotype), and late events (24-48 h) mediating more specialized function, including immune modulators, in the human endometrial stromal cell.