Decidual Cells Block Inflammation-Mediated Inhibition of 15-Hydroxyprostaglandin Dehydrogenase in Trophoblasts.

Chorioamnionitis generates prostaglandin (PG) E2 and F2α, promoting fetal membrane rupture, cervical ripening, and uterine contractions. 15-Hydroxyprostaglandin dehydrogenase (HPGD) contributes to pregnancy maintenance by inactivating PGs. The role of decidual cells in regulating HPGD expression at the maternal-fetal interface was investigated. HPGD immunostaining was primarily detected in anchoring villi and choriodecidual extravillous trophoblasts (EVTs) in the first, second, and third trimesters. Chorionic EVTs adjacent to decidua parietalis exhibited significantly higher HPDG levels than those adjacent to amnion. HPGD histologic score levels were significantly lower in choriodecidua from chorioamnionitis versus gestational age-matched controls (means ± SEM, 132.6 ± 3.8 versus 31.2 ± 7.9; P < 0.05). Conditioned media supernatant (CMS) from in vitro decidualized term decidual cells (TDCs) up-regulated HPGD levels in EVTs differentiated from human trophoblastic stem cells, primary trophoblasts, and HTR8/SVneo cells. However, CMS from 5 µg/mL lipopolysaccharide or 10 ng/mL IL-1ß pretreated TDC cultures down-regulated HPGD levels in HTR8/SVneo cultures. Similarly, direct treatment of HTR8/SVneo cultures with lipopolysaccharide or IL-1ß significantly reduced HPGD levels versus control (0.57 ± 0.1 or 0.47 ± 0.1 versus 1.03 ± 0.03; P < 0.05) but not in TDC-CMS pretreated HTR8/SVneo cultures. Collectively, the results uncover a novel decidual cell-mediated paracrine mechanism that stimulates levels of trophoblastic HPGD, whose function is to inactivate labor-inducing PGs, thereby promoting uterine quiescence during pregnancy. However, infectious/inflammatory stimuli in decidual cells cause a paracrine inhibition of trophoblastic HPGD expression, increasing PGE2/PGF2α levels, thereby contributing to preterm birth.

Dysregulated expression of GATA2 and GATA6 transcription factors in adenomyosis: implications for impaired endometrial receptivity.

OBJECTIVE: To study the effect of adenomyosis on the localized expression of the GATA binding proteins 2 and 6 (GATA2 and GATA6) zinc-finger transcription factors that are involved in proliferation of hematopoietic and endocrine cell lineages, cell differentiation, and organogenesis, potentially leading to impaired endometrial implantation. DESIGN: Laboratory based experimental study. SETTING: Academic hospital and laboratory. PATIENTS: Human endometrial stromal cells (HESCs) of reproductive age patients, 18-45 years of age, with adenomyosis were compared with patients with no pathology and leiomyomatous uteri as controls (n = 4 in each group, respectively). Additionally, midsecretory phase endometrial sections were obtained from patients with adenomyosis and control patients with leiomyoma (n = 8 in each group, respectively). INTERVENTIONS: GATA2 and GATA6 immunohistochemistry and H-SCORE were performed on the midsecretory phase endometrial sections from adenomyosis and leiomyoma control patients (n = 8 each, respectively). Control and adenomyosis patient HESC cultures were treated with placebo or 10-8 M estradiol (E2), or decidualization media (EMC) containing 10-8 M E2, 10-7 M medroxyprogesterone acetate, and 5 × 10-5 M cAMP for 6 and 10 days. Additionally, control HESC cultures (n = 4) were transfected with scrambled small interfering RNA (siRNA) (control) or GATA2-specific siRNAs for 6 days while adenomyosis HESC cultures (n = 4) were transfected with human GATA2 expression vectors to silence or induce GATA2 overexpression. MAIN OUTCOME MEASURES: Immunohistochemistry was performed to obtain GATA2 and GATA6 H-SCORES in adenomyosis vs. control patient endometrial tissue. Expression of GATA2, GATA6, insulin-like growth factor-binding protein 1 (IGFBP1), prolactin (PRL), progesterone receptor (PGR), estrogen receptor 1 (ESR1), leukemia inhibitory factor (LIF), and Interleukin receptor 11 (IL11R) messenger RNA (mRNA) levels were analyzed using by qPCR with normalization to ACTB. Silencing and overexpression experiments also had the corresponding mRNA levels of the above factors analyzed. Western blot analysis was performed on isolated proteins from transfection experiments. RESULTS: Immunohistochemistry revealed an overall fourfold lower GATA2 and fourfold higher GATA6 H-SCORE level in the endometrial stromal cells of patients with adenomyosis vs. controls. Decidual induction with EMC resulted in significantly lower GATA2, PGR, PRL and IGFBP1 mRNA levels in HESC cultures from patients with adenomyosis patient vs. controls. Leukemia inhibitory factor and IL11R mRNA levels were also significantly dysregulated in adenomyosis HESCs compared with controls. . Silencing of GATA2 expression in control HESCs induced an adenomyosis-like state with significant reductions in GATA2, increases in GATA6 and accompanying aberrations in PGR, PRL, ESR1 and LIF levels. Conversely, GATA2 overexpression via vector in adenomyosis HESCs caused partial restoration of the defective decidual response with significant increases in GATA2, PGR, PRL and LIF expression. CONCLUSION: In-vivo and in-vitro experiment results demonstrate that there is an overall inverse relationship between endometrial GATA2 and GATA6 levels in patients with adenomyosis who have diminished GATA2 levels and concurrently elevated GATA6 levels. Additionally, lower GATA2 and higher GATA6 levels, together with aberrant levels of important receptors and implantation factors, such as ESR1, PGR, IGFBP1, PRL, LIF, and IL11R mRNA in HESCs from patients with adenomyosis or GATA2-silenced control HESCs, support impaired decidualization. These effects were partially restored with GATA2 overexpression in adenomyosis HESCs, demonstrating a potential therapeutic target.

Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

Progestin-only long-acting reversible-contraceptive (pLARC)-exposed endometria displays decidualized human endometrial stromal cells (HESCs) and hyperdilated thin-walled fragile microvessels. The combination of fragile microvessels and enhanced tissue factor levels in decidualized HESCs generates excess thrombin, which contributes to abnormal uterine bleeding (AUB) by inducing inflammation, aberrant angiogenesis, and proteolysis. The- zinc finger and BTB domain containing 16 (ZBTB16) has been reported as an essential regulator of decidualization. Microarray studies have demonstrated that ZBTB16 levels are induced by medroxyprogesterone acetate (MPA) and etonogestrel (ETO) in cultured HESCs. We hypothesized that pLARC-induced ZBTB16 expression contributes to HESC decidualization, whereas prolonged enhancement of ZBTB16 levels triggers an inflammatory milieu by inducing pro-inflammatory gene expression and tissue-factor-mediated thrombin generation in decidualized HESCs. Thus, ZBTB16 immunostaining was performed in paired endometria from pre- and post-depo-MPA (DMPA)-administrated women and oophorectomized guinea pigs exposed to the vehicle, estradiol (E2), MPA, or E2 + MPA. The effect of progestins including MPA, ETO, and levonorgestrel (LNG) and estradiol + MPA + cyclic-AMP (E2 + MPA + cAMP) on ZBTB16 levels were measured in HESC cultures by qPCR and immunoblotting. The regulation of ZBTB16 levels by MPA was evaluated in glucocorticoid-receptor-silenced HESC cultures. ZBTB16 was overexpressed in cultured HESCs for 72 h followed by a ± 1 IU/mL thrombin treatment for 6 h. DMPA administration in women and MPA treatment in guinea pigs enhanced ZBTB16 immunostaining in endometrial stromal and glandular epithelial cells. The in vitro findings indicated that: (1) ZBTB16 levels were significantly elevated by all progestin treatments; (2) MPA exerted the greatest effect on ZBTB16 levels; (3) MPA-induced ZBTB16 expression was inhibited in glucocorticoid-receptor-silenced HESCs. Moreover, ZBTB16 overexpression in HESCs significantly enhanced prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP1), and tissue factor (F3) levels. Thrombin-induced interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels in control-vector-transfected HESCs were further increased by ZBTB16 overexpression. In conclusion, these results supported that ZBTB16 is enhanced during decidualization, and long-term induction of ZBTB16 expression by pLARCs contributes to thrombin generation through enhancing tissue factor expression and inflammation by enhancing IL-8 and PTGS2 levels in decidualized HESCs.

SINE RNA of the imprinted miRNA clusters mediates constitutive type III interferon expression and antiviral protection in hemochorial placentas.

Hemochorial placentas have evolved defense mechanisms to prevent the vertical transmission of viruses to the immunologically underdeveloped fetus. Unlike somatic cells that require pathogen-associated molecular patterns to stimulate interferon production, placental trophoblasts constitutively produce type III interferons (IFNL) through an unknown mechanism. We demonstrate that transcripts of short interspersed nuclear elements (SINEs) embedded in miRNA clusters within the placenta trigger a viral mimicry response that induces IFNL and confers antiviral protection. Alu SINEs within primate-specific chromosome 19 (C19MC) and B1 SINEs within rodent-specific microRNA cluster on chromosome 2 (C2MC) produce dsRNAs that activate RIG-I-like receptors (RLRs) and downstream IFNL production. Homozygous C2MC knockout mouse trophoblast stem (mTS) cells and placentas lose intrinsic IFN expression and antiviral protection, whereas B1 RNA overexpression restores C2MCΔ/Δ mTS cell viral resistance. Our results uncover a convergently evolved mechanism whereby SINE RNAs drive antiviral resistance in hemochorial placentas, placing SINEs as integral players in innate immunity.

Leptin-Mediated Induction of IL-6 Expression in Hofbauer Cells Contributes to Preeclampsia Pathogenesis.

Leptin plays a crucial role in regulating energy homoeostasis, neuroendocrine function, metabolism, and immune and inflammatory responses. The adipose tissue is a main source of leptin, but during pregnancy, leptin is also secreted primarily by the placenta. Circulating leptin levels peak during the second trimester of human pregnancy and fall after labor. Several studies indicated a strong association between elevated placental leptin levels and preeclampsia (PE) pathogenesis and elevated serum interleukin-6 (IL-6) levels in PE patients. Therefore, we hypothesized that a local increase in placental leptin production induces IL-6 production in Hofbauer cells (HBCs) to contribute to PE-associated inflammation. We first investigated HBCs-specific IL-6 and leptin receptor (LEPR) expression and compared their immunoreactivity in PE vs. gestational age-matched control placentas. Subsequently, we examined the in vitro regulation of IL-6 as well as the phosphorylation levels of intracellular signaling proteins STAT3, STAT5, NF-κB, and ERK1/2 by increasing recombinant human leptin concentrations (10 to 1000 ng/mL) in primary cultured HBCs. Lastly, HBC cultures were incubated with leptin ± specific inhibitors of STAT3 or STAT5, or p65 NF-κB or ERK1/2 MAPK signaling cascades to determine relevant cascade(s) involved in leptin-mediated IL-6 regulation. Immunohistochemistry revealed ~three- and ~five-fold increases in IL-6 and LEPR expression, respectively, in HBCs from PE placentas. In vitro analysis indicated that leptin treatment in HBCs stimulate IL-6 in a concentration-dependent manner both at the transcriptional and secretory levels (p < 0.05). Moreover, leptin-treated HBC cultures displayed significantly increased phosphorylation levels of STAT5, p65 NF-κB, and ERK1/2 MAPK and pre-incubation of HBCs with a specific ERK1/2 MAPK inhibitor blocked leptin-induced IL-6 expression. Our in situ results show that HBCs contribute to the pathogenesis of PE by elevating IL-6 expression, and in vitro results indicate that induction of IL-6 expression in HBCs is primarily leptin-mediated. While HBCs display an anti-inflammatory phenotype in normal placentas, elevated levels of leptin may transform HBCs into a pro-inflammatory phenotype by activating ERK1/2 MAPK to augment IL-6 expression.

Preeclampsia is Associated With Reduced ISG15 Levels Impairing Extravillous Trophoblast Invasion.

Among several interleukin (IL)-6 family members, only IL-6 and IL-11 require a gp130 protein homodimer for intracellular signaling due to lack of intracellular signaling domain in the IL-6 receptor (IL-6R) and IL-11R. We previously reported enhanced decidual IL-6 and IL-11 levels at the maternal-fetal interface with significantly higher peri-membranous IL-6 immunostaining in adjacent interstitial trophoblasts in preeclampsia (PE) vs. gestational age (GA)-matched controls. This led us to hypothesize that competitive binding of these cytokines to the gp130 impairs extravillous trophoblast (EVT) differentiation, proliferation and/or invasion. Using global microarray analysis, the current study identified inhibition of interferon-stimulated gene 15 (ISG15) as the only gene affected by both IL-6 plus IL-11 vs. control or IL-6 or IL-11 treatment of primary human cytotrophoblast cultures. ISG15 immunostaining was specific to EVTs among other trophoblast types in the first and third trimester placental specimens, and significantly lower ISG15 levels were observed in EVT from PE vs. GA-matched control placentae (p = 0.006). Induction of primary trophoblastic stem cell cultures toward EVT linage increased ISG15 mRNA levels by 7.8-fold (p = 0.004). ISG15 silencing in HTR8/SVneo cultures, a first trimester EVT cell line, inhibited invasion, proliferation, expression of ITGB1 (a cell migration receptor) and filamentous actin while increasing expression of ITGB4 (a receptor for hemi-desmosomal adhesion). Moreover, ISG15 silencing further enhanced levels of IL-1ß-induced pro-inflammatory cytokines (CXCL8, IL-6 and CCL2) in HTR8/SVneo cells. Collectively, these results indicate that ISG15 acts as a critical regulator of EVT morphology and function and that diminished ISG15 expression is associated with PE, potentially mediating reduced interstitial trophoblast invasion and enhancing local inflammation at the maternal-fetal interface. Thus, agents inducing ISG15 expression may provide a novel therapeutic approach in PE.

Regulation of Proinflammatory Molecules and Tissue Factor by SARS-CoV-2 Spike Protein in Human Placental Cells: Implications for SARS-CoV-2 Pathogenesis in Pregnant Women.

SARS-CoV-2 infects cells via binding to ACE2 and TMPRSS2, which allows the virus to fuse with host cells. The viral RNA is detected in the placenta of SARS-CoV-2-infected pregnant women and infection is associated with adverse pregnancy complications. Therefore, we hypothesize that SARS-CoV-2 infection of placental cells induces pro-inflammatory cytokine release to contribute to placental dysfunction and impaired pregnancy outcomes. First, expression of ACE2 and TMPRSS2 was measured by qPCR in human primary cultured term cytotrophoblasts (CTBs), syncytiotrophoblast (STBs), term and first trimester decidual cells (TDCs and FTDCs, respectively), endometrial stromal cells (HESCs) as well as trophoblast cell lines HTR8, JEG3, placental microvascular endothelial cells (PMVECs) and endometrial endothelial cells (HEECs). Later, cultured HTR8, JEG3, PMVECs and HEECs were treated with 10, 100, 1000 ng/ml of recombinant (rh-) SARS-CoV-2 S-protein ± 10 ng/ml rh-IFNγ. Pro-inflammatory cytokines IL-1ß, 6 and 8, chemokines CCL2, CCL5, CXCL9 and CXCL10 as well as tissue factor (F3), the primary initiator of the extrinsic coagulation cascade, were measured by qPCR as well as secreted IL-6 and IL-8 levels were measured by ELISA. Immunohistochemical staining for SARS-CoV-2 spike protein was performed in placental specimens from SARS-CoV-2-positive and normal pregnancies. ACE2 levels were significantly higher in CTBs and STBs vs. TDCs, FTDCs and HESCs, while TMPRSS2 levels were not detected in TDCs, FTDCs and HESCs. HTR8 and JEG3 express ACE2 and TMPRSS2, while PMVECs and HEECs express only ACE2, but not TMPRSS2. rh-S-protein increased proinflammatory cytokines and chemokines levels in both trophoblast and endothelial cells, whereas rh-S-protein only elevated F3 levels in endothelial cells. rh-IFNγ ± rh-S-protein augments expression of cytokines and chemokines in trophoblast and endothelial cells. Elevated F3 expression by rh-IFNγ ± S-protein was observed only in PMVECs. In placental specimens from SARS-CoV-2-infected mothers, endothelial cells displayed higher immunoreactivity against spike protein. These findings indicated that SARS-CoV-2 infection in placental cells: 1) induces pro-inflammatory cytokine and chemokine release, which may contribute to the cytokine storm observed in severely infected pregnant women and related placental dysfunction; and 2) elevates F3 expression that may trigger systemic or placental thrombosis.

FKBP51 Contributes to Uterine Leiomyoma Pathogenesis by Inducing Cell Proliferation and Extracellular Matrix Deposition.

The FK506-binding protein 51 (FKBP51) binds progesterone receptor (PR), glucocorticoid receptor (GR), and androgen receptor (AR) to coregulate their transcriptional activity. We evaluated FKBP51 expression and function in human leiomyoma vs. myometrial tissues and primary cultures to discover FKBP51 role(s) in the pathogenesis of leiomyomas. Quantification of in situ FKBP51 mRNA and protein levels inpaired myometrial vs. leiomyoma tissues from proliferative and secretory phases were analyzed by qPCR (n = 14), immunoblotting (n = 20), and immunohistochemistry (n = 12). Control (scramble) vs. FKBP5 siRNA-transfected leiomyoma cell cultures were assessed for proliferation, apoptosis, and mRNA levels of genes involved in cell survival and extracellular matrix (ECM) formation. Significantly higher FKBP5 mRNA levels were detected in leiomyoma vs. paired myometrium (P < 0.001). Immunoblot (P = 0.001) and immunostaining (P ≤ 0.001) confirmed increased FKBP51 levels in leiomyoma vs. paired myometrium. Compared to control siRNA transfection, FKBP5-silenced leiomyoma cell cultures displayed significantly decreased cell survival factors and reduced proliferation (P < 0.05). Moreover, qPCR analysis revealed significantly lower mRNA levels of ECM, TIPM1, and TIPM3 proteins in FKBP5-silenced leiomyoma cell cultures (P < 0.05). Increased FKBP51 expression in leiomyoma likely involves dysregulation of steroid signaling by blocking GR and PR action and promoting proliferation and ECM production. Evaluating the effect of FKBP51 inhibition in preclinical studies will clarify its significance as a potential therapeutic approach against leiomyoma.

Decidual cell FKBP51-progesterone receptor binding mediates maternal stress-induced preterm birth.

Depression and posttraumatic stress disorder increase the risk of idiopathic preterm birth (iPTB); however, the exact molecular mechanism is unknown. Depression and stress-related disorders are linked to increased FK506-binding protein 51 (FKBP51) expression levels in the brain and/or FKBP5 gene polymorphisms. Fkbp5-deficient (Fkbp5-/-) mice resist stress-induced depressive and anxiety-like behaviors. FKBP51 binding to progesterone (P4) receptors (PRs) inhibits PR function. Moreover, reduced PR activity and/or expression stimulates human labor. We report enhanced in situ FKBP51 expression and increased nuclear FKBP51-PR binding in decidual cells of women with iPTB versus gestational age-matched controls. In Fkbp5+/+ mice, maternal restraint stress did not accelerate systemic P4 withdrawal but increased Fkbp5, decreased PR, and elevated AKR1C18 expression in uteri at E17.25 followed by reduced P4 levels and increased oxytocin receptor (Oxtr) expression at 18.25 in uteri resulting in PTB. These changes correlate with inhibition of uterine PR function by maternal stress-induced FKBP51. In contrast, Fkbp5-/- mice exhibit prolonged gestation and are completely resistant to maternal stress-induced PTB and labor-inducing uterine changes detected in stressed Fkbp5+/+ mice. Collectively, these results uncover a functional P4 withdrawal mechanism mediated by maternal stress-induced enhanced uterine FKBP51 expression and FKPB51-PR binding, resulting in iPTB.

Catecholestradiol Activation of Adrenergic Receptors Induces Endometrial Cell Survival via p38 MAPK Signaling.

CONTEXT: Enhanced levels of catecholestradiols, 2-hydroxyestradiol (2-OHE2) or 4-hydroxyestradiol (4-OHE2), are reported in endometriosis. During gestation, catecholestradiol activation of adrenergic receptors (AR) elevates estrogen receptor (ER)-independent proliferation of uterine arterial endothelial cells. OBJECTIVE: To investigate ß-AR-mediated catecholestradiol effects on human endometrial stromal cell (HESC) and epithelial cell survival in endometriosis. DESIGN: ß-AR immunostaining of eutopic and ectopic endometria (n = 9). Assays for cell viability, 5-bromo-2'-deoxyuridine proliferation, apoptosis, quantitative PCR, and estrogenicity (alkaline phosphatase activity), as well as siRNA ß-AR silencing and immunoblot analyses of cultured HESCs or Ishikawa cells treated with control or 2-OHE2 or 4-OHE2 ±ß-AR antagonist or ±p38 MAPK inhibitor. SETTING: University research institution. PATIENTS: Women with or without endometriosis. INTERVENTIONS: None. MAIN OUTCOME MEASURES: ß-AR expression in eutopic vs ectopic endometria and regulation of HESC survival by 2-OHE2 and 4-OHE2. RESULTS: Eutopic and ectopic endometrial stromal and epithelial cells displayed ß2-AR immunoreactivity with increased staining in the functionalis vs basalis layer (P < 0.05). Both 2-OHE2 and 4-OHE2 enhanced HESC and Ishikawa cell survival (P < 0.05), an effect abrogated by ß-AR antagonist propranolol, but not ER antagonist ICI182,780. 2-OHE2 or 4-OHE2 failed to induce cell survival and estrogenic activity in ADRB2-silenced HESCs and in Ishikawa cells, respectively. Although 2-OHE2 inhibited apoptosis and BAX mRNA expression, 4-OHE2 induced proliferation and decreased apoptosis (P < 0.05). Both catecholestradiols elevated phospho-p38 MAPK levels (P < 0.05), which was blocked by propranolol, and p38 MAPK inhibitor reversed catecholestradiol-enhanced HESC survival. CONCLUSIONS: Catecholestradiols increase endometrial cell survival by an ER-independent ß-AR-mediated p38 MAPK activation, suggesting that agents blocking ß-AR (e.g., propranolol) or inhibiting 2-OHE2- or 4-OHE2-generating enzymes (i.e., CYP1A1/B1) could treat endometriosis.

The role of unfolded protein response in the pathogenesis of endometriosis: contribution of peritoneal fluid.

RESEARCH QUESTION: Endoplasmic reticulum stress (ERS) is caused by the accumulation of the misfolded or unfolded proteins in the endoplasmic reticulum and induces the unfolded protein response (UPR). Peritoneal fluid is important in the pathogenesis of endometriosis. In this study, the role of UPR associated with ERS in endometriosis, and peritoneal fluid, were investigated. DESIGN: Normal, eutopic and ectopic endometrium tissues were divided into menstrual cycle phases, and endometrial stromal cells (ESC) were treated with 10-20% concentration of control peritoneal fluid and peritoneal fluid obtained from women with endometriosis for 10, 30 and 60 min, and 24 and 48 h. The UPR signalling proteins were analysed immunohistochemically and immunocytochemically. Data were compared statistically. RESULTS: p-IRE1 was increased in ectopic glandular and stromal cells in the early proliferative phase compared with normal and eutopic endometrium. p-PERK increased in ectopic glandular and stromal cells in the late proliferative phase compared with normal endometrium. ATF6 was increased in ectopic glandular epithelium compared with normal endometrium in the proliferative phases, versus eutopic endometrium in the late secretory phase. p-IRE1 and p-PERK were increased in high concentrations of ESC treated with peritoneal fluid obtained from women with endometriosis for 10, 30 and 60 min compared with controls. In ESC treated with peritoneal fluid from women with endometriosis, p-IRE1 decreased at 24-48 h compared with 30 min. CONCLUSIONS: In endometriosis, UPR pathways are activated as highly dependent on cell type and phase. Also, p-PERK and p-IRE1 increased because of exposure to high-dose peritoneal fluid from women with endometriosis in stromal cells. Our findings provide a basis for further studies searching for a potential biomarker for the diagnosis of endometriosis.

Zika Virus-Infected Decidual Cells Elicit a Gestational Age-Dependent Innate Immune Response and Exaggerate Trophoblast Zika Permissiveness: Implication for Vertical Transmission.

Vertical transmission of the Zika virus (ZIKV) causes severe fetal defects, but the exact pathogenic mechanism is unclear. We identified up to a 10,480-fold higher expression of viral attachment factors AXL, GAS6, and PROS1 and a 3880-fold increase in ZIKV infectiousness/propagation in human term decidual stromal cells versus trophoblasts. Moreover, levels of viral attachment factors and ZIKV are significantly increased, whereas expression of innate immune response genes are significantly decreased, in human first trimester versus term decidual cells. ZIKV-infected decidual cell supernatants increased cytotrophoblasts infection up to 252-fold compared with directly infected cytotrophoblasts. Tizoxanide treatment efficiently inhibited Zika infection in both maternal and fetal cells. We conclude that ZIKV permissiveness, as well as innate immune responsiveness of human decidual cells, are gestational age dependent, and decidual cells augment ZIKV infection of primary human cytotrophoblast cultures, which are otherwise ZIKV resistant. Human decidual cells may act as reservoirs for trimester-dependent placental transmission of ZIKV, accounting for the higher Zika infection susceptibility and more severe fetal sequelae observed in early versus late pregnancy. Moreover, tizoxanide is a promising agent in preventing perinatal Zika transmission as well as other RNA viruses such as coronavirus.

Chromosome 19 microRNA cluster enhances cell reprogramming by inhibiting epithelial-to-mesenchymal transition.

During implantation, cytotrophoblasts undergo epithelial-to-mesenchymal transition (EMT) as they differentiate into invasive extravillous trophoblasts (EVTs). The primate-specific microRNA cluster on chromosome 19 (C19MC) is exclusively expressed in the placenta, embryonic stem cells and certain cancers however, its role in EMT gene regulation is unknown. In situ hybridization for miR-517a/c, a C19MC cistron microRNA, in first trimester human placentas displayed strong expression in villous trophoblasts and a gradual decrease from proximal to distal cell columns as cytotrophoblasts differentiate into invasive EVTs. To investigate the role of C19MC in the regulation of EMT genes, we employed the CRISPR/dCas9 Synergistic Activation Mediator (SAM) system, which induced robust transcriptional activation of the entire C19MC cistron and resulted in suppression of EMT associated genes. Exposure of human iPSCs to hypoxia or differentiation of iPSCs into either cytotrophoblast-stem-like cells or EVT-like cells under hypoxia reduced C19MC expression and increased EMT genes. Furthermore, transcriptional activation of the C19MC cistron induced the expression of OCT4 and FGF4 and accelerated cellular reprogramming. This study establishes the CRISPR/dCas9 SAM as a powerful tool that enables activation of the entire C19MC cistron and uncovers its novel role in suppressing EMT genes critical for maintaining the epithelial cytotrophoblasts stem cell phenotype.

Thrombin-Induced Decidual Colony-Stimulating Factor-2 Promotes Abruption-Related Preterm Birth by Weakening Fetal Membranes.

Preterm premature rupture of membranes (PPROM) and thrombin generation by decidual cell-expressed tissue factor often accompany abruptions. Underlying mechanisms remain unclear. We hypothesized that thrombin-induced colony-stimulating factor-2 (CSF-2) in decidual cells triggers paracrine signaling via its receptor (CSF2R) in trophoblasts, promoting fetal membrane weakening and abruption-associated PPROM. Decidua basalis sections from term (n = 10), idiopathic preterm birth (PTB; n = 8), and abruption-complicated pregnancies (n = 8) were immunostained for CSF-2. Real-time quantitative PCR measured CSF2 and CSF2R mRNA levels. Term decidual cell (TDC) monolayers were treated with 10-8 mol/L estradiol ± 10-7 mol/L medroxyprogesterone acetate (MPA) ± 1 IU/mL thrombin pretreatment for 4 hours, washed, and then incubated in control medium with estradiol ± MPA. TDC-derived conditioned media supernatant effects on fetal membrane weakening were analyzed. Immunostaining localized CSF-2 primarily to decidual cell cytoplasm and cytotrophoblast cell membranes. CSF-2 immunoreactivity was higher in abruption-complicated or idiopathic PTB specimens versus normal term specimens (P < 0.001). CSF2 mRNA was higher in TDCs versus cytotrophoblasts (P < 0.05), whereas CSF2R mRNA was 1.3 × 104-fold higher in cytotrophoblasts versus TDCs (P < 0.001). Thrombin enhanced CSF-2 secretion in TDC cultures fourfold (P < 0.05); MPA reduced this effect. Thrombin-pretreated TDC-derived conditioned media supernatant weakened fetal membranes (P < 0.05), which MPA inhibited. TDC-derived CSF-2, acting via trophoblast-expressed CSFR2, contributes to thrombin-induced fetal membrane weakening, eliciting abruption-related PPROM and PTB.

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.

Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration.

Preeclampsia (PE) is a common cause of maternal morbidity, characterized by impaired trophoblast invasion and spiral artery transformation resulting in progressive uteroplacental hypoxia. Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation, and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to PE. Here we show that LIN28B is expressed ∼1300-fold higher than LIN28A in human term placenta and is the predominant paralog expressed in primary human trophoblast cultures. The expression of LIN28B mRNA and protein levels are significantly reduced in gestational age-matched preeclamptic vs. normal placentas, whereas LIN28A expression is not different. First trimester human placental sections displayed stronger LIN28B immunoreactivity in extravillous (invasive) cytotrophoblasts and syncytial sprouts vs. villous trophoblasts. LIN28B overexpression increased HTR8 cell proliferation, migration, and invasion, whereas LIN28B knockdown in JEG3 cells reduced cell proliferation. Moreover, LIN28B knockdown in JEG3 cells suppressed syncytin 1 (SYN-1), apelin receptor early endogenous ligand (ELABELA), and the chromosome 19 microRNA cluster, and increased mRNA expression of ITGß4 and TNF-α. Incubation of BeWo and JEG3 cells under hypoxia significantly decreased expression of LIN28B and LIN28A, SYN-1, and ELABELA, whereas TNF-α is increased. These results provide the first evidence that LIN28B is the predominant paralog in human placenta and that decreased LIN28B may play a role in PE by reducing trophoblast invasion and syncytialization, and by promoting inflammation.-Canfield, J., Arlier, S., Mong, E. F., Lockhart, J., VanWye, J., Guzeloglu-Kayisli, O., Schatz, F., Magness, R. R., Lockwood, C. J., Tsibris, J. C. M., Kayisli, U. A., Totary-Jain, H. Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration.

Tumor necrosis factor alfa and interleukin 1 alfa induced phosphorylation and degradation of inhibitory kappa B alpha are regulated by estradiol in endometrial cells.

OBJECTIVE: When bound to the inhibitory kappa B (IкB) protein, the transcription factor nuclear factor kappa B (NF-кB) remains inactively in the cytoplasm. Activated NF-кB upregulates the gene expression of many chemokines including monocyte chemoattractant protein-1 and interleukin (IL)-8. We hypothesized that estrogen may regulate IкB phosphorylation and degradation thus influencing NF-кB-dependent gene expression. Regulation of chemokines by estrogen is different in uterine endometrial cells when compared to ectopic endometrial cells of endometriosis. MATERIALS AND METHODS: We investigated the in vivo expression of IкB in normal endometrium and in eutopic and ectopic endometrium of women with endometriosis. We then studied in cultured endometrial cells to assess the effects of estradiol on IкB and NF-кB function. RESULTS: Normal endometrium from mid-late proliferative phase revealed the strongest IкB immunoreactivity throughout the cycle (p<0.05). When compared to paired homologous eutopic endometrium, ectopic endometrium revealed significantly less immunoreactivity for IкB (p<0.05). Moreover, estradiol induced a decrease in tumor necrosis factor-and IL-1-induced IкB phosphorylation, and also decreased the levels of active-NF-кB (p<0.05). CONCLUSION: Our results support the conclusion that one pathway for estradiol-mediated NF-кB inhibition occurs through the down-regulation of IкB phosphorylation. We propose that the estradiol-induced regulation of IкB and consequent reduction in active-NF-кB may affect inflammatory responses in human endometrial cells.

p38 Mitogen-Activated Protein Kinase is Involved in the Pathogenesis of Endometriosis by Modulating Inflammation, but not Cell Survival.

BACKGROUND: Local pro-inflammatory environment and enhanced cell survival contribute to the endometriosis development. A serine/threonine kinase p38 mitogen-activated protein kinase (MAPK) mediates intracellular signaling of cytokine production, cell proliferation, and apoptosis in different cell types. The current study compares p38 MAPK activity in normal endometrium and endometriosis, and assesses role(s) of p38 MAPK on cytokine production and cell survival in endometriosis. METHODS: Immunohistochemical levels of total and phosphorylated (active) p38 MAPK as well as its correlation with interleukin 8 (IL-8) expression, and cell proliferation and apoptosis were compared in normal human endometrium and endometriosis. The action of p38 MAPK on pro-inflammatory cytokine-induced IL-8 and monocyte chemotactic protein (MCP)-1 expression in endometriotic cells were assessed by enzyme-linked immunosorbent assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival, 5-bromo-2'-deoxyuridine incorporation, and Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assays were used to determine the function of p38 MAPK in cultured human endometriotic stromal cell proliferation and apoptosis. RESULTS: p38 MAPK activity was significantly higher in both eutopic and ectopic endometria compared to normal endometria during late proliferative and early secretory phases ( P < .05). Increased p38 MAPK activity in endometriotic cells correlated with IL-8 expression (Pearson correlation coefficient r = 0.83, P < .01), but not with apoptosis in vivo. The pro-inflammatory cytokines IL-1ß and tumor necrosis factor (TNF)-α induced activation of p38 MAPK. Inhibition of p38 MAPK activity blocked IL-1ß and TNF-α-induced IL-8 and MCP-1 secretion in cultured endometriotic stromal cells ( P < .05), but did not impact on endometriotic cell survival. CONCLUSIONS: These results suggest that rather than modulating cell survival, increased p38 MAPK activity in endometriotic cells contributes to the pathogenesis of endometriosis by promoting the local inflammatory milieu.

The extracellular signal-regulated kinase 1/2 triggers angiogenesis in human ectopic endometrial implants by inducing angioblast differentiation and proliferation.

PROBLEM: The role of extracellular signal-regulated kinase (ERK)1/2-mediated angiogenesis during endometriotic nidation is unknown. We posit that ERK1/2-induced angioblast differentiation and proliferation promotes ectopic endometrial angiogenesis. METHODS OF STUDY: Human eutopic and ectopic endometria were immunostained for total- (T-) or phosphorylated- (P-) ERK1/2 or double-immunostained for P-ERK1/2-CD34 and PCNA-CD34. Estradiol (E2 ), cytokines, normal peritoneal fluid (NPF) or endometriotic peritoneal fluid (EPF) ±PD98059, an ERK1/2 inhibitor, treaded primary human endometrial endothelial cells (HEECs) were evaluated by T-/P-ERK1/2 immunoblotting, MTT viability and tube formation assays. RESULTS: HEECs exhibited higher endothelial P-ERK1/2 immunoreactivity in ectopic vs eutopic endometria. Double-immunostained ectopic endometria displayed abundant CD34-positive angioblasts exhibiting strong P-ERK1/2 and PCNA immunoreactivity. EPF and vascular growth factor (VEGF)-A significantly increased HEEC proliferation and P-ERK1/2 levels. PD98059 reduced basal, EPF, and VEGF-induced HEEC proliferation and promoted vascular stabilization following tube formation. CONCLUSION: Enhanced ERK1/2 activity in angioblasts by such peritoneal factors as VEGF, E2 induces proliferation to trigger ectopic endometrial angiogenesis.

hCG: Biological Functions and Clinical Applications.

Human chorionic gonadotropin (hCG) is produced primarily by differentiated syncytiotrophoblasts, and represents a key embryonic signal that is essential for the maintenance of pregnancy. hCG can activate various signaling cascades including mothers against decapentaplegic homolog 2 (Smad2), protein kinase C (PKC), and/or protein kinase A (PKA) in several cells types by binding to luteinizing hormone/chorionic gonadotropin receptor (LHCGR) or potentially by direct/indirect interaction with transforming growth factor beta receptor (TGFßR). The molecule displays specialized roles in promoting angiogenesis in the uterine endothelium, maintaining myometrial quiescence, as well as fostering immunomodulation at the maternal-fetal interface. It is a member of the glycoprotein hormone family that includes luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and follicle-stimulating hormone (FSH). The α-subunit of hCG displays homologies with TSH, LH, and FSH, whereas the ß subunit is 80-85% homologous to LH. The hCG molecule is produced by a variety of organs, exists in various forms, exerts vital biological functions, and has various clinical roles ranging from diagnosis and monitoring of pregnancy and pregnancy-related disorders to cancer surveillance. This review presents a detailed examination of hCG and its various clinical applications.