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.

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.

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.

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.

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.

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.

Long-acting progestin-only contraceptives impair endometrial vasculature by inhibiting uterine vascular smooth muscle cell survival.

Molecular mechanisms responsible for abnormal endometrial vasculature in women receiving long-acting progestin-only contraceptives (LAPCs) are unknown. We hypothesize that LAPCs impair vascular smooth muscle cell (VSMC) and pericyte proliferation and migration producing thin-walled hyperdilated fragile microvessels prone to bleeding. Proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (αSMA) double-immunostaining assessed VSMC differentiation and proliferation in endometria from women before and after DepoProvera (Depo) treatment and from oophorectomized guinea pigs (OVX-GPs) treated with vehicle, estradiol (E2), medroxyprogesterone acetate (MPA), or E2+MPA. Whole-genome profiling, proliferation, and migration assays were performed on cultured VSMCs treated with MPA or etonogestrel (ETO). Endometrial vessels of Depo-administered women displayed reduced αSMA immunoreactivity and fewer PCNA (+) nuclei among αSMA (+) cells (P < 0.008). Microarray analysis of VSMCs identified several MPA- and ETO-altered transcripts regulated by STAT1 signaling (P < 2.22 × 10(-6)), including chemokine (C-C motif) ligand 2 (CCL2). Both MPA and ETO reduce VSMC proliferation and migration (P < 0.001). Recombinant CCL2 reversed this progestin-mediated inhibition, whereas a STAT1 inhibitor abolished the CCL2 effect. Similarly, the endometria of MPA treated OVX-GPs displayed decreased αSMA staining and fewer PCNA (+) nuclei in VSMC (P < 0.005). In conclusion, LAPCs promote abnormal endometrial vessel formation by inhibiting VSMC proliferation and migration.

Enhanced Human Decidual Cell-Expressed FKBP51 May Promote Labor-Related Functional Progesterone Withdrawal.

Sustained plasma progesterone (P4) levels suggest initiation of human term labor by functional P4 withdrawal, reflecting reduced progesterone receptor (PR) and/or glucocorticoid receptor (GR) expression or activity. The steroid-induced immunophilin cochaperone FKBP51 inhibits PR- and GR-mediated transcription, suggesting a labor-initiating role. Gestational age-matched decidual sections were immunostained for FKBP51 and decidual cell (DC) and interstitial trophoblast (IT) markers, vimentin and cytokeratin, respectively. Term DC cultures were incubated with vehicle (control), estradiol (E2) with or without medroxyprogesterone acetate, dexamethasone (Dex), or Organon 2058. FKBP51 histologic scoring was significantly higher in DC nuclei during labor versus prelabor decidua, whereas FKBP51 immunostaining was undetected in interstitial trophoblasts (P < 0.05). In term DC cultures, E2 + medroxyprogesterone acetate or E2 + Dex enhanced FKBP51 expression (P < 0.01), whereas E2 + Organon 2058 inhibited PR expression (P < 0.05), and E2 + Dex inhibited GR expression (P < 0.05). Unlike term DCs, FKBP51 was undetected in control or Dex-treated cultured third-trimester trophoblasts. Electrophoretic mobility shift assays revealed that FKPB51 overexpression or silencing in cultured DCs altered PR-DNA binding. Increased FKBP51 levels in term DCs during labor complement our prior in situ observations of significantly lower PR in labor versus prelabor DCs. In a milieu of sustained plasma P4 levels, these reciprocal changes will amplify functional P4 withdrawal in DCs via FKBP51-mediated PR resistance coupled with declining PR levels, whereas the lack of FKBP51 expression in interstitial trophoblasts suggests unopposed constitutive GR action.

Mechanisms of chorioamnionitis-associated preterm birth: interleukin-1ß inhibits progesterone receptor expression in decidual cells.

In chorioamnionitis (CAM), a major cause of preterm birth (PTB), maternal-fetal inflammation of the decidua and amniochorion cause the release of cytokines that elicit cervical ripening, fetal membrane rupture and myometrial activation. We posit that this inflammatory milieu triggers PTB by inhibiting progesterone receptor (PR) expression and increasing decidual prostaglandin (PG) production. Immunohistochemical staining of decidua detected significantly lower PR levels in decidual cells (DCs) from CAM-complicated PTB. Incubation of DCs with IL-1ß decreased PR expression and significantly increased PGE2 and PGF2α production and COX-2 expression. The addition of PGF2α to DC cultures also suppressed PR expression. However, the COX inhibitor, indomethacin, did not reverse IL-1ß suppression of PR expression in DC cultures. Although IL-1ß treatment activated the NF-KB, ERK1/2 and p38 MAPK signalling cascades in DCs, inhibition of ERK1/2 MAPK signalling alone was sufficient to completely reverse the suppression of PR levels by IL-1ß. These findings suggest that CAM-associated PTB is induced at least in part by IL-1ß-mediated functional progesterone withdrawal.

Interferon-γ protects first-trimester decidual cells against aberrant matrix metalloproteinases 1, 3, and 9 expression in preeclampsia.

Human extravillous trophoblast (EVT) invades the decidua via integrin receptors and subsequently degrades extracellular matrix proteins. In preeclampsia (PE), shallow EVT invasion elicits incomplete spiral artery remodeling, causing reduced uteroplacental blood flow. Previous studies show that preeclamptic decidual cells, but not interstitial EVTs, display higher levels of extracellular matrix-degrading matrix metalloproteinase (MMP)-9, but not MMP-2. Herein, we extend our previous PE-related assessment of MMP-2 and MMP-9 to include MMP-1, which preferentially degrades fibrillar collagens, and MMP-3, which can initiate a local proteolytic cascade. In human first-trimester decidual cells incubated with estradiol, tumor necrosis factor-α (TNF-α) significantly enhanced MMP-1, MMP-3, and MMP-9 mRNA and protein levels and activity measured by real-time quantitative RT-PCR, ELISA, immunoblotting, and zymography, respectively. In contrast, interferon γ (IFN-γ) reversed these effects and medroxyprogesterone acetate elicited further reversal. Immunoblotting revealed that p38 mitogen-activated protein kinase signaling mediated TNF-α enhancement of MMP-1, MMP-3, and MMP-9, whereas IFN-γ inhibited p38 mitogen-activated protein kinase phosphorylation. Unlike highly regulated MMP-1, MMP-3, and MMP-9, MMP-2 mRNA and protein expression was constitutive in decidual cells. Because inflammation underlies PE-associated shallow EVT invasion, these results suggest that excess macrophage-derived TNF-α augments expression of MMP-1, MMP-3, and MMP-9 in decidual cells to interfere with normal stepwise EVT invasion of the decidua. In contrast, decidual natural killer cell-derived IFN-γ reverses such TNF-α-induced MMPs to protect against PE.

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.

Abruption-induced preterm delivery is associated with thrombin-mediated functional progesterone withdrawal in decidual cells.

Plasma progesterone levels remain elevated throughout human pregnancy, suggesting that reduced reproductive-tract progesterone receptor (PR) initiates labor. Placental abruption and excess thrombin generation elicit preterm delivery (PTD). PR, glucocorticoid receptor (GR), and total and p-ERK1/2 in decidual cells (DCs) and interstitial trophoblasts (IT) were assessed via immunohistochemical staining in abruption-associated PTD versus gestational-age matched control placentas, and in cultured DCs incubated with estradiol (E2) ± medroxyprogesterone acetate (MPA) ± thrombin. Immunostaining for PR was lower in DC nuclei in abruption versus control decidua and was absent from ITs; GR was higher in IT than DCs, with no abruption-related changes in either cell type; p-ERK1/2 was higher in DCs in abruption than control decidua, with total ERK 1/2 unchanged. Immunoblotting of cultured DCs demonstrated strong E2, weak MPA, and intermediate E2+MPA mediated elevation of PR-A and PR-B levels, with constitutive GR expression. In cultured DCs, thrombin inhibited PR but not GR mRNA levels, reduced PR binding to DNA and [(3)H]progesterone binding to PR, and enhanced phosphorylated but not total ERK1/2 levels. Coincubation with a specific p-ERK1/2 inhibitor reversed thrombin-enhanced p-ERK1/2 and lowered PR levels. Thus, abruption-associated PTD is initiated by functional progesterone withdrawal, as indicated by significantly reduced DC nuclear expression of PR-A and PR-B. Functional withdrawal of progesterone results in increased p-ERK1/2, and is thus one pathway initiating abruption-associated PTD.

Genistein inhibits cell proliferation and stimulates apoptosis in human coronary artery endothelial cells.

BACKGROUND/AIMS: Isoflavone genistein is a plant-derived compound structurally similar to estradiol, which behaves weakly estrogenic or anti-estrogenic in a cell- and concentration-dependent manner. Genistein has been hypothesized to have beneficial effects on vascular diseases, although the mechanism has been unclear. Here, we investigated whether genistein may play a role in atherogenesis by regulating human coronary artery endothelial cell (HCAEC) survival. METHODS: HCAECs obtained from 48- to 53-year-old women (n = 3) were used and immunocytochemistry, cell proliferation assay and apoptosis assay were carried on HCAECs treated by genistein. RESULTS: Immunocytochemistry confirmed that HCAECs in culture express predominantly ESR2. Cell proliferation assay revealed that following 72 h of genistein treatment, HCAEC proliferation decreased in a concentration-dependent (10(-10) to 10(-6)M) manner compared to control (p < 0.01). The anti-proliferative effect of genistein is inhibited by estradiol. Genistein (10(-8)M) also induced a time-dependent increase in the number of apoptotic HCAECs after 24-, 48- and 72-hour treatments as detected by TUNEL and morphological analyses. CONCLUSION: These findings suggest that genistein acts as an anti-proliferative agent on HCAECs. The anti-proliferative and proapoptotic effects of genistein on vascular cells underlie the proposed anti-atherogenic and cardioprotective role of genistein.

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.

Alteration of gene expression in human cumulus cells as a potential indicator of oocyte aneuploidy.

BACKGROUND: Human female meiosis is particularly error prone, leading to the generation of aneuploidy, a problem which increases dramatically with advancing age. Despite being of great clinical importance, the genesis of oocyte aneuploidy remains poorly understood. Cumulus cells (CCs) surround oocytes in antral follicles and play a crucial role in regulating their maturation. During this investigation, we aimed to obtain an insight into the follicular environment of oocytes that become aneuploid and identify non-invasive markers of oocyte chromosome status and competence. METHODS: Microarray comparative genomic hybridization was used to assess oocyte ploidy. Expression of 96 genes was examined via a large real-time PCR experiment in 51 CC samples, whereas an additional 26 CC clumps were used for a more focused real-time PCR experiment assessing just three genes. Gene selection was based on the results of a previous microarray analysis comparing the transcriptome of CCs from normal and aneuploid oocytes. RESULTS: Fifty eight of the investigated genes were confirmed to be expressed in CCs. Patterns of expression were generally similar among all CC samples, regardless of the chromosomal status of the corresponding oocyte. However, two genes, SPSB2 and TP53I3, were both significantly down-regulated in CCs from chromosomally abnormal oocytes (P<0.05). The expression of SPSB2 was also found to display a strong trend towards up-regulation in the CCs of oocytes that produced a healthy live birth (P=0.054). CONCLUSIONS: In the current study, SPSB2 and TP53I3 exhibited highly significant differences in their expression between CCs of normal and chromosomally abnormal oocytes. SPSB2 is involved in intracellular signalling and homeostasis, whereas TP53I3 regulates carbohydrate metabolism and apoptosis. We speculate that both these genes might have the potential to serve as non-invasive markers of oocyte aneuploidy.

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.

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.

Bidirectional interaction between unfolded-protein-response key protein HSPA5 and estrogen signaling in human endometrium.

The human endometrium is a dynamic tissue that undergoes cyclic changes under the influence of steroid hormones as well as numerous local paracrine and autocrine factors. Heat shock 70 kDa protein (HSPA5; also known as GRP78/BiP), a molecular chaperone within the endoplasmic reticulum, plays crucial roles in normal cellular processes as well as in stress conditions, in which it is a central regulator for the unfolded protein response (UPR). We hypothesized that HSPA5 expression level is variable throughout the menstrual cycle in human endometrium and that estrogen signaling cross-talks with UPR signaling by interacting with HSPA5. HSPA5 expression throughout the menstrual cycle was evaluated in vivo in normal human endometrium. Using in vitro techniques, we then assessed the bidirectional regulation of HSPA5 and estrogen signaling in human endometrial glandular (Ishikawa) and stromal cells (ESC). HSPA5 immunoreactivity in endometrial glandular and stromal cells was cycle-dependent, and was significantly higher in phases of the menstrual cycle when estradiol (E(2)) levels are known to be the lowest compared with the rest of the cycle (P < 0.001). E(2) did not affect HSPA5 expression after 8-24 h incubation in Ishikawa cells and ESC in vitro. However, tunicamycin-induced HSPA5 expression was significantly lowered in these cells when pretreated with E(2) (P < 0.01 and P < 0.05, respectively). On the other hand, tunicamycin decreased E(2) up-regulated alkaline phosphatase activity (P < 0.001). In conclusion, there is cycle-dependent HSPA5 expression with a possible inverse correlation between HSPA5 expression and E(2) levels in human endometrium. We suggest that estrogen signaling cross-talks with the UPR cascade by interacting with HSPA5, as supported by our in vitro findings.

Regulation of interleukin-6 expression in human decidual cells and its potential role in chorioamnionitis.

Chorioamnionitis frequently precedes both genital tract and placental inflammation and is both a primary cause of maternal morbidity and a major antecedent of preterm premature rupture of the membranes (PPROM) as well as preterm delivery (PTD). In most cases of chorioamnionitis, neutrophils dominate the decidua. In a subset of these cases, a predominance of monocytes is uniquely associated with both neonatal intraventricular hemorrhage and death. The multifunctional cytokine, interleukin-6, promotes local monocyte dominance via several mechanisms. In this study, immunostaining of placental sections revealed significantly higher interleukin-6 HSCOREs in decidual cells (DCs) but not in interstitial trophoblasts, in chorioamnionitis versus gestational age-matched control placentas (P < 0.05). In confluent leukocyte-free term DCs, secreted interleukin-6 levels in incubations with estradiol-17ß were increased 2500-fold by IL-1ß (P < 0.05). This up-regulation was inhibited by more than 50% in parallel incubations that included medroxyprogesterone acetate (n = 12, P < 0.05). Western blotting data confirmed these enzyme-linked immunosorbent assay results; quantitative RT-PCR findings demonstrated corresponding changes in interleukin-6 mRNA levels. Specific inhibitors of signaling for both nuclear factor-κB activation and p38-mitogen-activated protein kinase, but not for protein kinase C, significantly decreased IL-1ß-enhanced interleukin-6 expression levels in cultured DCs. In conclusion, in situ and in vitro results indicate that significantly enhanced interleukin-6 expression levels in DCs during chorioamnionitis could be pivotal in skewing decidual monocyte differentiation to macrophages.