Mammalian target of rapamycin signaling is a mechanistic link between increased endoplasmic reticulum stress and autophagy in the placentas of pregnancies complicated by growth restriction.

INTRODUCTION: Increased endoplasmic reticulum (ER) stress and autophagy have been noted in the placentas of pregnancies complicated by idiopathic intrauterine growth restriction (IUGR); however, the cause of these phenomena remains unclear. We surmised that oxygen-glucose deprivation (OGD) may increase ER stress and autophagy and that mammalian target of rapamycin (mTOR) signaling is involved in regulating placental ER stress and autophagy in pregnancies complicated by IUGR. METHODS: We obtained placentas from women with normal term pregnancies and pregnancies complicated by IUGR to compare ER stress, mTOR signaling, and levels of autophagy-related proteins between the two groups and used primary cytotrophoblast cells treated with or without salubrinal (an ER stress inhibitor), MHY1485 (an mTOR activator), or rapamycin (an mTOR inhibitor) to investigate the effects of OGD on ER stress, mTOR activity, and autophagy levels in vitro. RESULTS: Women with pregnancies complicated by IUGR displayed higher placental ER stress and autophagy levels but lower mTOR activity than women with normal pregnancies. Furthermore, OGD increased ER stress, regulated in development and DNA damage responses-1 (REDD1), phosphorylated tuberous sclerosis complex 2 (TSC2), and autophagy levels and decreased mTOR activity compared to the standard culture condition; however, the salubrinal treatment attenuated these changes. Moreover, the administration of MHY1485 or rapamycin to OGD-treated cells decreased or increased autophagy levels, respectively. DISCUSSION: Based on our results, mTOR is a mechanistic link between OGD-induced ER stress and autophagy in cytotrophoblast cells; thus, mTOR plays an essential role in the pathogenesis of pregnancies complicated by IUGR.

Micronized progesterone pretreatment affects the inflammatory response of human gestational tissues and the cervix to lipopolysaccharide stimulation.

INTRODUCTION: Vaginal administration of micronized progesterone (utrogestan capsule, UG) reduces the risk of preterm birth (PTB) in asymptomatic women with a sonographic short cervix at mid-trimester or with a prior history of spontaneous PTB; however, its exact mechanisms remain unclear. We hypothesized that UG limits the inflammatory processes within the gestational tissues and the cervix. METHODS: Fetal membranes and villous tissues were obtained from normal term placentas from women with cesarean delivery before labor onset. Ectocervical tissues were obtained from premenopausal women undergoing hysterectomies for uterine fibroids. Explant tissue cultures were pretreated with UG for 24 h and then exposed to UG with or without lipopolysaccharide (LPS) for 48 h. Tumor necrosis factor-α, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1, interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-10, IL-13, interferon-γ, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 levels in tissue homogenates and culture medium were measured by enzyme-linked immunosorbent assays. Real-time quantitative PCR, Western blot, and gelatine zymography were used to measure matrix metalloproteinase (MMP)-9 and MMP-2 mRNA, protein, and activity levels, respectively. RESULTS: UG pretreatment did not cause a significant change in basal levels or in LPS-induced production and secretion of cytokines, chemokines, and TIMPs in the three tissues. However, UG pretreatment significantly reduced MMP-9 and MMP-2 expression and activity in fetal membranes stimulated with LPS but not in villous or cervical tissues. DISCUSSION: UG pretreatment significantly reduced MMP-9 and MMP-2 expression and activity in fetal membranes stimulated with LPS, suggesting a possible protective mechanism of micronized progesterone in preventing infection-associated PTB.

Autophagy in the human placenta throughout gestation.

BACKGROUND: Autophagy has been reported to be essential for pre-implantation development and embryo survival. However, its role in placental development and regulation of autophagy during pregnancy remain unclear. The aims of this study were to (1) study autophagy by characterizing changes in levels of beclin-1, DRAM, and LC3B in human placenta throughout gestation; (2) determine whether autophagy is involved in regulation of trophoblast invasion in JEG-3 cells (a choriocarcinoma cell line); (3) examine the effects of reduced oxygen and glucose on the autophagic changes; and (4) investigate the effect of reoxygenation and supplementation of glucose after oxygen-glucose deprivation (OGD) on the autophagic changes in primary cytotrophoblasts obtained from normal term pregnancy. METHODOLOGY/PRINCIPAL FINDINGS: An analysis of 40 placental samples representing different gestational stages showed (1) no significant differences in beclin-1, DRAM, and LC3B-II levels in placentas between early and mid-gestation, and late gestation with vaginal delivery; (2) placentas from late gestation with cesarean section had lower levels of LC3B-II compared to early and mid-gestation, and late gestation with vaginal delivery; levels of DRAM were also lower compared to placentas from early and mid-gestation; and (3) using explant cultures, villous tissues from early and late gestation had similar rates of autophagic flux under physiological oxygen concentrations. Knockdown of BECN1, DRAM, and LC3B had no effects on viability and invasion activity of JEG-3 cells. On the other hand, OGD caused a significant increase in the levels of LC3B-II in primary cytotrophoblasts, while re-supplementation of oxygen and glucose reduced these changes. Furthermore, there were differential changes in levels of beclin-1, DRAM, and LC3B-II in response to changes in oxygen and glucose levels. CONCLUSIONS/SIGNIFICANCE: Our results indicate that autophagy is involved in development of the human placenta and that changes in oxygen and glucose levels participate in regulation of autophagic changes in cytotrophoblast cells.

Increased autophagy in placentas of intrauterine growth-restricted pregnancies.

BACKGROUND: Unexplained intrauterine growth restriction (IUGR) may be a consequence of placental insufficiency; however, its etiology is not fully understood. We surmised that defective placentation in IUGR dysregulates cellular bioenergic homeostasis, leading to increased autophagy in the villous trophoblast. The aims of this work were (1) to compare the differences in autophagy, p53 expression, and apoptosis between placentas of women with normal or IUGR pregnancies; (2) to study the effects of hypoxia and the role of p53 in regulating trophoblast autophagy; and (3) to investigate the relationship between autophagy and apoptosis in hypoxic trophoblasts. METHODOLOGY/PRINCIPAL FINDINGS: Compared with normal pregnant women, women with IUGR had higher placental levels of autophagy-related proteins LC3B-II, beclin-1, and damage-regulated autophagy modulator (DRAM), with increased p53 and caspase-cleaved cytokeratin 18 (M30). Furthermore, cytotrophoblasts cultured under hypoxia (2% oxygen) in the presence or absence of nutlin-3 (a p53 activity stimulator) had higher levels of LC3B-II, DRAM, and M30 proteins and increased Bax mRNA expression compared with controls cultured under standard conditions. In contrast, administration of pifithrin-α (a p53 activity inhibitor) during hypoxia resulted in protein levels that were similar to those of the control groups. Moreover, cytotrophoblasts transfected with LC3B, beclin-1, or DRAM siRNA had higher levels of M30 compared with the controls under hypoxia. However, transfection with Bcl-2 or Bax siRNA did not cause any significant change in the levels of LC3B-II in hypoxic cytotrophoblasts. CONCLUSIONS/SIGNIFICANCE: Together, these results suggest that there is a crosstalk between autophagy and apoptosis in IUGR and that p53 plays a pivotal and complex role in regulating trophoblast cell turnover in response to hypoxic stress.

The association between maternal oxidative stress at mid-gestation and subsequent pregnancy complications.

OBJECTIVE: To investigate the association between maternal oxidative stress at mid-gestation and subsequent development of pregnancy complications. STUDY DESIGN: A total of 503 healthy pregnant women provided their blood and urine samples at 24 to 26 weeks of gestation and were prospectively followed through postpartum. These samples were used to assess a variety of oxidative stress markers, including plasma total antioxidant capacity, 8-isoprostane, erythrocyte glutathione peroxidase and superoxide dismutase activity, and urinary 8-hydroxydeoxyguanosine (8-OHdG). RESULTS: Compared with women with uncomplicated pregnancies, significantly higher plasma 8-isoprostane levels were noted in women who developed preeclampsia (P = .008) and small-for-gestational age infants (P = .002), while higher urinary 8-OHdG concentrations were noted in women who subsequently had low-birth-weight neonates (<2500 g, P = .043). CONCLUSION: Increased maternal oxidative stress at mid-gestation was associated with subsequent pregnancy complications.

Differential effects of concomitant use of vitamins C and E on trophoblast apoptosis and autophagy between normoxia and hypoxia-reoxygenation.

BACKGROUND: Concomitant supplementation of vitamins C and E during pregnancy has been reportedly associated with low birth weight, the premature rupture of membranes and fetal loss or perinatal death in women at risk for preeclampsia; however, the cause is unknown. We surmise that hypoxia-reoxygenation (HR) within the intervillous space due to abnormal placentation is the mechanism and hypothesize that concomitant administration of aforementioned vitamin antioxidants detrimentally affects trophoblast cells during HR. METHODOLOGY/PRINCIPAL FINDINGS: Using villous explants, concomitant administration of 50 microM of vitamins C and E was observed to reduce apoptotic and autophagic changes in the trophoblast layer at normoxia (8% oxygen) but to cause more prominent apoptosis and autophagy during HR. Furthermore, increased levels of Bcl-2 and Bcl-xL in association with a decrease in the autophagy-related protein LC3-II were noted in cytotrophoblastic cells treated with vitamins C and E under standard culture conditions. In contrast, vitamin treatment decreased Bcl-2 and Bcl-xL as well as increased mitochondrial Bak and cytosolic LC3-II in cytotrophoblasts subjected to HR. CONCLUSIONS/SIGNIFICANCE: Our results indicate that concomitant administration of vitamins C and E has differential effects on the changes of apoptosis, autophagy and the expression of Bcl-2 family of proteins in the trophoblasts between normoxia and HR. These changes may probably lead to the impairment of placental function and suboptimal growth of the fetus.

A longitudinal study of oxidative stress and antioxidant status in women with uncomplicated pregnancies throughout gestation.

Pregnancy is associated with increased oxidative stress, and exaggeration of oxidative damage is considered important in pregnancy complications such as preeclampsia. There is, however, only scattered information regarding the longitudinal change of oxidative stress during pregnancy. We surmised that the placenta is the cause of the increased oxidative stress during pregnancy and hypothesized that the amount of placenta-derived cell-free fetal DNA in maternal plasma is correlated with maternal oxidative stress status. Blood and urine samples were collected at different stages of gestation and 6 to 8 weeks postpartum from 105 women with uncomplicated pregnancies. Additionally, 40 healthy women of reproductive age, but not pregnant, were recruited as controls. All oxidative stress markers, including urinary 8-hydroxydeoxyguanosine (8-OHdG), plasma 8-isoprostane, total antioxidant capacity (TAC), and erythrocyte glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities, were increased in the third trimester, and most of them returned to nonpregnant levels postpartum. Furthermore, the DYS14 gene of the Y chromosome was quantified from plasma samples obtained at 26 to 30 weeks' gestation in 54 women who later delivered a male infant. Significant associations were noted between plasma concentrations of the DYS14 gene and the levels of urinary 8-OHdG and plasma 8-isoprostane, suggesting an association between the breakdown of syncytiotrophoblast and maternal oxidative stress during pregnancy.

Tumor necrosis factor-alpha converting enzyme in the human placenta throughout gestation.

Ectodomain shedding of epidermal growth factor receptor ligands such as transforming growth factor- alpha (TGF-alpha), heparin-binding epidermal growth factor-like growth factor (HBEGF), and amphiregulin (AREG) is considered to be important during implantation. Tumor necrosis factor-alpha converting enzyme (TACE) has been suggested as the major sheddase for these molecules. The objectives of this study are (1) to characterize the expression of TACE in the human placenta throughout gestation; (2) to determine the association between the expression of TACE with TGF-alpha, HBEGF, and AREG; (3) to ascertain whether TACE mediates TGF-alpha, HBEGF, and AREG shedding; and (4) to examine the effect of hypoxia on the expression of TACE. By analyzing a total of 55 villous samples representing different gestational ages, the authors found that TACE was continuously expressed in the placentas throughout gestation and that the levels of TACE were positively correlated with the levels of TGF-alpha, HBEGF, and AREG. Preadministration of a TACE inhibitor in villous explant cultures or transfection of cytotrophoblastic cells with TACE-specific small interference RNA decreased the shedding of HBEGF and AREG. Moreover, hypoxia (2% O(2)) caused an increase in the levels of TACE mRNA and protein in villous explants and primary cytotrophoblastic cells in vitro. These results indicate that oxygen regulates the expression of TACE and that TACE may be important for placental development during human pregnancy.

Covariance control with observed-state feedback gains for continuous nonlinear systems using T-S fuzzy models.

The design problem of state variance constrained control for stochastic systems has received rather extensive attention in recent years. This paper solves the state variance constrained controller design problem by using the covariance control theory, with observed-state feedback gains for continuous Takagi-Sugeno (TS) fuzzy models. By incorporating the technique of state estimation into the practical covariance control theory, a variance constrained control methodology is developed for the continuous TS fuzzy models. Finally, a numerical example is shown to demonstrate the efficiency and applicability of the proposed approach.