Intrauterine Tissues Research Articles

Localisation and Expression of FoxO1 Proteins in Human Gestational Tissues

In non-gestational tissues, emerging data indicate that the FoxO1 family of Forkhead transcription factors play diverse roles in many cellular processes coordinating programs of gene expression that regulate apoptosis, oxidative stress resistance, and immune cell homeostasis. Successful outcome of human parturition rely on many of these processes, however there is no data available on FoxO1 proteins in human intrauterine tissues, nor their role in pregnancy complications such as pre-eclampsia. Thus the aim of this study was (i) to characterises the localisation and expression of FoxO1, acetylated (ac)-FoxO1 and phosphorylated (p)-FoxO1 in human placenta and fetal membranes obtained from term Caesarean sections ( n = 5); and (ii) to compare the expression of FoxO1 proteins in term placental samples from normal and pre-eclamptic pregnancies ( n = 5 per group). In placenta, weak FoxO1 staining was localised to the syncytiotrophoblast layer, whereas ac-FoxO1 and p-FoxO1 staining was mainly localised in the syncytiotrophoblasts and cytotrophoblasts. In fetal membranes, FoxO1, ac-FoxO1 and p-FoxO1 were localised to the trophoblast layer of the chorion, amnion epithelium and decidual cells. Quantitative RT-PCR (qRT-PCR) analysis showed a 6-fold and 12-fold higher mRNA expression in the choriodecidua compared to placenta and amnion, respectively. In both amnion and choriodecidua, FoxO1 protein expression was higher in the cytoplasmic fractions than in the nuclear fractions. On the otherhand, ac-FoxO1 and p-FoxO1 protein expression was higher in the nuclear fractions for all three tissues. There was no difference in the mRNA or protein expression of FoxO1 proteins in placental samples from normal and pre-eclamptic term pregnancies. The exact role of FoxO1 proteins in human pregnancy are unknown, however the finding that they are expressed in human gestational tissues warrants further research into their function in these tissues.

AKR1C1andSRD5A1Messenger RNA Expression at Term in the Human Myometrium and Chorioamniotic Membranes

We sought to determine relative mRNA expression of AKR1C1 and SRD5A1, which respectively encode for the key progesterone metabolizing enzymes, 20alpha-hydroxysteroid dehydrogenase and 5alpha-reductase type 1, in the myometrium and chorioamniotic membranes during human spontaneous or induced labor and nonlabor. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to compare relative mRNA expression of AKR1C1 and SRD5A1 in the myometrium and chorioamniotic membranes from 20 subjects during three different states of labor: not in labor ( N = 10), spontaneous labor ( N = 5), or induced labor ( N = 5). Labor was defined as regular uterine contractions that resulted in cervical dilation. Myometrial AKR1C1 mRNA expression was significantly greater in spontaneously laboring subjects compared with those not in labor (2.4-fold [1.97 to 2.98], P = 0.02). There was no difference in myometrial AKR1C1 mRNA expression between those with induced labor compared with those not in labor. Regardless of labor status, no differences were observed in the chorioamniotic membrane AKR1C1 mRNA expression between the groups. SRD5A1 mRNA expression was significantly lower in the membranes of both laboring groups when compared with those not in labor (spontaneous: 0.10-fold [0.06 to 0.18], P = 0.007; induced: 0.09-fold [0.03 to 0.25], P = 0.013). Regardless of labor status, there was no difference in SRD5A1 mRNA expression in the myometrium. Our study demonstrated tissue-specific changes in progesterone metabolizing enzyme mRNA expression in human intrauterine tissue at term associated with labor status. These observed changes in mRNA expression may have important implications for progesterone metabolism at those specific sites and thereby may differentially regulate the tissue-specific progesterone concentration and/or the level of specific progesterone metabolites.

Inflammation in Pregnancy: Its Roles in Reproductive Physiology, Obstetrical Complications, and Fetal Injury

Fetal development and growth occur in a sterile amniotic cavity while first exposure to microorganisms happens at birth. However, at least 25% of all preterm births, the leading cause of perinatal morbidity and mortality worldwide, occur in mothers with microbial invasion of the amniotic cavity. Microbial attack of the fetus takes place in approximately 10% of pregnancies with intra-amniotic infection, and the human fetus is capable of deploying an inflammatory response (cellular and humoral) in the mid-trimester of pregnancy. The onset of premature labor in the context of infection is mediated by pro-inflammatory cytokines, such as interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha), as these cytokines are produced by intrauterine tissues in response to microbial products, can stimulate prostaglandin production, and induce labor in animals. Moreover, knockout experiments suggest that infection is less likely to lead to premature labor when the IL-1 and TNF signaling pathways are disrupted. A fetal inflammatory systemic response occurs in a fraction of fetuses exposed to microorganisms in utero, and is associated with the impending onset of labor as well as multisystem organ involvement. Neonates born with funisitis, the histologic marker of such inflammation, are at increased risk for neurologic handicap and cerebral palsy. Evidence has begun to accumulate that gene-environment interactions determine the likelihood of preterm labor and delivery and, probably, the risk of fetal injury. Fetal inflammation has emerged as a major mechanism of disease responsible for complications in the perinatal period (in utero and in the first 28 days of life), as well as in infancy. Moreover, reprogramming of the fetal immune response may predispose to diseases in adulthood.

Differential Regulation of Prostaglandin Production Mediated by Corticotropin-Releasing Hormone Receptor Type 1 and Type 2 in Cultured Human Placental Trophoblasts

Prostaglandin (PG) production by intrauterine tissues plays a key part in the control of pregnancy and parturition. The present study was to investigate the role of placenta-derived CRH and CRH-related peptides in the regulation of PG synthesis and metabolism. We found that placental trophoblasts expressed both CRH-R1 and CRH-R2. Treatment of cultured placental cells with either a CRH or urocortin I (UCNI) antibody resulted in a significant decrease in PGE2 release. Both CRH and UCNI antibodies significantly decreased mRNA and protein expression of synthetic enzymes cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2 and increased mRNA and protein expression of 15-hydroxyprostaglandin dehydrogenase (PGDH), the key enzyme of PG metabolism. CRH-R1/-R2 antagonist astressin and CRH-R1 antagonist antalarmin significantly inhibited PGE2 release, whereas CRH-R2 antagonist astressin-2b had no effect on PGE(2) release. Administration of astressin decreased expression of cPLA2 but had no effect on COX-2 expression. Antalarmin reduced cPLA2 and COX-2 expression, whereas astressin-2b did not alter cPLA2 expression but increased COX-2 expression. PGDH expression was enhanced by these three antagonists. Cells treated with exogenous CRH and UCNI showed an increase in PGE(2) release and expression of cPLA2 and COX-2 but a decrease in PGDH expression. UCNII and UCNIII had no effect on PGE2 release but decreased COX-2 and PGDH expression. Our results suggested CRH and CRH-related peptides act on CRH-R1 and CRH-R2 to exert different effects on PG biosynthetic enzymes cPLA2 and COX-2 and thereby modulate output of PGs from placenta, which would be important for controlling pregnancy and parturition.

Dose-Dependent Effects of Meloxicam Administration on Cyclooxygenase-1 and Cyclooxygenase-2 Protein Expression in Intrauterine Tissues and Fetal Tissues of a Sheep Model of Preterm Labor

Meloxicam (MEL), a cyclooxygenase (COX)-2 inhibitor, decreases prostaglandin production and blocks preterm labor in sheep. The objective of this study is to investigate MEL dosage regimens on COX-1, COX-2, and prostaglandin dehydrogenase (PGDH) expression in ovine intrauterine and fetal tissues. Animals in preterm labor received maternal infusions of saline or MEL at maintained high or graded doses (study 1) or acute graded doses (study 2). MEL blocked preterm labor. In study 1, MEL decreased COX-2 expression in the endometrium, myometrium, and amnion but not placenta or fetal tissues. In study 2, COX-2 expression was unchanged. COX-1/PGDH expression was unaffected. While MEL is an effective tocolytic, reductions in COX-2 protein occurred only with maintained MEL exposure. MEL effects are tissue specific and do not affect COX-1 or PGDH expression. Maternal MEL does not affect fetal COX expression in the sheep, possibly contributing to its lack of fetal side effects.

Polycystic ovary syndrome and its developmental origins

The prenatal testosterone (T)-treated adult female rhesus monkey is one animal model of polycystic ovary syndrome (PCOS) in women, with early prenatal T excess programming a permanent PCOS-like phenotype characterized by luteinizing hormone (LH) hypersecretion from reduced hypothalamic sensitivity to steroid negative feedback and relative insulin excess from increased abdominal adiposity. These combined reproductive and metabolic abnormalities are associated with ovarian hyperandrogenism and follicular arrest in adulthood, as well as premature follicle differentiation and impaired embryo development during gonadotropin therapy for in vitro fertilization (IVF). A second animal model for PCOS, the prenatal T-treated sheep also is characterized by LH hypersecretion from reduced hypothalamic sensitivity to steroid negative feedback, persistent follicles and insulin resistance, but also is associated with intrauterine growth retardation and compensatory growth after birth. The ability of prenatal T excess in both species to alter the developmental trajectory of multiple organ systems in utero provides evidence that the hormonal environment of intrauterine life programs target tissue differentiation, raising the possibility that T excess in human fetal development promotes PCOS in adulthood. Such a hypothesis must include data from clinical studies of PCOS women to clarify the homology between these PCOS-like animal models and PCOS per se in reproductive and metabolic function. Future studies should develop new clinical strategies that improve pregnancy outcome and minimize pregnancy loss in women with disorders of insulin action, including PCOS, obesity and diabetes mellitus as well as minimize transgenerational susceptibility to adult PCOS and its metabolic derangements in male close relatives.

Separate and synergistic effect of progesterone and estradiol on induction of annexin 2 and its interaction protein p11 in pregnant sheep myometrium

To search for myometrial candidate genes regulated by progesterone, we isolated annexin 2 cDNA by subtractive hybridization and cloning. We also examined the effect of estradiol and/or progesterone, individually or combined, on expressions of annexin 2 and its ligand protein, p11 in pregnant sheep intrauterine tissues. Annexin 2 is a Ca(2+)-dependent phospholipid-binding protein which interacts with p11 to form a bivalent heterotetramer. The heterotetramer was indicated in the production of prostaglandins through the regulation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Thus, annexin 2 and p11 could be the important players in Ca(2+) signaling and prostaglandin production in uterine smooth muscles. Twenty-two ewes were treated with vehicle (n = 6), or 5 mg estradiol administered intramuscularly twice a day for 2 days (n = 6), or 100 mg progesterone administered intramuscularly twice a day for 14 days (n = 5), or estradiol plus progesterone with 100 mg progesterone administered intramuscularly twice a day for 10 days (n = 5) and then with vehicle for 2 days followed by estradiol for 2 days (5 mg administered intramuscularly twice a day). At 121 days of gestation age (dGA), endometrium, myometrium, placenta, and cervix were obtained under halothane anesthesia. Subtractive hybridization was used to isolate differentially expressed mRNAs from myometrial tissues treated with progesterone, which was confirmed by western blot at protein level. Annexin 2 and p11 interaction was validated by immunoprecipitation and their expressions in intrauterine tissues were determined by western blot analysis. Data were analyzed by ANOVA. A cDNA clone from myometrial-subtracted library representing differentially expressed mRNA in the pregnant sheep myometrium with progesterone treatment was identified as annexin 2 by sequence analysis and BLASTN search. Annexin 2 was present in both cytosolic and membrane-associated fractions of intrauterine tissues. In contrast, p11 was detectable only in myometrial cytosolic fraction. Both annexin 2 and p11 significantly increased in myometrial cytosolic fraction after progesterone or progesterone plus estradiol treatment. Furthermore, estradiol and progesterone combined had a more profound effect on induction of annexin 2 than progesterone alone. Annexin 2 was immunoprecipitated specifically by p11 antibody from the myometrium. This study indicates that progesterone may play an important role in the control of myometrial contractility by modifying protein expression associated with Ca2+ and prostaglandin signaling systems during pregnancy. Enhanced expression of myometrial annexin 2 in progesterone plus estradiol group supports our hypothesis that estrogen's stimulation is optimized by progesterone's priming in the pregnant sheep uterus.

Quantitative profiling of epoxyeicosatrienoic, hydroxyeicosatetraenoic, and dihydroxyeicosatetraenoic acids in human intrauterine tissues using liquid chromatography/electrospray ionization tandem mass spectrometry

A reversed-phase liquid chromatography negative ion electrospray tandem mass spectrometry (LC/ESI–MS/MS) method was developed and validated to quantify a range of physiologically relevant eicosanoids, including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs); 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs), and 5,6-, 8,15-, and 12,20-dihydroxyeicosatetraenoic acids (DiHETEs) in human intrauterine tissues. A solid-phase extraction method was employed to extract the eicosanoids, and gradient LC separation was performed on a Kromasil C 18 column. Mass spectrometric detection was performed by multiple reaction monitoring over a 31-min run time. The calibration curves were linear over the range of 4–400 pmol/g tissue, and the intra- and interday precision and accuracy were within a coefficient of variation of 2.0 to 27.4% and 4.6 to 17.9%, respectively. The lower limit of quantitation was 1.0 pmol/g tissue. The method was applied successfully to the characterization and quantitation of eicosanoids in the different compartments of human intrauterine tissues. Our results demonstrate significantly greater amounts of HETEs than of either the EETs or DiHETEs ( P < 0.001), irrespective of tissue type. Specifically, the metabolite 12-HETE was significantly more abundant ( P < 0.001) than all other HETEs. Of the EET metabolites, 5,6-EET predominated ( P < 0.001). A significant negative correlation between EETs and HETEs for all tissues ( ρ = −0.390, P < 0.001) was identified, implying a biological feedback mechanism between these two arachidonate metabolite classes.

The role of CRH receptors and their agonists in myometrial contractility and quiescence during pregnancy and labour

The mechanism of human labor remains a scientific enigma. Corticotropin-releasing hormone (CRH), a hypothalamic peptide that controls the response of the body to stress and which is also produced by the placenta and intrauterine tissues during pregnancy is potentially involved in the onset of labor. CRH is part of a family of mammalian peptides that includes the urocortins (UCNs), which are also expressed by the placenta and intrauterine tissues. During human pregnancy, CRH appears to target multiple feto-maternal tissues, including the myometrium, implicating CRH in the regulation of the transition from relaxation to active uterine contractions. The myometrial actions of CRH are mediated via a wide network of specific G-protein coupled membrane-bound receptors. These receptors have various functional properties, depending on the receptor subtype, the ability of agonists to activate specific signalling cascades and the stage of pregnancy. In addition, their function is dependent upon other intracellular signals via communication between signalling cascades, suggesting potential multiple roles of CRH and other CRH-like peptides during pregnancy and labor. This review will provide the current concepts about the role of CRH and UCNs and their myometrial receptors during pregnancy, labor and delivery.

Prostaglandin F2α and Its Receptor as Activators of Human Decidua

Prostaglandins produced by the intrauterine tissues of both mother and fetus (myometrium, decidua, placenta, chorion, and amnion) are involved with all of the physiologies of parturition (membrane rupture, cervical dilatation, myometrial contractility, placental separation, and uterine involution). For parturition to occur, however, the intrauterine tissues need to first be activated to prepare for the work of labor, then stimulated to initiate labor. Prostaglandins normally are considered to be stimulators of the physiologies of labor. This review presents evidence that one prostaglandin, PGF2alpha, and its receptor, FP, are also activators, especially of the decidua. Stimulated by cytokines, the decidual synthesis of PGF2alpha and the expression of FP lead to increased matrix metalloproteinase activity, further enhancement of cytokine activity, increased decidual oxytocin and oxytocin receptor expression, decreased progesterone responsiveness, and possibly, enhanced expression of vascular endothelial growth factor. These collective actions prepare the decidua for its role in parturition.

The Pattern of Glucocorticoid and Estrogen Receptors May Explain Differences in Steroid Dependency of Intrauterine Prostaglandin Production at Parturition in Sheep

We have recently described two distinct pathways of intrauterine prostaglandin (PG) synthesis: a cortisol-dependent/estradiol-independent mechanism within trophoblast tissue leading to elevations in fetal plasma PGE2, and an estradiol-dependent mechanism within maternal endometrium that leads to increased maternal plasma PGF2(2alpha). We hypothesized that the differential effects of cortisol and estradiol on intrauterine PGH synthase-II (PGHS-II) expression and PG production may be because of the tissue specific expression of the glucocorticoid and estradiol receptors (GR and ER, respectively) within the intrauterine tissues. In addition, we suggest that these two pathways of PG production are linked through the expression of P450(C17hydroxylase) (P450(C17)) and subsequent increase in placental estradiol synthesis. To test the hypotheses, we infused singleton, chronically catheterized fetal sheep beginning at day 125 of gestation (term 147 to 150 days) with (1) cortisol (0.45 mg/mL; n = 5); (2) cortisol and 4-hydroxyandrostenedione, a P450(aromatase) inhibitor (4-OHA: 1.44 mg/h; n = 5); (3) saline (n = 5); or (4) saline and 4-OHA (n = 5). PGHS-II, ER alpha, ER beta, and GR alpha were localized using immunohistochemistry. ER alpha, ER beta, P450(C17), and GR alpha protein expressions were determined by Western blot analysis. Data were analyzed by analysis of variance (ANOVA) (P < or =.05). Fetal cortisol infusion in the presence or absence of a rise in placental estrogen synthesis increased placental expression of GR alpha; both PGHS-II and GR alpha localized to the uninucleate trophoblast cells of the placentome and were excluded from the maternal stroma and binucleate cells. Both forms of ER were excluded from the trophoblast tissue of the placentome. ER alpha, ER beta, and PGHS-II showed a similar pattern of distribution within the luminal epithelium of the endometrium; there were no alterations in the level of the ER in the presence of cortisol +/- 4-OHA. Placental P450(C17) protein expression was increased in the presence of a rise in fetal cortisol independent of changes in placental estrogen synthesis. We concluded that the differential effects of cortisol and estradiol on intrauterine PGHS-II expression and PG production may be due to the tissue-specific expression of the GR and ER within the intrauterine tissues. Glucocorticoid effects on trophoblast PG production may be mediated in a positive feed-forward manner. We further suggest that either cortisol or a cortisol-stimulated intermediate, like PGE2, increased P450(C17) expression, leading to a rise in placental estradiol synthesis and triggering maternal intrauterine tissue PG production.

Diabetic Pregnancies: The Challenge of Developing in a Pro-Inflammatory Environment

The maternal diabetic environment alters the embryo and the feto-placental development. The results of these alterations are: increased embryo resorption and malformation rates, placental dysfunction, fetal alterations that lead to increased neonatal morbidity and mortality rates, and also diseases that will be evident later in the adult life of the newborn. The etiology of these many maternal diabetes-induced complications are not yet understood in full. In this review the role of maternal diabetes as an inductor of a pro-inflammatory environment that impairs embryo and placental development is discussed. An overproduction of pro-inflammatory agents is found in the uterus during implantation and the developing embryo and placenta from experimental models of diabetes, as well as in placenta from diabetic women. In these tissues there are increases in reactive oxygen species, pro-inflammatory cytokines and prostaglandins, nitric oxide and peroxynitrites. These pro-inflammatory agents lead to the intrauterine activation of matrix metalloproteinases, proteases involved in remodeling the extracellular matrix during implantation and feto-placental development. Many of these pro-inflammatory agents have overlapping mechanisms of action and cross regulatory pathways that propagate the inflammatory processes. Antioxidants, PPARgamma activators, and NF-kappaB inhibitors are able to reduce the concentrations of these agents in intrauterine gestational tissues. This article reviews the current understanding of maternal diabetes-induced changes in pro-inflammatory and anti-inflammatory pathways that affect the embryo and placental development in maternal diabetes, and stresses the need of a strict maternal control of the pathology to prevent deleterious consequences in the offspring.

Phorbol ester treatment of human myometrial cells suppresses expression of oxytocin receptor through a mechanism that does not involve activator protein-1

Oxytocin (OT) is a potent uterine agonist. Its receptor (OTR) is a G protein-coupled receptor that is downregulated by prolonged exposure to OT. We hypothesized that activation of PKC mediated this OT-induced decrease in OTR expression. Diminished PKC activity in late pregnancy could underlie the increased expression of uterine OTR preceding labor onset. Using cell cultures of transformed human uterine myocytes, we determined the effects of PKC agonists and antagonists on the expression of OTR. We also explored the effects of overexpression of activator protein-1 (AP-1, a mediator of many PKC- and phorbol ester-induced effects) using adenoviral expression vectors for the AP-1 subunits c-Jun and c-Fos. Stimulation of PKC using the phorbol ester 12-O-tetradecanoylphorbol 13-acetate caused a rapid, significant (P < or = 0.05) increase in c-Jun and c-Fos concentrations but a significant decrease in mRNA for OTR within 6 h followed by a significant decrease in OT binding by 24 h. Adenoviral infection of the cells with expression vectors for c-Jun and c-Fos increased the AP-1 subunits but had no effect on OTR expression. Furthermore, there were no changes in c-Fos or c-Jun levels in human intrauterine tissues around the time of labor onset, as measured by Western analyses. We conclude that phorbol ester treatment decreases OTR expression, likely through a mechanism that does not involve AP-1.

Regulation of Membrane-Associated Prostaglandin E2 Synthase 1 in Pregnant Sheep Intrauterine Tissues by Glucocorticoid and Estradiol

This study was designed to determine the regulatory effect of glucocorticoid and estradiol on expression of ovine intrauterine membrane-associated prostaglandin E(2) synthase 1 (mPTGES1) in late gestation and at labor. For gestational and labor groups, 16 pregnant ewes from 95-147 d gestational age (dGA) and four pregnant ewes at spontaneous term labor were used. The fetal glucocorticoid group, 14 pregnant ewes at 123-125 dGA with fetuses, was divided into the following groups: after sham adrenalectomy (n = 5), adrenalectomy (n = 4), and adrenalectomy with fetal cortisol replacement to late gestation levels (n = 5). For the maternal glucocorticoid group, nine pregnant ewes were treated with saline (n = 4) and three courses of maternal dexamethasone (n = 5). For the estradiol group, 10 pregnant ewes at 119-121 dGA were treated with sesame oil (n = 5) or estradiol (n = 5) to produce labor levels of estradiol in maternal plasma. Endometrial, myometrial, and placental mRNA and proteins were analyzed by Northern and Western blot and immunocytochemistry for mPTGES1. Data were analyzed by Student's t test and ANOVA. There was a significant increase of placental mPTGES1 in late gestation. Glucocorticoids, given to the mother or fetus, significantly stimulated mPTGES1 in placenta. mPTGES1 was elevated only in the endometrium during spontaneous term labor and after estradiol treatment. The mPTGES1 was localized in the myometrial smooth muscle cells, endometrial stromal cells, and placental trophoblast cells. Our study suggested that increased expression of placental mPTGES1 throughout late gestation might result from the increased fetal and maternal circulating glucocorticoids, whereas elevated maternal plasma estradiol concentration might be responsible for the induced mPTGES1 expression in the endometrium during labor.

Macrophage migration inhibitory factor expression in the intrauterine endometrium of women with endometriosis varies with disease stage, infertility status, and pelvic pain

To evaluate the concentrations of macrophage migration inhibitory factor (MIF) in the eutopic endometrial tissue of women with and without endometriosis. Retrospective study using ELISA to measure MIF concentrations in total endometrial tissue proteins extracts. Gynecology clinic and human reproduction research laboratory. Forty-five women with endometriosis and 25 normal women. Endometrial biopsies were obtained a few days before laparoscopy. Concentrations of MIF in tissue protein extracts. Levels of MIF were significantly higher in women with endometriosis, increased with disease stage, and were cycle phase dependent. Of note is the significant increase in MIF levels occurring in the midsecretory phase in women with endometriosis as compared with controls, particularly in infertile patients, as well as in the late secretory phase preceding menstruation. Furthermore, MIF levels seemed to be particularly elevated in women with endometriosis who were infertile and who suffered from pelvic pain. This study showed a significant increase in MIF concentrations in the intrauterine endometrial tissue of women with endometriosis, occurring at specific phases of the menstrual cycle, a relationship between MIF concentrations and disease stage, and a possible role for this factor in endometriosis-associated pain and infertility.

Enhancement of Cortisol-Induced 11β-Hydroxysteroid dehydrogenase Type 1 Expression by Interleukin 1β in Cultured Human Chorionic Trophoblast Cells

Chorion is the most abundant site of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) expression within intrauterine tissues. It is important to study the regulation of 11beta-HSD1 expression in the chorion in terms of local cortisol production during pregnancy. Using real-time PCR and enzyme activity assay, we found that cortisol (1 mum) and IL-1beta (10 ng/ml) for 24 h significantly increased 11beta-HSD1 mRNA expression and reductase activity in cultured human chorionic trophoblasts. A further significant increase of 11beta-HSD1 mRNA expression and reductase activity was observed with cotreatment of cortisol and IL-1beta. To explore the mechanism of induction, 11beta-HSD1 promoter was cloned into pGL3 plasmid expressing a luciferase reporter gene. By transfecting the constructed vector into WISH cells, an amnion-derived cell line, we found that cortisol (1 microM) or IL-1beta (10 ng/ml) significantly increased reporter gene expression. Likewise, an additional increase in reporter gene expression was observed with cotreatment of cortisol and IL-beta. To explore the physiological significance of 11beta-HSD1 induction in the chorion, we studied the effect of cortisol on cytosolic phospholipase A(2) and cyclooxygenase 2 expression. We found that treatment of chorionic trophoblast cells with cortisol (1 microM) induced both cytosolic phospholipase A(2) and cyclooxygenase 2 mRNA expression. We conclude that cortisol up-regulates 11beta-HSD1 expression through induction of promoter activity, and the effect was enhanced by IL-1beta, suggesting that more biologically active glucocorticoids could be generated in the fetal membranes in the presence of infection, which may consequently feed forward in up-regulation of prostaglandin synthesis.

Maternal IL-10 and IL-6 are elevated in response to localized intrauterine inflammation

INTRAUTERINE INFLAMMATION MICHAL ELOVITZ, CONJEEVARAM MRINALINI, University of Pennsylvania, Philadelphia, Pennsylvania, University of Pennsylvania, Obstetrics and Gynecology, Philadelphia, Pennsylvania OBJECTIVE: Preterm birth is associated with a pro-inflammatory response in maternal tissues. Using a mouse model of localized intrauterine inflammation, we have demonstrated that, just prior to parturition, there is a potent TH1/TH2 response in the uterus and cervix. These studies were performed to elucidate which of these cytokines may play a primary role in activating an inflammatory response in the uterus and whether cytokine levels in the mother could predict the presence of a localized inflammatory state. STUDY DESIGN: On day 15 of gestation, CD-1 mice were randomized to intrauterine LPS or saline. At 1, 3, and 6 hours after intrauterine infusion, maternal serum, uterine and cervical tissues were collected. Real-time PCR was performed to determine message expression of IL-1b, TNF-a, IL-6, L-10, and IL-13 in the uterus and cervix. ELISAs were performed to correlate uterine mRNA and protein expression as well as determine cytokine levels in maternal serum. Statistical analysis using ANOVA and pairwise comparison was performed. RESULTS: TH1 and TH2 cytokine mRNA expression was significantly increased in the uterus and cervix rapidly after intrauterine infusion of LPS. Increased protein expression of IL-1, TNF, and IL-10 in the uterus was evident at 3 hrs. TNF and IL-1b levels in maternal serumwere not significantly elevated. In contrast, 3 hrs after intrauterine LPS, IL-10 and IL-6 were increased 18 and 6-fold respectively over controls. By 6 hrs, systemic IL-10 levels were still significantly elevated from controls but on a decreased compared to the 3 hr levels while IL-6 levels continued to increase and were O100-fold elevated compared to controls. CONCLUSION: Despite a potent proinflammatory response in the uterus and cervix in response to localized intrauterine inflammation, systemic levels of IL-1b and TNF-a are not reliable predictors of this inflammatory state. In contrast, elevated maternal IL-10 levels may be an early indicator of intrauterine inflammation and serial systemic IL-6 levels may predict localized inflammatory response in the uterus.

Unique Suppression of Prostaglandin H Synthase-2 Expression by Inhibition of Histone Deacetylation, Specifically in Human Amnion but Not Adjacent Choriodecidua

The key molecular regulatory mechanisms that govern and coordinate the molecular alterations that underpin the process of human labor remain incompletely understood although enhanced intrauterine prostaglandin production is known to be requisite. Studies from cancer tissues have indicated that at least one key enzyme of prostaglandin biosynthesis can have its activity severely reduced by increased histone deacetylation and enhanced DNA methylation status. We have advanced the hypothesis that similar regulation may occur in intrauterine tissues during pregnancy to prevent inadvertent activation of this powerful initiating signal by dampening responses to premature activation by agents such as cytokines. Our studies have shown that responsiveness of amnion, a key intrauterine tissue, to interleukin-1beta is abrogated by inhibition of histone deacetylation, whereas PGDH amounts were increased basally. The findings do integrate well with others concerning progesterone (inhibitory) actions such that a decrease in the level of histone acetylation in human gestational tissues near term might herald a coordinated series of events that all result in a positive drive for parturition. Hence, a new level of regulatory action and potential therapeutic targets for pathologies such as preterm labor can flow from these findings.

The Promise of Prostaglandins: Have They Fulfilled Their Potential as Therapeutic Targets for the Delay of Preterm Birth?

The elucidation some 30 years ago by Sir Mont Liggins that the activation of the hypothalamic-pituitary-adrenal-placental axis in fetal sheep led to elevated maternal prostaglandin (PG) concentrations and the initiation of labor provided hope that targeting PG synthesis or action would lead to effective tocolysis and lowering of the human preterm birth rate. This was the "promise of PGs." Although early trials showed that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit PG H synthase (PGHS), delayed preterm birth by 48 hours, other trials revealed an association between NSAIDs and adverse fetal effects, including oligohydramnios, patent ductus arteriosus, necrotizing enterocolitis, intraventricular hemorrhage, and persistent pulmonary hypertension of the newborn (PPHN). Hope was revived when studies in the mid 1990s demonstrated that much of the PGs synthesized by intrauterine tissues at preterm labor were derived from the inducible isoenzyme PGHS-2. Unfortunately, administration of specific PGHS-2 inhibitors led to the same adverse fetal effects displayed by the mixed PGHS-1 and -2 NSAIDs, causing interest in the promise of PGs to wane. This led to the development of new strategies for specific PG inhibition or antagonism. One of these is the application of a specific PGF2alpha receptor blocker, Theratechnologies (THG)113.31. THG113.31 decreases the in vitro contractile activity of mouse, sheep, and human myometrium in response to exogenous PGF2alpha, delays lipopolysaccharide (LPS)-induced preterm birth in mice, and lowers uterine electromyographic activity and delays preterm birth in sheep administered RU486. There have been no observable maternal or fetal side effects with its use. By developing new strategies based on other therapeutic targets, the promise of PGs may once again offer hope for delaying preterm birth.

Prostaglandin E 2 Inhibits Proliferation and Migration of HTR-8/SVneo Cells, a Human Trophoblast-derived Cell Line

Normal placentation requires a highly coordinated control of proliferation, migration and invasiveness of extravillous trophoblast cells. Since prostaglandin E2 is a major prostanoid synthesized by intrauterine tissues and highly involved in pregnancy homeostasis, we examined the possibility that it modulates extravillous trophoblast cell functions. Here, we report the presence of mRNAs for prostaglandin E2 EP2 and EP4 receptor isoforms and of proteins in both first-trimester human chorionic villi and in the human trophoblast-derived HTR-8/SVneo cells. Moreover we found that: (i) this cell line releases prostaglandin E2 and the output is enhanced by interleukin-1beta; (ii) the prostanoid consistently inhibits serum- or epidermal growth factor-induced cell proliferation and also migration. An involvement of cAMP in the prostaglandin E2 antiproliferative action is suggested by the observation that the prostanoid greatly enhances cAMP level in HTR-8/SVneo cells and that forskolin inhibits cell proliferation; moreover the administration of prostaglandin E2 plus forskolin, a condition which evokes a synergistic enhancement of cAMP, induces a major impairment of cell growth. Provided that our data are applicable to the trophoblast tissue in vivo, we suggest that prostaglandin E2 exerts an important control on extravillous trophoblast cell functions, preventing an excessive proliferation and migration.