Regulation of Multidrug Resistance P-Glycoprotein in the Developing Blood-Brain Barrier: Interplay between Glucocorticoids and Cytokines.

P-glycoprotein (P-gp) encoded by Abcb1 provides protection to the developing brain from xenobiotics. P-gp in brain endothelial cells (BECs) derived from the developing brain microvasculature is up-regulated by glucocorticoids and inhibited by pro-inflammatory cytokines in vitro. However, little is known about how prenatal maternal glucocorticoid treatment can affect Abcb1/P-gp function and subsequent cytokine regulation in foetal BECs. We hypothesised that glucocorticoid exposure increases Abcb1/P-gp in the foetal brain microvasculature and enhances the sensitivity of Abcb1/P-gp in BECs to the inhibitory effects of cytokines. BECs isolated from dexamethasone- or vehicle-exposed foetal guinea pigs were cultured and treated with interleukin-1ß, interleukin-6 or tumour necrosis factor-α, and Abcb1/P-gp expression and function were assessed. Prenatal dexamethasone exposure significantly increased Abcb1/P-gp expression/activity and cytokine receptor levels in BECs of the foetal brain microvasculature. Foetal dexamethasone exposure in vivo also increased the subsequent responsiveness of BECs to pro-inflammatory cytokines in vitro. In conclusion, maternal treatment with synthetic glucocorticoids appears to prematurely mature P-gp mediated drug resistance at the foetal BBB in vivo and profoundly impact the subsequent responsiveness of P-gp to pro-inflammatory cytokines in the foetal BEC. The significance of these findings to foetal brain protection against xenobiotics and other P-gp substrates in vivo requires further elaboration. However, the results of the present study may have implications for human pregnancy and foetal brain protection, particularly in cases of preterm birth combined with infection.

ATP-binding cassette transporters in reproduction: a new frontier.

BACKGROUND: The transmembrane ATP-binding cassette (ABC) transporters actively efflux an array of clinically relevant compounds across biological barriers, and modulate biodistribution of many physiological and pharmacological factors. To date, over 48 ABC transporters have been identified and shown to be directly and indirectly involved in peri-implantation events and fetal/placental development. They efflux cholesterol, steroid hormones, vitamins, cytokines, chemokines, prostaglandins, diverse xenobiotics and environmental toxins, playing a critical role in regulating drug disposition, immunological responses and lipid trafficking, as well as preventing fetal accumulation of drugs and environmental toxins. METHODS: This review examines ABC transporters as important mediators of placental barrier functions and key reproductive processes. Expression, localization and function of all identified ABC transporters were systematically reviewed using PubMed and Google Scholar websites to identify relevant studies examining ABC transporters in reproductive tissues in physiological and pathophysiological states. Only reports written in English were incorporated with no restriction on year of publication. While a major focus has been placed on the human, extensive evidence from animal studies is utilized to describe current understanding of the regulation and function of ABC transporters relevant to human reproduction. RESULTS: ABC transporters are modulators of steroidogenesis, fertilization, implantation, nutrient transport and immunological responses, and function as 'gatekeepers' at various barrier sites (i.e. blood-testes barrier and placenta) against potentially harmful xenobiotic factors, including drugs and environmental toxins. These roles appear to be species dependent and change as a function of gestation and development. The best-described ABC transporters in reproductive tissues (primarily in the placenta) are the multidrug transporters p-glycoprotein and breast cancer-related protein, the multidrug resistance proteins 1 through 5 and the cholesterol transporters ABCA1 and ABCG1. CONCLUSIONS: The ABC transporters have various roles across multiple reproductive tissues. Knowledge of efflux direction, tissue distribution, substrate specificity and regulation of the ABC transporters in the placenta and other reproductive tissues is rapidly expanding. This will allow better understanding of the disposition of specific substrates within reproductive tissues, and facilitate development of novel treatments for reproductive disorders as well as improved approaches to protecting the developing fetus.

Effect of oxygen on multidrug resistance in term human placenta.

INTRODUCTION: The placenta contains efflux transporters, including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), that limit the passage of xenobiotics, certain hormones and nutrients from the maternal to the fetal circulation. The expression of these transporters changes with gestational age, yet the mechanisms involved remain unknown. However, the changes in P-gp and BCRP transporter expression coincide with those of oxygen tension in the placenta, and oxygen tension has been shown to modulate P-gp and BCRP expression in other tissues. The objective of this study was to investigate the effects of oxygen tension on P-gp and BCRP expression in the term human placenta. METHODS: Following equilibration in culture (96 h), term placental explants (n = 7) were cultured in 3% or 20% oxygen for 24 and 48 h. Culture medium was collected every 24 h to measure lactate dehydrogenase (LDH; explant viability) and human chorionic gonadotropin (hCG; syncytiotrophoblast function). P-gp (encoded by ABCB1) and BCRP (encoded by ABCG2) protein and mRNA, as well as VEGFA mRNA were measured using western blot and qRT-PCR. P-gp localization was determined using immunofluorescence. RESULTS: Oxygen tension had a significant effect on P-gp expression, with ABCB1/P-gp mRNA and protein levels increased in the hypoxic condition (3% O2) after 48 h (p < 0.05). VEGFA mRNA was elevated by hypoxia at both 24 and 48 h (p < 0.05). In contrast, placental ABCG2/BCRP mRNA and protein expression were stable with changes in oxygen tension. We identified profound differences in the glycosylation of P-gp between cultured and non-cultured placental tissue, with cultured explants expressing deglycosylated P-gp. CONCLUSIONS: These findings demonstrate that, at term, the expression of placental P-gp, is regulated by oxygen tension. This suggests that changes in oxygenation of the placenta in the third trimester may alter levels of placental P-gp, and in doing so alter fetal exposure to P-gp substrates, including xenobiotics and certain hormones.

Effect of oxygen on multidrug resistance in the first trimester human placenta.

INTRODUCTION: The multidrug resistance proteins, P-glycoprotein (P-gp, encoded by the ABCB1 gene) and breast cancer resistance protein (BCRP, encoded by ABCG2) are highly expressed in the first trimester placenta. These transporters protect the fetus from exposure to maternally derived toxins and xenobiotics. Since oxygen is a regulator of multidrug resistance in various tissues, we hypothesized that changes in oxygen tension alter placental ABCB1/P-gp and ABCG2/BCRP expression in the first trimester. METHODS: Placental specimens were collected from first (n = 7), second (n = 5) and term pregnancies (n = 5). First trimester placental villous explants were incubated (24 or 48 h) in different oxygen tension (3-20%). ABCB1, ABCG2 and VEGFA mRNA expression levels were assessed by RT-PCR and protein was localized by IHC. RESULTS: ABCB1 is expressed most highly in the first trimester placenta (p < 0.05), whereas ABCG2 expression does not change significantly over pregnancy. P-gp and BCRP staining is present in the syncytiotrophoblast and in cytotrophoblasts. ABCG2 mRNA is increased in hyperoxic (20%) conditions after 48 h (p < 0.05). In contrast, hypoxia (3%) did not change ABCB1 mRNA expression but significantly increased VEGFA mRNA (p < 0.05). Hypoxia resulted in increased BCRP staining in cytotrophoblasts and in the microvillous membrane of the syncytium. Whereas, hypoxia resulted in increased P-gp staining in proliferating cytotrophoblasts. CONCLUSION: We conclude that placental multidrug resistance expression, specifically ABCG2, is regulated by oxygen tension in the first trimester. It is possible that changes in placental oxygen supply are capable of altering fetal drug exposure especially during early pregnancy.

Placental drug transporters and their role in fetal protection.

The human placenta has a number of protective mechanisms that help to prevent potentially harmful compounds from entering the fetal compartment. Two important transporter proteins are phospho-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) which are mainly expressed in the syncytiotrophoblast where they actively extrude a wide range of xenobiotics. The expression profile of these transporters varies with advancing gestation. P-gp has been shown to decline near term, leaving the fetus susceptible to potentially teratogenic drugs commonly administered to pregnant women (i.e., synthetic glucocorticoids, selective serotonin reuptake inhibitors, glyburide, antiretrovirals, etc.). Drug transporter expression is regulated by a number of transcription factors, and steroid hormones present during pregnancy, such as progesterone, estrogen and corticosteroids. Drug transporter levels have also been found to be altered in pathological pregnancies (preterm, pre-eclampsia, growth restriction and infection). Genetic variation in the genes that encode these drug transporters can significantly alter transporter function and may play a significant role in determining the susceptibility of a fetus to maternally-administered therapeutic drugs. Understanding the regulation of placental drug transporters in normal and pathological pregnancies is critical to further our knowledge of fetal development, and may lead to the development of more selectively-targeted maternal and fetal drug treatments.

Effect of glucocorticoids on regulation of placental multidrug resistance phosphoglycoprotein (P-gp) in the mouse.

Previously, we and others have shown that placental Abcb1 mRNA and phosphoglycoprotein (P-gp; encoded by Abcb1 mRNA) decreases over the second half of gestation, resulting in increased accumulation of P-gp substrates in the fetal compartment. Very little is known pertaining to the regulation of placental Abcb1 mRNA and P-gp. In non-placental adult murine tissues, synthetic glucocorticoids have been shown to regulate Abcb1 (Abcb1a and Abcb1b) mRNA in an isoform and tissue-specific manner. Furthermore, given that maternal and fetal endogenous glucocorticoid levels increase dramatically in late gestation, we hypothesized that synthetic glucocorticoids down-regulate placental Abcb1 and P-gp expression, consequently decreasing placental P-gp mediated fetal protection. Pregnant FVB mice were treated with dexamethasone (0.1 mg/kg or 1 mg/kg; s.c.) or vehicle (saline) from either embryonic day (E)9.5-15.5 or E12.5-E18.5 and then injected with [3H]digoxin (i.v.) to assess placental P-gp function. Dexamethasone treatment from E12.5-E18.5 significantly up-regulated Abcb1a mRNA (1 mg/kg) and P-gp (0.1 mg/kg and 1 mg/kg) expression on E18.5; however, this did not correlate to changes in drug accumulation in the fetal compartment. Similarly, dexamethasone (1 mg/kg) treatment during mid-gestation (E9.5-E15.5) significantly increased placental Abcb1a mRNA. In conclusion, glucocorticoids exhibit complex regulation of the P-gp transport system at the level of gene transcription and translation. Dexamethasone exposure up-regulates Abcb1a mRNA and P-gp protein, particularly in late gestation. However, these changes do not appear to be reflected by changes in P-gp mediated drug transfer. While the latter is somewhat reassuring with respect to antenatal use of glucocorticoids for management of preterm labour, further studies are required to understand regulation of these important drug transporters in the placenta.

Expression and localisation of breast cancer resistance protein (BCRP) in human fetal membranes and decidua and the influence of labour at term.

Breast cancer resistance protein (BCRP) is a multidrug resistant ABC transport protein (ABCG-2). It extrudes a wide range of substrates, including many chemotherapy drugs, steroids and folate. It is present in many cancers, as well as normal tissues, in particular barrier tissues such as the blood-brain barrier, the intestine, blood vessels and the human placenta. Human fetal membranes (amnion and chorion laeve) provide the barrier between the maternal uterine environment and the fetus. In the present study, we defined the expression and localisation of BCRP mRNA and protein in human fetal membranes (amnion and chorion) and attached decidua obtained before and following labour at term. BCRP protein and mRNA was expressed in all tissues examined and the levels of expression were not altered by labour. BCRP was localised to the amnion epithelial cells, chorion trophoblast cells and decidua stromal cells, as well as the endothelial cells of maternal blood vessels in the decidua, but was absent from mesenchymal cells. In the amnion epithelium, BCRP protein was localised to the apical surface, cytoplasm and membrane between cells. In the chorion trophoblast and decidua stromal cells, BCRP protein was localised to the plasma membrane. However, in the chorion trophoblast, BCRP protein was also highly expressed in the nucleus. The level of BCRP protein in the membranes was comparable to that in the placenta. These high levels raise the possibility that this transporter plays an important role in the physiological function of the tissues.

Breast cancer resistance protein (Bcrp1/Abcg2) in mouse placenta and yolk sac: ontogeny and its regulation by progesterone.

Breast Cancer Resistance Protein (BCRP), a recently-discovered transporter belonging to ABC superfamily, is highly expressed within the labyrinth of the placenta, the primary site of exchange between the maternal and fetal circulation. It has been proposed to function as an efflux pump protecting the fetus from a wide range of xenobiotics. It has also been recently shown that the yolk sac, in addition to the placenta, may be involved in transport of certain substances to and from the fetus. We hypothesised that there are changes in placental Bcrp1 (the mouse orthologue of human BCRP) expression during pregnancy and that these correlate with changes in progesterone production that occur in late gestation. We also hypothesised that Bcrp1 is expressed in the yolk sac, and that levels change with advancing gestation. Either whole concepti, or placenta and yolk sac, were collected from pregnant mice and analysed at embryonic (E) day 9.5, 12.5, 15.5 and 18.5 (term approximately E19.5). Peak expression of Bcrp1 mRNA was detected using in situ hybridisation within the placenta at E9.5 and the yolk sac at E12.5. There was a significant decrease thereafter in both tissues (p<0.001). In contrast, expression of Bcrp1 protein as assessed by immunohistochemistry and Western immunoblots did not change significantly during gestation either in the placenta nor the yolk sac, and no sex difference in Bcrp1 protein expression in either tissue was observed at E12.5. Daily progesterone treatment starting at E14.5 and continuing until E18.5 significantly increased maternal progesterone levels, but did not elicit any changes in the Bcrp1 mRNA or Bcrp1 protein expression either in the placenta or the yolk sac. Significant expression of Bcrp1 protein in fetal tissue was evident at the end of gestation, while expression in the fetal brain endothelium was evident as early as E12.5. We suggest that the placenta and the yolk sac, both of which express Bcrp1, may limit fetal exposure to the potentially adverse effects of xenobiotics including therapeutic drugs which the mother may be exposed to during pregnancy. The significant decrease in Bcrp1 mRNA expression in both the yolk sac and the placenta from mid to late gestation may be counter-balanced by an increase in Bcrp1 expression in fetal organs involved in absorption, excretion and protection.

The effect of tobacco exposure on the fetal hypothalamic-pituitary-adrenal axis.

OBJECTIVE: Our objective was to determine if maternal smoking is associated with programming of the fetal hypothalamic-pituitary-adrenal (HPA) axis. Cigarette smoking, which induces a state of hypoxia in the fetus, may promote in utero'programming' of the HPA axis. In utero, adaptations to the HPA axis, which become maladaptive later in life, have been hypothesised to contribute to the development of adult cardiovascular disease and metabolic disorders. DESIGN: This was a prospective cohort study of term infants. POPULATION AND SETTING: The study involved 104 infants born by elective caesarean section, 21 of whom were exposed to in utero tobacco and 83 were nonexposed. METHODS: Healthy women with healthy pregnancies were recruited if they were undergoing elective caesarean section. Maternal blood was drawn for cortisol and cotinine in the morning, and the umbilical blood was drawn immediately after delivery of the baby. MAIN OUTCOME MEASURES: Umbilical arterial cortisol and adrenocorticotropin hormone (ACTH) levels. RESULTS: ACTH levels were significantly elevated in smoke-exposed infants [17 (4-22) pmol/l versus 4 (2-11) pmol/l, respectively, P= 0.005], while cortisol levels were similar [182 (130-240) nmol/l versus 192 (127-265) nmol/l, respectively, P= 0.541]. CONCLUSIONS: For the first time, it was shown that infants exposed to in utero tobacco smoke have significantly elevated ACTH levels compared with nonexposed infants. The results of this study warrant further exploration of the effect of smoking on the neonatal HPA axis as a potential set up for 'programming'.

The pattern of glucocorticoid and estrogen receptors may explain differences in steroid dependency of intrauterine prostaglandin production at parturition in sheep.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Expression of the multidrug resistance P-glycoprotein, (ABCB1 glycoprotein) in the human placenta decreases with advancing gestation.

The multidrug resistance p-glycoprotein (P-gp), encoded by the ABCB1 gene, is a plasma membrane protein that actively extrudes a wide variety of substances from cells. Preliminary studies in mice have shown that the ABCB1/P-gp can protect the fetus from a number of toxic substances. ABCB1/P-gp is expressed in the human placenta and is potentially capable of protecting the fetus from a large number of drugs and toxins, including herbicides and pesticides. The protein can also extrude various steroids including certain glucocorticoids and may therefore play an important role in regulating fetal access of glucocorticoids. The aim of the present study was to examine the expression profile and cellular localization of ABCB1/P-gp in human placenta throughout gestation. We hypothesized that there would be gestational age-related changes in the expression of the protein. ABCB1/P-gp mRNA was measured by Real-Time PCR using specific probes in tissues obtained from 6 weeks gestation to term. ABCB1/P-gp mRNA levels in placental tissue obtained at 6-10 weeks (n=5) and 24-35 weeks (n=5) were significantly higher than in tissues obtained at term (38-41 weeks gestation) by elective C-section (n=6) or following labor (n=6). The profile of ABCB1/P-gp protein levels, quantified using Western analysis, demonstrated a similar decrease with advancing gestation. At all gestational ages ABCB1/P-gp was localized by immunohistochemistry to the syncytiotrophoblast. In term tissues, it appeared to be localized to some areas of the villi and not others. Together, these data indicate that with advancing gestation there is a decrease in the level of ABCB1/P-gp in the human placenta indicating that the fetus may be more susceptible to toxic insults in the latter part of gestation. Further, the reduction in ABCB1/P-gp expression may contribute to the increased transfer of maternal cortisol to the fetus that is known to occur in late gestation.

Expression of breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout gestation and at term before and after labor.

Breast cancer resistance protein, BCRP, is a multidrug resistance protein that is highly expressed in the human placenta. In cancer tissues, this protein actively extrudes a wide variety of chemically and structurally unrelated chemotherapeutic drugs and other compounds. Studies in mice have shown that in the absence of BCRP activity in the placenta, there is a 2-fold increase in the uptake in BCRP substrates into fetus. This suggests that in the placenta, BCRP extrudes compounds that would otherwise cross the syncytiotrophoblast cells into fetal circulation. The purpose of this study was to examine the expression and localization of BCRP in the human placenta throughout gestation. Tissues from 6-13, 16-19, 24-29, 32-35, and 38-41 weeks of gestation were used. Real time RT-PCR analysis demonstrated that the mRNA levels of BCRP in the placenta do not change significantly as gestation progressed. However, Western blot analysis revealed that the protein levels increased towards the end of gestation. We demonstrated that BCRP is localized to the syncytiotrophoblast of the placenta and in some fetal blood vessels within the placenta. Tissues from the early stages of pregnancy (6-13 weeks) showed fewer BCRP positive blood vessels than term tissues (38-41 weeks).

Effects of cortisol and oestradiol on hepatic 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus.

In the late-gestation sheep, increased fetal plasma cortisol concentration and placental oestradiol (E(2)) output contribute to fetal organ maturation, in addition to the onset of parturition. Both cortisol and E(2) are believed to regulate the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which interconverts bioactive 11-hydroxy glucocorticoids and their inactive 11-keto metabolites. 11beta-HSD1, abundantly expressed in fetal liver, operates primarily as a reductase enzyme to produce bioactive cortisol and thus regulates local hepatic glucocorticoid concentrations. Cortisol acts through the glucocorticoid receptor (GR) present in the liver. In this study, we examined the effects of cortisol and E(2) on hepatic 11beta-HSD1 and GR in the liver of chronically catheterized sheep fetuses treated with saline (n=5), cortisol (1.35 mg/h; n=5), saline+4-hydroxyandrostendione, a P450 aromatase inhibitor (4-OHA; 1.44 mg/h; n=5), or cortisol+4-OHA (n=5). Cortisol infusion resulted in increased plasma concentrations of fetal cortisol and E(2); concurrent administration of 4-OHA attenuated the increase in plasma E(2) concentrations. Using immunohistochemistry, we showed that fetal hepatocytes expressed both 11beta-HSD1 and GR proteins. Cortisol treatment increased GR in both cytosol and nuclei of hepatocytes; concurrent administration of 4-OHA was associated with distinct nuclear GR staining. Western blot revealed that cortisol, in the absence of increased E(2) concentrations, significantly increased concentrations of 11beta-HSD1 (34 kDa) and GR (95 kDa) proteins. 11beta-HSD1 enzyme activity was measured in the liver microsomal fraction in the presence of [(3)H]cortisone (10(-)(6) M) or [(3)H]cortisol (10(-)(6) M) and NADPH (reductase activity) or NADP(+) (dehydrogenase activity) respectively. 11beta-HSD1 reductase activity was significantly greater in the presence of cortisol. In summary, we found that, in sheep during late gestation, cortisol increased both 11beta-HSD1 and GR in the fetal liver, and these effects were accentuated in the absence of increased E(2).

Mechanisms of term and preterm birth.

Labour at term and preterm results from activation and then stimulation of the myometrium. Activation can occur through mechanical stretch of the uterus, and by endocrine pathways resulting from increased activity of the fetal hypothalamic-pituitary-adrenal axis. In women and in experimental animals, cortisol likely contributes to increased prostaglandin production in fetal tissues through up-regulation of the type 2 prostaglandin H2, synthase-2 (PGHS-2) and down-regulation of 15-OH prostaglandin dehydrogenase. Cortisol increases expression of prostaglandin dehydrogenase in the chorion by reversing the stimulatory effect of progesterone, and may represent "progesterone withdrawal" in the primate. By competing with progesterone inhibition, cortisol also increases expression of placental corticotropin-releasing hormone. Other agents, such as pro-inflammatory cytokines, similarly up-regulate PGHS-2 and decrease expression of prostaglandin dehydrogenase. Oxytocin, produced locally within the intrauterine tissues, is also thought to be involved in parturition, and there is a marked increase in oxytocin receptor expression at term. There are thus several mechanisms by which labour at term or preterm may be initiated. These different mechanisms need to be considered in the development of strategies for the detection and management of women in preterm labour. Ongoing studies are investigating the use of oxytocin receptor antagonists, PGHS-2 inhibitors, and nitric oxide to prevent or regulate preterm labour. The presence of fibronectin in vaginal secretions, and elevated maternal serum levels of corticotropin-releasing hormone, estrogens, and cytokines have been examined as possible markers of preterm labour. However, at the present time, we do not have the ability to accurately predict or diagnose preterm labour, nor do we have specific or efficient methods to inhibit labour once it has started.

Expression of prostaglandin I(2) synthase, but not prostaglandin E synthase, changes in myometrium of women at term pregnancy.

Prostaglandins (PGs) act as potent uterotonins at the time of labor. Prostaglandin E synthase (PGES) is responsible for the formation of PGE(2), a uterotonin. PGI(2) is synthesized by the prostaglandin I synthase enzyme (PGIS) and contributes to relaxation in the lower uterine segment. We examined the expression of membrane-bound PGES and PGIS in myometrium from pregnant women during preterm and term labor. Tissues were collected from the lower uterine segment from preterm no labor, preterm labor, term no labor, and term labor patients and used for immunohistochemistry and Western blot analysis using specific antibodies. Immunoreactive (ir-) PGES and PGIS proteins were localized to the cytoplasm of myocytes of the myometrium and vascular smooth muscle cells. Ir-PGES was also detected in vascular endothelial cells. Western blot analyses revealed a predominant protein band of 180 kDa, and a second 16-kDa band for ir-PGES and 56-kDa band for ir-PGIS. There was no significant change in ir-PGES protein (180 or 16 kDa) or mRNA levels with preterm or term labor or gestational age. There was a significant decrease in PGIS mRNA and protein with advancing gestational age. We conclude that the gestational age decrease in the inhibitory PGIS is consistent with lessening of its influence in myometrium at the time of labor. The lack of change in PGES indicates that alterations at other points along the pathway of arachidonic acid metabolism may be of greater importance in affecting local changes in PGE(2).

Ontogeny and regulation of ovine placental prostaglandin E2 synthase.

Recent evidence suggests that ovine placental output of prostaglandin (PG) E2 rises through late gestation partly because of a direct effect of cortisol on PGH2 synthase 2 (PGHS-2) expression and activity within trophoblast tissue. Synthesis of PGE2 is also dependent, however, on PGE2 synthase (PGES), which converts PGH2 to PGE2. We hypothesized that PGES is expressed in the ovine placenta, and that, similar to PGHS-2, expression increases through gestation and is regulated positively by cortisol. Placental tissues from pregnant ewes in mid and late gestation, at term, and during early and active labor were analyzed to determine the gestational profile of PGES. The regulation of PGES expression was assessed in placental tissues from pregnant ewes in which intrafetal cortisol infusion was administered in late gestation, in the presence or absence of an aromatase inhibitor, to block the cortisol-stimulated rise in estradiol. Expression of PGES was analyzed by in situ hybridization, Western blot analysis, and immunohistochemistry. In the placentome, PGES localized to fetal trophoblast cells and endothelial cells in maternal blood vessels, consistent with its contribution to the rise in placental PGE2 output toward the onset of labor and with a role of PGE2 in the local regulation of uteroplacental blood flow, respectively. Expression of PGES mRNA and protein increased with gestation. However, there was no significant further change with labor or during cortisol infusion in the presence or absence of a rise in fetal plasma estradiol, in contrast to reported changes in PGHS-2. These results suggest that PGES is not coregulated with PGHS-2 in the sheep placenta at term. The progressive increase in PGES, however, likely contributes to the rise in circulating PGE2 in the fetus in late pregnancy.

Localization of nuclear factor-kappa B (NF kappa B) and inhibitory factor-kappa B (I kappa B) in human fetal membranes and decidua at term and preterm delivery.

The human fetal membranes and decidua are thought to be involved in the onset of human parturition. These tissues produce and respond to various cytokines, which may be involved in preterm labour and possibly term labour. They also show increasing production of prostaglandins (PGs) with advancing gestation and labour. The expression of PGHS-2, a rate limiting enzyme in PG synthesis, is increased in the fetal membranes at labour. The gene for PGHS-2 and many of the cytokine genes (e.g. TNFalpha, IL-1, IL-6) are stimulated by the transcription factor NF kappa B. This factor is composed of two subunits, p50 and p65, which are localized in the cytoplasm bound to I kappa B. When activated I kappa B is metabolized, and p50, p65 translocate to the nucleus to activate various genes. The purpose of the present study was to examine the tissue and cellular distribution of p65 and I kappa B in the human fetal membranes and decidua throughout gestation. Term tissues were obtained prior to labour by elective caesarean section (n=10) or following vaginal delivery (n=10) and 10 preterm tissues were obtained following labour prior to 37 weeks gestation. None of the tissues had any evidence of infection. The immunoreactive NF kappa B and I kappa B were localized in the tissues. p65 protein was found in the nucleus and cytoplasm of cells in the amnion, chorion laeve and decidua. In the amnion and chorion laeve, no changes occurred in subcellular localization with advancing gestation or term labour. However, in the decidua, there was a marked increase in the nuclear localization of i.r. p 65 in tissues obtained at term when compared with tissues delivered preterm. In the case of I kappa B, it was localized to the cytoplasm of cells in all tissues and there was an increase i.r. I kappa B in decidua at term compared to preterm but no change occurred in the amnion or chorion. The increase in nuclear localization of p65 in the decidua that occurs with advancing gestation, highlights the potential importance of this factor in the regulation of parturition related genes in this tissue.

Understanding preterm labor.

Increased uterine contractility at term and preterm results from activation and then stimulation of the myometrium. Activation can be provoked by mechanical stretch of the uterus and by an endocrine pathway resulting from increased activity of the fetal hypothalamic-pituitary-adrenal (HPA) axis. In fetal sheep, increased cortisol output during pregnancy regulates prostaglandin H synthase type 2 (PGHS2) expression in the placenta in an estrogen-independent manner, resulting in increased levels of PGE2 in the fetal circulation. Later increases in maternal uterine expresssion of PGHS2 require elevations of estrogen and lead to increased concentrations of PGF2alpha in the maternal circulation. Thus, regulation of PGHS2 at term is differentially controlled in fetal (trophoblast) and maternal (uterine epithelium) tissue. This difference may reflect expression of the glucocorticoid receptor (GR), but not estrogen receptor (ER), in placental trophoblast cells. In women, cortisol also contributes to increased PG production in fetal tissues through upregulation of PGHS2 (amnion and chorion) and downregulation of 15-OH PG dehydrogenase (chorion trophoblasts). The effect of cortisol on chorion expression of PGDH reverses a tonic stimulatory effect of progesterone, potentially through a paracrine or autocrine action. We have interpreted this interaction as a reflection of "progesterone withdrawal" in the primate, in relation to birth. Other agents, such as proinflammatory cytokines, similarly upregulate PGHS2 and decrease expression of PGDH, indicating the presence of several mechanisms by which labor at term or preterm may be initiated. These different mechanisms need to be considered in the development of strategies for the detection and management of the patient in preterm labor.