Sex-specific and lasting effects of a single course of antenatal betamethasone treatment on human placental 11ß-HSD2.

INTRODUCTION: We have previously shown that even a single course of antenatal betamethasone (BET) as an inductor for lung maturity reduces birth weight and head circumference. Moreover, animal studies link BET administration to alterations of the hypothalamic-pituitary-adrenal-gland-axis (HPA). The unhindered development of the fetal HPA axis is dependent on the function and activity of 11ß-hydroxysteroiddehydrogenase type 2 (11ß-HSD2), a transplacental cortisol barrier. Therefore, we investigated the effects of BET on this transplacental barrier and fetal growth. METHODS: Pregnant women treated with a single course of BET between 23 + 5 to 34 + 0 weeks of gestation were compared to gestational-age-matched controls. Placental size and neonatal anthropometrics were taken. Cortisol and ACTH levels were measured in maternal and umbilical cord blood samples. Placental 11ß-hydroxysteroiddehydrogenase type 1 (11ß-HSD1) protein levels and 11ß-HSD2 protein and activity levels were determined. Parameters were analyzed independent of sex, and in subgroups divided by gender and gestational age. RESULTS: In term born females, BET administration was associated with reduced head circumference and decreased 11ß-HSD2 protein levels and enzyme activity. Males treated with BET, especially those born prematurely, showed increased 11ß-HSD2 protein levels. CONCLUSION: A single course of BET alters placental glucocorticoid metabolism in a sex-specific manner. Decreased 11ß-HSD2 levels in term born females may lead to an increased placental transfer of maternal cortisol and therefore result in a reduced head circumference and a higher risk for altered stress response in adulthood. Further research is needed to conclude the significance of increased 11ß-HSD2 levels in males.

Effects of maternal dexamethasone treatment early in pregnancy on glucocorticoid receptors in the ovine placenta.

The effects of endogenous cortisol on binucleate cells (BNCs), which promote fetal growth, may be mediated by glucocorticoid receptors (GRs), and exposure to dexamethasone (DEX) in early pregnancy stages of placental development might modify this response. In this article, we have investigated the expression of GR as a determinant of these responses. Pregnant ewes carrying singleton fetuses (n = 119) were randomized to control (2 mL saline/ewe) or DEX-treated groups (intramuscular injections of 0.14 mg/kg ewe weight per 12 hours) at 40 to 41 days of gestation (dG). Placental tissue was collected at 50, 100, 125, and 140 dG. Total glucocorticoid receptor protein (GRt) was increased significantly by DEX at 50 and 125 dG in females only, but decreased in males at 125 dG as compared to controls. Glucocorticoid receptor α (GRα) protein was not changed after DEX treatment. Three BNC phenotypes were detected regarding GRα expression (++, +-, --), DEX increased the proportion of (++) and decreased (--) BNC at 140 dG. Effects were sex- and cell type dependent, modifying the responsiveness of the placenta to endogenous cortisol. We speculate that 3 maturational stages of BNCs exist and that the overall activity of BNCs is determined by the distribution of these 3 cell types, which may become altered through early pregnancy exposure to elevated glucocorticoids.

Growth restricting effects of a single course of antenatal betamethasone treatment and the role of human placental lactogen.

UNLABELLED: Betamethasone (BET) is a widely used treatment for women who are at high risk of preterm delivery. In sheep, BET-induced growth restriction was found to be associated with reduced placenta lactogen (PL), a key regulator of fetal growth. We therefore hypothesized that also in humans a single course of BET administration is associated with a reduction of PL, associated with a deceleration in fetal growth. OBJECTIVE: To investigate effects of a single course of antenatal BET in humans on birth weight and PL. METHODS: Women exposed to BET (2 × 12 mg; n = 44) with normally grown fetuses between 23 + 5 and 34 + 0 wks (weeks + days of gestation) who delivered between 23 + 5 to 42 + 0 wks were compared to gestational age-matched controls (n = 49). Maternal gestational blood samples were obtained before, during and after BET treatment and at the time of birth. MAIN OUTCOME MEASURES: BET effects on fetal anthropometrics, placental morphometry and placental PL-protein and maternal plasma levels. RESULTS: The mean duration of days between BET administration and birth was 52 days. BET treatment was associated with decreased birth weight (-18.2%), head circumference (-8.6%), body length (-6.0%), and placental width (-5.5%), as compared to controls. These changes were irrespective of possible maternal confounders (gestational age at birth, maternal age, maternal BMI gain during pregnancy, smoking etc.). However, neither PL-plasma levels within 48 h after BET treatment nor placental PL-protein levels and maternal plasma levels at birth were changed after BET treatment. In central regions of the placenta, BET treatment increased the circumference of syncytiotrophoblast nuclei by +4.7% and nucleus surface area by +9.4% compared to controls, but these changes were not related to placental PL-protein or maternal PL-plasma levels at birth. CONCLUSION: A single course of BET treatment was accompanied with reduced fetal growth, but this growth restricting effect was not associated with altered placental or maternal plasma PL levels. Altered expression of PL appears not to be causal for BET-induced fetal growth restriction in the human.

The impact of maternal synthetic glucocorticoid administration in late pregnancy on fetal and early neonatal hypothalamic-pituitary-adrenal axes regulatory genes is dependent upon dose and gestational age at exposure.

In this study, we determined the gene and/or protein expression of hypothalamic-pituitary-adrenal (HPA) axis regulatory molecules following synthetic glucocorticoid exposures. Pregnant sheep received intramuscular saline or betamethasone (BET) injections at 104 (BET-1), 104 and 111(BET-2) or 104, 111 and 118 (BET-3) days of gestation (dG). Samples were collected at numerous time-points between 75 dG and 12 weeks postnatal age. In the BET-3 treatment group, fetal plasma cortisol levels were lower at 145 dG than controls and gestational length was lengthened significantly. The cortisol:adrenocorticotropic hormone (ACTH) ratio in fetal plasma of control and BET-3 fetuses rose significantly between132 and 145 dG, and remained elevated in lambs at 6 and 12 weeks of age; this rise was truncated at day 145 in fetuses of BET-3 treated mothers. After BET treatment, fetal and postnatal pituitary proopiomelanocortin mRNA levels were reduced from 109 dG to 12 weeks postnatal age; pituitary prohormone convertase 1 and 2 mRNA levels were reduced at 145 dG and postnatally; hypothalamic arginine vasopressin mRNA levels were lowered at all time-points, but corticotrophin-releasing hormone mRNA levels were reduced only in postnatal lambs. Maternal BET increased late fetal and/or postnatal adrenal mRNA levels of ACTH receptor and 3ß hydroxysteroid dehydrogenase but decreased steroidogenic acute regulatory protein and P450 17-α hydroxylase. The altered mRNA levels of key HPA axis regulatory proteins after maternal BET injections suggests processes that may subserve long-term changes in HPA activity in later life after prenatal exposure to synthetic glucocorticoids.

Effects of glucocorticoid treatment given in early or late gestation on growth and development in sheep.

Antenatal corticosteroids are used to augment fetal lung maturity in human pregnancy. Dexamethasone (DEX) is also used to treat congenital adrenal hyperplasia of the fetus in early pregnancy. We previously reported effects of synthetic corticosteroids given to sheep in early or late gestation on pregnancy length and fetal cortisol levels and glucocorticoids alter plasma insulin-like growth factor (IGF) and insulin-like growth factor binding protein (IGFBP) concentrations in late pregnancy and reduce fetal weight. The effects of administering DEX in early pregnancy on fetal organ weights and betamethasone (BET) given in late gestation on weights of fetal brain regions or organ development have not been reported. We hypothesized that BET or DEX administration at either stage of pregnancy would have deleterious effects on fetal development and associated hormones. In early pregnancy, DEX was administered as four injections at 12-hourly intervals over 48 h commencing at 40-42 days of gestation (dG). There was no consistent effect on fetal weight, or individual fetal organ weights, except in females at 7 months postnatal age. When BET was administered at 104, 111 and 118 dG, the previously reported reduction in total fetal weight was associated with significant reductions in weights of fetal brain, cerebellum, heart, kidney and liver. Fetal plasma insulin, leptin and triiodothyronine were also reduced at different times in fetal and postnatal life. We conclude that at the amounts given, the sheep fetus is sensitive to maternal administration of synthetic glucocorticoid in late gestation, with effects on growth and metabolic hormones that may persist into postnatal life.

Differential appearance of placentomes and expression of prostaglandin H synthase type 2 in placentome subtypes after betamethasone treatment of sheep late in gestation.

UNLABELLED: Inappropriate fetal exposure to maternal glucocorticoid (GC) has been proposed as a mechanism for fetal programming where the effects of GC may be mediated by the placenta. However, the consequences of maternal GC on placental morphology and enzyme expression are unclear. OBJECTIVES: We used betamethasone (BET) to determine effects on placentome subtype distribution and expression of prostaglandin H synthase type 2 (PGHS-2) enzyme. METHODS: Pregnant sheep carrying male fetuses were randomized to receive injections of saline (n = 30) or one (104 days of gestation, (dG); n = 6), two (104, 111 dG; n = 6) or three (104, 111, 118 dG; n = 11) doses of BET (0.5 mg/kg). Placental tissue was collected prior to (75, 84, 101 dG), during (109, 116 dG) and after BET (122, 132, 146 dG). RESULTS: Total number of placentomes was not different between gestational ages. A- and B-subtypes were most affected by prenatal BET exposure; numbers of A-subtypes were increased and numbers of B-subtypes were decreased compared to controls at 116 dG. At term numbers of A-subtypes were lower after BET, but the weight range distribution was similar to controls. In controls, placental PGHS-2 protein levels increased with gestational age and PGHS-2 localized primarily to uninuclear trophoblast cells. After BET, PGHS-2 protein in C-subtypes at term was significantly increased compared to A-subtypes. CONCLUSIONS: Maternal BET treatment in late gestation affects the proportions of placentome subtypes and their differential expression of PGHS-2. Our data do not support previous hypotheses that A-subtypes develop into B-, C- and D-subtypes over the course of gestation.

Dexamethasone stimulates placental system A transport and trophoblast differentiation in term villous explants.

Synthetic glucocorticoids (GCs) are given to women with threatened preterm labour but their administration has been linked to reduced infant birthweight. The underlying mechanisms are unknown, but impaired placental development and/or function has been implicated. The activity of the system A amino acid transporter is decreased in placentas from pregnancies complicated by fetal growth restriction. Whether GCs adversely affect placental amino acid transport is unknown. The objective of this study was to determine the regulatory effects of GCs on system A transport using a human in vitro placental explant model. Term explants (n=7) were treated with dexamethasone (DEX 10(-8)M or 10(-6)M) or vehicle for 48 h. System A activity was measured by the uptake of (14)C-N-methylated aminoisobutyric acid by explants. Explants were also processed for electron microscopy (EM), immunohistochemistry, and qRT-PCR. Lactate dehydrogenase (LDH), human chorionic gonadotropin (hCG) and human placental lactogen (hPL) release into the culture medium was measured. DEX (10(-6)M) stimulated system A activity compared to vehicle (p<0.05). System A transporter proteins were localized to the newly regenerating syncytiotrophoblast layer, but mRNA levels were unchanged with DEX treatment. DEX did not adversely affect explant viability as determined by reduced LDH release (p<0.05). DEX treatment was associated with morphological (accelerated apical microvilli formation, nuclear maturation, and increased cell organelle number) and functional (elevated hCG secretion, increased 11beta-HSD2 mRNA expression and reduced cytotrophoblast proliferation (p<0.05 for all)) markers of syncytiotrophoblast differentiation. These findings suggest that DEX stimulates system A activity and promotes syncytiotrophoblast differentiation and maturation.

Innate immune defences in the human uterus during pregnancy.

The prevention of uterine infection is critical to appropriate fetal development and term delivery. The innate immune system is one component of the uterine environment and has a role in prevention of uterine infection. Natural antimicrobials are innate immune molecules with anti-bacterial, anti-viral and anti-fungal activity. We discuss two groups of natural antimicrobials in relation to pregnancy: (i) the defensins; and (ii) the whey acidic protein motif containing proteins, secretory leukocyte protease inhibitor (SLPI) and elafin. Human beta-defensins (HBD) 1-3 are expressed by placental and chorion trophoblast, amnion epithelium and decidua in term and preterm pregnancy. Elafin shows a similar pattern of localisation while SLPI is produced only by amnion epithelium and decidua. Evidence suggests that there is aberrant production of some natural antimicrobials in pathologic conditions of pregnancy. In preterm premature rupture of membranes (PPROM) levels of SLPI and elafin are reduced in amniotic fluid and fetal membranes, respectively. Elafin and HBD3 increase in chorioamnionitis and levels of the alpha-defensins, HNP1-3, increase in maternal plasma and amniotic fluid in women affected by microbial invasion of the uterus. In vitro culture studies have suggested a mechanism for increased production of natural antimicrobials in chorioamnionitis. Elafin, SLPI, HBD2 and 3 are all upregulated by inflammatory molecules in cells derived from gestational tissues. In summary, production of natural antimicrobials at key sites within the pregnant uterus suggests an important role in prevention of uterine infection during pregnancy and labour. Aberrant production of these molecules in PPROM and chorioamnionitis suggests that they also have a role in pathologic conditions. In particular, upregulation of these molecules by inflammatory molecules present in chorioamnionitis will ensure a robust response to infection.

Expression and localization of cellular prion and COMMD1 proteins in human placenta throughout pregnancy.

Copper is an essential trace element for successful pregnancy. However, the mechanisms by which copper is transported from maternal circulation to the fetus have not been clearly elucidated. Two proteins, cellular prion (PrP(C)) and COMMD1, are known to be responsible for prion diseases and canine copper toxicosis, respectively, and are thought to play a role in copper homeostasis. However, their placental expression and localization throughout human gestation are still unknown. In this study, we used quantitative RT-PCR, western blotting and immunohistochemistry to investigate in detail the expression and localization of PrP(C) and COMMD1 proteins in human placenta throughout pregnancy. Our results show that both proteins are expressed in human placenta. PrP(C) showed the highest mRNA and protein expression levels during the first trimester of pregnancy. PrP(C) and COMMD1 proteins are similarly localized within the placental villi. Both proteins are present in the syncytiotrophoblast, the cytotrophoblast, vascular endothelial cells and Hofbauer cells. These data offer some insights into possible roles for PrP(C) and COMMD1 within the placenta.

Expression of natural antimicrobials by human placenta and fetal membranes.

Preterm birth associated with infection is a major clinical problem. We hypothesized that this condition is associated with altered expression of natural antimicrobial molecules (beta-defensins (HBD), elafin). Therefore, we examined expression of these molecules and their regulation by proinflammatory cytokines in placentae and fetal membranes from term pregnancy. HBD1-3 and elafin were localized by immunohistochemistry in fetal membranes and placenta. Real-time quantitative PCR was used to examine mRNA expression in primary trophoblast cells treated with inflammatory molecules. HBD1-3 and elafin were immunolocalized to placental and chorion trophoblast layers of fetal membranes and placenta. Immunoreactivity was also observed in amnion epithelium and decidua. No differences were noted between samples from women who were not in labour compared to those in active labour. In in vitro cultures of primary trophoblast cells, HBD2 and elafin mRNA expression was upregulated by the proinflammatory cytokine, IL-1beta. These results suggest that the chorion and placental trophoblast layers may be key barriers to the progression of infection in the pregnant uterus. Natural antimicrobial expression may be altered in response to inflammatory mediator expression associated with the onset of labour and/or uterine infection, providing increased protection when the uterus may be particularly susceptible to infection.

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.

Fetal exposure to excess glucocorticoid is unlikely to explain the effects of periconceptional undernutrition in sheep.

Periconceptional undernutrition alters fetal growth, metabolism and endocrinology in late gestation. The underlying mechanisms remain uncertain, but fetal exposure to excess maternal glucocorticoids has been hypothesized. We investigated the effects of periconceptional undernutrition on maternal hypothalamic-pituitary-adrenal axis function and placental 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) activity. Ewes received maintenance feed (N, n= 20) or decreased feed from -60 to +30 days from mating to achieve 15% weight loss after an initial 2-day fast (UN, n= 21). Baseline plasma samples and arginine vasopressin (AVP)-corticotrophin-releasing hormone (CRH) challenges were performed on days -61, -57, -29, -1, +29, 33, and 49 from mating (day 0). Maternal adrenal and placental tissue was collected at 50 days. Baseline plasma levels of adrenocorticotrophic hormone (ACTH) and cortisol decreased in the UN group (P < 0.0001). ACTH response to AVP-CRH was greater in UN ewes during undernutrition (P= 0.03) returning to normal levels after refeeding. Cortisol response to AVP-CRH was greater in UN ewes after the initial 2-day fast, but thereafter decreased and was lower in UN ewes from mating until the end of the experiment (P= 0.007). ACTH receptor, StAR and p450c17 mRNA levels were down-regulated in adrenal tissue from UN ewes. Placental 11betaHSD2 activity was lower in UN than N ewes at 50 days (P= 0.014). Moderate periconceptional undernutrition results in decreased maternal plasma cortisol concentrations during undernutrition and after refeeding, and adrenal resistance to ACTH for at least 20 days after refeeding. Fetal exposure to excess maternal cortisol is unlikely during the period of undernutrition, but could occur later in gestation if maternal plasma cortisol levels return to normal while placental 11betaHSD2 activity remains low.

IL-1beta treatment does not co-ordinately up-regulate mPGES-1 and COX-2 mRNA expression, but results in higher degree of cellular and intracellular co-localization of their immunoreactive proteins in human placenta trophoblast cells.

PGE2 is involved in initiation and progression of labor in many species. Biosynthesis of PGE2 is mediated by cyclooxygenases (COX) and prostaglandin E synthases (PGES). mPGES-1 and COX-2 form an inducible pathway for PGE2 production in many cell systems. In this study we investigated whether mPGES-1 is involved in cytokine induced PGE2 biosynthesis in human trophoblast cells. We have evaluated the cellular and intracellular co-localization of mPGES-1 and COX-2, as well as cPGES and COX-1 in human trophoblast cells by dual immunofluorescent staining. The effect of IL-1beta on mPGES-1 and COX-2 co-localization, such as would occur with infection, and the regulatory effects of pro-inflammatory cytokines IL-1beta and TNF-alpha on transcriptional activity of mPGES-1 and COX-2 in these cells were also studied. We found that in cultured unstimulated trophoblasts, some cells expressed predominantly either mPGES-1 or COX-2, though there were cells co-expressing both enzymes. With IL-1beta treatment, mPGES-1 and COX-2 became more consistently co-localized. mPGES-1 was not transcriptionally co-induced with COX-2 by the cytokine treatment. We conclude that mPGES-1 is not involved in the inducible COX-2 mediated pathway for PGE2 biosynthesis at the transcriptional level, however, the treatment with IL-1beta results in a higher degree of co-ordination of the mPGES-1 and COX-2 protein immunolocalization, eliciting PGE2 synthesis.

Localization of prostaglandin H synthase, prostaglandin dehydrogenase, corticotropin releasing hormone and glucocorticoid receptor in rhesus monkey fetal membranes with labor and in the presence of infection.

Prostaglandins (PGs) play a central role in primate parturition by their actions on uterine contractility and on cervical ripening. Rhesus monkey placentation is hemochorial and the endocrine events surrounding parturition are qualitatively similar to human pregnancy. Although there is an increase in PG production before the onset of labor, little is known about the cellular localization of the PGH synthase (PGHS) or the 15-hydroxy PG dehydrogenase (PGDH) in the fetal membranes of nonhuman primates and whether it changes at term in spontaneous labor or during preterm labor associated with infection. Placental corticotropin releasing hormone (CRH) and the glucocorticoid receptor (GR) have also been implicated as mediators in parturition by virtue of their roles in PG production. We utilized immunohistochemical methods to localize the inducible isoform PGHS-2, PGDH, GR and CRH in rhesus monkey amnion, chorion and attached decidua. Tissues were obtained at cesarean section during late pregnancy, in spontaneous labor at term and in premature labor induced by Group B streptococcal intraamniotic infection. Specific staining for immunoreactive (ir)-PGHS-2 was observed in amnion epithelial and mesenchymal cells and to a lesser extent in chorion and decidua. In contrast, ir-PGDH was localized primarily to the extravillous trophoblast layer of chorion. GR was localized to both the cytoplasm and nucleus of amnion epithelial cells, subepithelial fibroblasts, chorion trophoblasts and in decidua. Immunostaining for CRH was found in amnion and in scattered decidual cells but was most intense in the chorion trophoblast layer. There was no demonstrable change in this overall pattern of immunostaining in association with the onset of labor at term except for a decrease in staining for ir-PGDH in chorion. Experimental Group B streptococcal chorioamnionitis resulted in preterm labor and extensive necrosis of extravillous trophoblast cells with subsequent loss of chorionic ir-PGDH and relative sparing of ir-PGHS-2 in amnion epithelium which favors the net production of PGs. The expression pattern of these effectors in the rhesus monkey fetal membranes points to a functional role of PGs and glucocorticoids in the process of term and preterm parturition which is similar to that in human pregnancy.

Synthetic glucocorticoids: antenatal administration and long-term implications.

A clear relationship between intrauterine development and later life predisposition to long-term disease is well established. Weight at birth provides a surrogate measure for fetal development and low birth weight predicts changes in most endocrine axes in adulthood. The exposure of the fetus to elevated levels of either endogenous or synthetic glucocorticoids, pre and periconceptional nutritional status and immediate postnatal development including catch-up growth all contribute substantially to the development of adult onset disease. Fetal exposure to high levels of glucocorticoids has direct clinical relevance. Synthetic glucocorticoids (betamethasone/ dexamethasone) are administered to women at risk of preterm delivery to advance fetal maturation and reduce neonatal morbidity and mortality. However, in human pregnancy, evidence suggests that fetal exposure to synthetic glucocorticoids has detrimental effects on birth outcome, childhood cognition and long-term behavior. Studies in animals have established a link between prenatal exposure to synthetic glucocorticoids and alterations in fetal development as well as changes in placental function. These developmental alterations appear to be permanent. Whether this is the case in humans awaits long-term follow-up of children enrolled in randomized controlled trials of prenatal glucocorticoid therapy. The research challenges in this field are now centered on uncovering the mechanisms by which glucocorticoids are involved in programming the fetus for its future life, and discovering ways in which the effectiveness and safety of antenatal glucocorticoids can be enhanced. The purpose of this mini-review is to provide a background into the use of antenatal synthetic corticosteroids and to highlight and summarize recently published clinical and animal-based studies.

Developmental changes in the distribution of corticosteroid-binding globulin in fetal and newborn sheep tissues.

Developmental changes in immunohistochemical localization of corticosteroid-binding globulin (CBG) in fetal and newborn sheep tissues were studied. Tissue samples have been harvested at days 63-64, 100-103, 125-128 and 142-144 of gestation or 2 postnatal days. In the liver, immunoreactive CBG (irCBG) has been identified in hepatocytes. The intensity of CBG staining was highest at 63-64th gestation days and then was lowered gradually down to negligible level in newborn lambs. Within kidney, irCBG was selectively localized to the epithelium of proximal and distal convoluted tubules. Its amount in the course of development followed a pattern similar to that in the liver. By contrast, fetal sheep lung and pancreas demonstrated noticeable rise of irCBG late in gestation. irCBG has been detected in respiratory epithelium of tertiary bronchi, bronchioles and terminal bronchioles, meanwhile alveoli and other lung tissues were CBG-immunonegative. In the pancreas, irCBG staining was associated with acinary cells, whereas Langerhans islets contained no irCBG at all examined stages of pregnancy. Developmental changes in irCBG did not follow reported triphasic profile of fetal sheep plasma CBG concentrations thereby showing the existence of independent cellular mechanisms regulating CBG level in the tissues. Peculiarities of intraorgan distribution and developmental changes in irCBG suggest that intracellular CBG may regulate bioavailable cortisol concentrations in the sheep tissues during fetal and early postnatal life.

Intramolecular interactions between the AF3 domain and the C-terminus of the human progesterone receptor are mediated through two LXXLL motifs.

The human progesterone receptor (PR) exists in two major forms, PRA and PRB, which differentially regulate gene transcription in a cell- and promoter-specific manner. The molecular mechanisms underlying this differential transcriptional activity have been attributed to the presence of a unique AF3 domain within PRB that may result in the two isoforms adopting different protein conformations. We demonstrate here that in myometrial cells, PRB exhibits strong progesterone-dependent transcriptional activity that is dependent on the presence of two LXXLL motifs within the AF3 domain. In vitro and in vivo protein interaction assays indicate that these motifs mediate the direct interaction between the AF3 domain and C-PR in a progesterone-dependent manner. Mutation of either of the LXXLL motifs or deletion of the last 30 amino acids within the C-terminus disrupts this interaction and progesterone-dependent transcriptional activity of PRB. Members of the p160 family of co-activators (such as GRIP-1) also interact with C-PR through their LXXLL motifs. However, GRIP-1 does not compete with AF3 but rather acts to synergize these two transactivation domains. Our data suggest that a failure to form an appropriate AF3-C-terminus interaction results in an inability of co-activators to induce maximal PR-dependent transactivation. The absence of an AF3 domain within PRA may account for its inability to activate progesterone-responsive genes, as well as its actions as a dominant trans-repressor.

Regulation of 15-hydroxy prostaglandin dehydrogenase by corticotrophin-releasing hormone through a calcium-dependent pathway in human chorion trophoblast cells.

Prostaglandins (PGs) play a crucial role in mediating parturition events, and their synthesis and metabolism are regulated by PG H synthase and 15-hydroxy-PG dehydrogenase (PGDH), respectively. Within the chorion tissue, it is the actions of PGDH that predominate. Throughout gestation, the fetal membranes secrete increasing amounts of CRH. We hypothesized that CRH, produced locally in the chorion, could act to modulate PGDH activity throughout gestation. To investigate this, we obtained Percoll-purified human chorion and placental trophoblast cells from uncomplicated term pregnancies and cultured them for 72 h. Activity of PGDH was assessed by incubation (4 h) with PGF(2alpha) (282 nM) and measurement of conversion to 13,14-dihydro-15-keto PG F(2alpha). Dose-response curves were constructed for the chorion cell cultures with CRH or 8-bromo-cAMP. To investigate the role of CRH and calcium, cells were treated with either astressin, a CRH antibody, BAPTA, or EGTA. CRH (0-1 micro M) had no effect on PGDH activity; however, cells treated with astressin (10 micro M), with or without exogenous CRH (1 micro M), and cells treated with a CRH antibody showed a significant decrease in PGDH activity. 8-Bromo-cAMP (0-1 mM) had no effect on 13,14-dihydro-15-keto PG F(2alpha) output in chorion trophoblast cells but significantly decreased output from placental trophoblast cells. Cells treated with either BAPTA-AM or EGTA had significantly reduced PGDH activity; and, at intermediate concentrations of chelator, exogenous CRH restored PGDH activity. We suggest that, in chorion trophoblast cells, endogenously produced CRH exerts a tonic stimulatory effect on PGDH activity and may help maintain a metabolic barrier, preventing the transfer of bioactive PGs from the chorioamnion to the myometrium.

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).