Melanie C. Audette

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.

Antenatal Dexamethasone Treatment in Midgestation Reduces System A-Mediated Transport in the Late-Gestation Murine Placenta

Clinically, approximately 30% of women who receive synthetic glucocorticoids (sGC) for risk of preterm labor carry to term. In vitro studies have shown that sGC acutely regulate the placental system A amino acid transporter, but there are no comparable data in vivo. Hence, the objective of our study was to examine the acute [embryonic day (E)15.5] and longer-term (E17.5 and E18.5) consequences of midgestation antenatal sGC [dexamethasone (DEX); 0.1 mg/kg on E13.5 and E14.5] on placental system A-mediated transfer in the mouse (measured in vivo as maternal-fetal unidirectional (14)C-methylaminoisobutyric acid transfer per gram of placenta). System A transfer and Slc38a mRNA expression significantly increased from E12.5 to E18.5 (P < 0.05), corresponding to increased fetal growth. DEX treatment had no acute effect at E15.5 or longer-term effect at E17.5 but significantly decreased system A-mediated transfer before term (E18.5; P < 0.05) in placentae of male and female fetuses. There was no effect of DEX on Slc38a gene expression. Administration of DEX in this regime had no effect on birth weight. We conclude that sGC treatment in midgestation leads to a substantial decrease in placental system A-mediated transport in late gestation, suggesting that prenatal sGC therapy may lead to a reduction in availability of neutral amino acids to the fetus if gestation persists to term.

Effects of Maternal Dexamethasone Treatment in Early Pregnancy on Pituitary-Adrenal Axis in Fetal Sheep

Fetal exposure to elevated levels of bioactive glucocorticoids early in gestation, as in suspected cases of congenital adrenal hyperplasia, may result in adverse neurological events. Fetal hypothalamic-pituitary-adrenal development and function may be involved. We investigated immediate and long-term effects of maternal dexamethasone (DEX) administration early in pregnancy on fetal growth and pituitary-adrenal activity in sheep. Pregnant ewes carrying singleton fetuses (total n = 119) were randomized to control (2 ml saline/ewe) or DEX-treated groups (im injections of 0.14 mg/kg ewe weight . 12 h) at 40-41 d gestation (dG). At 50, 100, 125, and 140 dG, fetal plasma and tissues were collected. DEX-exposed fetuses were lighter than controls at 100 dG (P < 0.05) but not at any other times. Fetal plasma ACTH levels and pituitary POMC and PC-1 mRNA levels were similar between groups. Fetal plasma cortisol levels were significantly reduced after DEX exposure in both male and female fetuses at 50 dG (P < 0.05), were similar at 100 and 125 dG, but were significantly higher than controls at 140 dG. At 140 dG, there was increased adrenal P450C(17) and 3beta-HSD mRNA in female fetuses and reduced expression of ACTH-R mRNA in males. Fetal hepatic CBG mRNA levels mimicked plasma cortisol patterns. DEX exposure reduced CBG only in males at 50 dG (P < 0.05). Placental mRNA levels of 11beta-HSD2 were increased after DEX in males (P < 0.05). Therefore, in sheep, early DEX may alter the developmental trajectory of the fetal hypothalamic-pituitary-adrenal axis, directly increasing fetal adrenal activation but not anterior pituitary function. In females, this effect may be attributed, in part, to increased fetal adrenal steroidogenic activity.

Prenatal endotoxemia and placental drug transport in the mouse: placental size-specific effects.

Lipopolysaccharide (LPS) in high doses inhibits placental multidrug resistance P-glycoprotein (P-gp - Abcb1a/b) and breast cancer resistance protein (BCRP - Abcg2). This potentially impairs fetal protection against harmful factors in the maternal circulation. However, it is unknown whether LPS exposure, at doses that mimic sub-lethal clinical infection, alters placental multidrug resistance. We hypothesized that sub-lethal (fetal) LPS exposure reduces placental P-gp activity. Acute LPS (n = 19;150 µg/kg; ip) or vehicle (n = 19) were given to C57BL/6 mice at E15.5 and E17.5. Placentas and fetal-units were collected 4 and 24 h following injection. Chronic LPS (n = 6; 5 µg/kg/day; ip) or vehicle (n = 5) were administered from E11.5–15.5 and tissues were collected 4 h after final treatment. P-gp activity was assessed by [3H]digoxin accumulation. Placental Abcb1a/b, Abcg2, interleukin-6 (Il-6), Tnf-α, Il-10 and toll-like receptor-4 (Tlr-4) mRNA were measured by qPCR. Maternal plasma IL-6 was determined. At E15.5, maternal IL-6 was elevated 4 h after single (p<0.001) and chronic (p<0.05) LPS, but levels had returned to baseline by 24 h. Placental Il-6 mRNA was also increased after acute and chronic LPS treatments (p<0.05), whereas Abcb1a/b and Abcg2 mRNA were unaffected. However, fetal [3H]digoxin accumulation was increased (p<0.05) 4 h after acute LPS, and maternal [3H]digoxin myocardial accumulation was increased (p<0.05) in mice exposed to chronic LPS treatments. There was a negative correlation between fetal [3H]digoxin accumulation and placental size (p<0.0001). Acute and chronic sub-lethal LPS exposure resulted in a robust inflammatory response in the maternal systemic circulation and placenta. Acute infection decreased placental P-gp activity in a time- and gestational age-dependent manner. Chronic LPS decreased P-gp activity in the maternal myocardium and there was a trend for fetuses with smaller placentas to accumulate more P-gp substrate than their larger counterparts. Collectively, we demonstrate that acute sub-lethal LPS exposure during pregnancy impairs fetal protection against potentially harmful xenobiotics in the maternal circulation.

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.

Synthetic Glucocorticoid Reduces Human Placental System A Transport in Women Treated With Antenatal Therapy

CONTEXT Synthetic glucocorticoids (sGCs) are routinely given to women with threatened preterm labor and have been linked to fetal growth restriction and developmental programming. Reductions in fetal growth are likely to be mediated by placental dysfunction, including altered nutrient transport. sGCs modify the system A neutral amino acid transporter in vitro, but there are no in vivo comparable data in human placenta. OBJECTIVE Because ∼ 30% of women who receive sGCs carry to term, our objective was to examine the short- and longer-term consequences of antenatal sGCs on placental system A transport. METHODS AND PATIENTS Placental tissue was collected from women treated with sGCs between 24 hours and 14 days before delivery (24h-14d), 14 days after treatment but before term (14d-term), or at term, compared with healthy term (control) deliveries to measure system A-mediated activity (Na(+)-dependent [(14)C]methylaminoisobutyric acid uptake per gram placenta) and mRNA expression. RESULTS After sGC treatment, system A activity was significantly reduced at term compared with both sGC placentas delivered 24h-14d and compared with controls. Placentae from women treated with sGCs who delivered between 14d-term also had significantly reduced system A activity compared with 24h-14d placentas. SLC38A1 and SLC38A2 mRNA expression was unaffected. However, SLC38A4 was significantly reduced by sGCs at term compared with placentas delivered between 14d-term. CONCLUSION We conclude that women who are at risk of preterm labor and receive sGCs but deliver at term have significantly reduced placental system A amino acid transporter activity. Altered placental transporter function could affect fetal growth and may contribute to developmental programming reported in both animal and clinical studies.

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‐1β, 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.

A placenta clinic approach to the diagnosis and management of fetal growth restriction

&NA; Effective detection and management of fetal growth restriction is relevant to all obstetric care providers. Models of best practice to care for these patients and their families continue to evolve. Since much of the disease burden in fetal growth restriction originates in the placenta, the concept of a multidisciplinary placenta clinic program, managed primarily within a maternal‐fetal medicine division, has gained popularity. In this context, fetal growth restriction is merely one of many placenta‐related disorders that can benefit from an interdisciplinary approach, incorporating expertise from specialist perinatal ultrasound and magnetic resonance imaging, reproductive genetics, neonatal pediatrics, internal medicine subspecialties, perinatal pathology, and nursing. The accurate diagnosis and prognosis for women with fetal growth restriction is established by comprehensive clinical review and detailed sonographic evaluation of the fetus, combined with uterine artery Doppler and morphologic assessment of the placenta. Diagnostic accuracy for placenta‐mediated fetal growth restriction may be enhanced by quantification of maternal serum biomarkers including placenta growth factor alone or combined with soluble fms‐like tyrosine kinase‐1. Uterine artery Doppler is typically abnormal in most instances of early‐onset fetal growth restriction and is associated with coexistent preeclampsia and underlying maternal vascular malperfusion pathology of the placenta. By contrast, rare but potentially more serious underlying placental diagnoses, such as massive perivillous fibrinoid deposition, chronic histiocytic intervillositis, or fetal thrombotic vasculopathy, may be associated with normal uterine artery Doppler waveforms. Despite minor variations in placental size, shape, and cord insertion, placental function remains, largely normal in the general population. Consequently, morphologic assessment of the placenta is not currently incorporated into current screening programs for placental complications. However, placental ultrasound can be diagnostic in the context of fetal growth restriction, for example in Breus’ mole and triploidy, which in turn may enhance diagnosis and management. Several examples are illustrated in our figures and supplementary videos. Recent advances in the ability of multiparameter screening and intervention programs to reduce the risk of severe preeclampsia will likely increase efforts to deliver similar improvements for women at risk of fetal growth restriction. Placental pathology is important because the underlying pathologies associated with fetal growth restriction have a wide range of recurrence risks. Rare conditions such as massive perivillous fibrinoid deposition or chronic histolytic intervillositis may recur in >50% of subsequent pregnancies. Postpartum care in a placenta‐focused program can provide effective counseling for modifiable maternal risk factors, and can assist in planning future pregnancy care based on the pathologic basis of fetal growth restriction.

Does Preeclampsia Reduce the Risk of Breast Cancer

J Obstet Gynaecol Can 2015;37(8):736–739 A the outset of their reproductive careers, today’s women face significant health challenges from two relatively common conditions, namely preeclampsia and breast cancer. Depending on the definition used, preeclampsia affects up to 5% of women in their first pregnancy,1 while today’s cohort of young women face an approximate 10% lifetime risk of breast cancer.2 An emerging body of evidence suggests an interaction between these conditions, in that women appear to have a reduced risk of breast cancer following a pregnancy that has been complicated by hypertension, including preeclampsia. Substantial efforts have been made in the past decade to understand the placental basis of transient hypertension and multi-organ dysfunction in preeclampsia, and to identify biomarkers to predict preeclampsia. This knowledge may thus prove invaluable in the fight against breast cancer.

Spatiotemporal distribution of small ubiquitin‐like modifiers during human placental development and in response to oxidative and inflammatory stress

The post‐translational modification of target proteins by SUMOylation occurs in response to stressful stimuli in a variety of organ systems. Small ubiquitin‐like modifier (SUMO) isoforms 1–4 have recently been identified in the human placenta, and are upregulated in the major obstetrical complication of pre‐eclampsia. This is the first study to characterize the spatiotemporal distribution of SUMO isoforms and their targets during placental development across gestation and in response to stress induced by pre‐eclampsia and chorioamnionitis. Keratins were identified as major targets of placental SUMOylation. The interaction with SUMOs and cytoskeletal filaments provides evidence for SUMOylation possibly contributing to underlying dysfunctional trophoblast turnover, which is a hallmark feature of pre‐eclampsia. Further understanding the role of individual SUMO isoforms and SUMOylation underlying placental dysfunction may provide a target for a novel therapeutic candidate as an approach for treating pre‐eclampsia complicated with placental pathology.