Association of intraplacental oxygenation patterns on dual-contrast MRI with placental abnormality and fetal brain oxygenation.

OBJECTIVES: Most human in-vivo placental imaging techniques are unable to distinguish and characterize various placental compartments, such as the intervillous space (IVS), placental vessels (PV) and placental tissue (PT), limiting their specificity. We describe a method that employs T2* and diffusion-weighted magnetic resonance imaging (MRI) data to differentiate automatically placental compartments, quantify their oxygenation properties and identify placental lesions (PL) in vivo. We also investigate the association between placental oxygenation patterns and fetal brain oxygenation. METHODS: This was a prospective study conducted between 2018 and 2021 in which dual-contrast clinical MRI data (T2* and diffusion-weighted MRI) were acquired from patients between 20 and 38 weeks' gestation. We trained a fuzzy clustering method to analyze T2* and diffusion-weighted MRI data and assign placental voxels to one of four clusters, based on their distinct imaging domain features. The new method divided automatically the placenta into IVS, PV, PT and PL compartments and characterized their oxygenation changes throughout pregnancy. RESULTS: A total of 27 patients were recruited, of whom five developed pregnancy complications. Total placental oxygenation level and T2* did not demonstrate a statistically significant temporal correlation with gestational age (GA) (R2 = 0.060, P = 0.27). In contrast, the oxygenation level reflected by T2* values in the placental IVS (R2 = 0.51, P = 0.0002) and PV (R2 = 0.76, P = 1.1 × 10-7 ) decreased significantly with advancing GA. Oxygenation levels in the PT did not show any temporal change during pregnancy (R2 = 0.00044, P = 0.93). A strong spatial-dependent correlation between PV oxygenation level and GA was observed. The strongest negative correlation between PV oxygenation and GA (R2 = 0.73, P = 4.5 × 10-7 ) was found at the fetal-vessel-dominated region close to the chorionic plate. The location and extent of the placental abnormality were automatically delineated and quantified in the five women with clinically confirmed placental pathology. Compared to the averaged total placental oxygenation, placental IVS oxygenation level best reflected fetal brain oxygenation level during fetal development. CONCLUSION: Based on clinically feasible dual-MRI, our method enables accurate spatiotemporal quantification of placental compartment and fetal brain oxygenation across different GAs. This information should improve our knowledge of human placenta development and its relationship with normal and abnormal pregnancy. © 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Structure dependence and species sensitivity of in vivo hepatobiliary toxicity with lysophosphatidic acid receptor 1 (LPA1) antagonists.

BMS-986020, BMS-986234 and BMS-986278, are three lysophosphatidic acid receptor 1 (LPA1) antagonists that were or are being investigated for treatment of idiopathic pulmonary fibrosis (IPF). Hepatobiliary toxicity (elevated serum AST, ALT, and ALP, plasma bile acids [BAs], and cholecystitis) was observed in a Phase 2 clinical trial with BMS-986020, and development was discontinued. In dogs and rats, the species used for the pivotal toxicology studies, there was no evidence of hepatobiliary toxicity in the dog while findings in the rat were limited to increased plasma BAs levels (6.1× control), ALT (2.9×) and bilirubin (3.4×) with no histopathologic correlates. Since neither rats nor dogs predicted clinical toxicity, follow-up studies in cynomolgus monkeys revealed hepatobiliary toxicity that included increased ALT (2.0× control) and GLDH (4.9×), bile duct hyperplasia, cholangitis, cholestasis, and cholecystitis at clinically relevant BMS-986020 exposures with no changes in plasma or liver BAs. This confirmed monkey as a relevant species for identifying hepatobiliary toxicity with BMS-986020. In order to assess whether the toxicity was compound-specific or related to LPA1 antagonism, two structurally distinct LPA1 antagonists (BMS-986234 and BMS-986278), were evaluated in rat and monkey. There were no clinical or anatomic pathology changes indicative of hepatobiliary toxicity. Mixed effects on plasma BAs in both rat and monkey has made this biomarker not a useful predictor of the hepatobiliary toxicity. In conclusion, the nonclinical data indicate the hepatobiliary toxicity observed clinically and in monkeys administered BMS-986020 is compound specific and not mediated via antagonism of LPA1.

Centennial-scale reductions in nitrogen availability in temperate forests of the United States.

Forests cover 30% of the terrestrial Earth surface and are a major component of the global carbon (C) cycle. Humans have doubled the amount of global reactive nitrogen (N), increasing deposition of N onto forests worldwide. However, other global changes-especially climate change and elevated atmospheric carbon dioxide concentrations-are increasing demand for N, the element limiting primary productivity in temperate forests, which could be reducing N availability. To determine the long-term, integrated effects of global changes on forest N cycling, we measured stable N isotopes in wood, a proxy for N supply relative to demand, on large spatial and temporal scales across the continental U.S.A. Here, we show that forest N availability has generally declined across much of the U.S. since at least 1850 C.E. with cool, wet forests demonstrating the greatest declines. Across sites, recent trajectories of N availability were independent of recent atmospheric N deposition rates, implying a minor role for modern N deposition on the trajectory of N status of North American forests. Our results demonstrate that current trends of global changes are likely to be consistent with forest oligotrophication into the foreseeable future, further constraining forest C fixation and potentially storage.

Studies of the dynamics of nuclear clustering in human syncytiotrophoblast.

Syncytial nuclear aggregates (SNAs), clusters of nuclei in the syncytiotrophoblast of the human placenta, are increased as gestation advances and in pregnancy pathologies. The origins of increased SNAs are unclear; however, a better appreciation of the mechanism may give insight into placental ageing and factors underpinning dysfunction. We developed three models to investigate whether SNA formation results from a dynamic process of nuclear movement and to generate alternative hypotheses. SNA count and size were measured in placental explants cultured over 16 days and particles released into culture medium were quantified. Primary trophoblasts were cultured for 6 days. Explants and trophoblasts were cultured with and without cytoskeletal inhibitors. An in silico model was developed to examine the effects of modulating nuclear behaviour on clustering. In explants, neither median SNA number (108 SNA/mm(2) villous area) nor size (283 µm(2)) changed over time. Subcellular particles from conditioned culture medium showed a wide range of sizes that overlapped with those of SNAs. Nuclei in primary trophoblasts did not change position relative to other nuclei; apparent movement was associated with positional changes of the syncytial cell membrane. In both models, SNAs and nuclear clusters were stable despite pharmacological disruption of cytoskeletal activity. In silico, increased nuclear movement, adhesiveness and sites of cytotrophoblast fusion were related to nuclear clustering. The prominence of SNAs in pregnancy disorders may not result from an active process involving cytoskeleton-mediated rearrangement of syncytial nuclei. Further insights into the mechanism(s) of SNA formation will aid understanding of their increased presence in pregnancy pathologies.

N-myc downstream-regulated gene 1 (NDRG1) mediates pomegranate juice protection from apoptosis in hypoxic BeWo cells but not in primary human trophoblasts.

INTRODUCTION: N-Myc downstream-regulated gene 1 (NDRG1) expression is increased in placentas of human pregnancies with intrauterine growth restriction and in hypoxic cultured primary trophoblasts. We previously showed that elevated NDRG1 decreases trophoblast apoptosis induced by hypoxia. Separately, we found that pomegranate juice (PJ) decreases cell death induced by hypoxia in trophoblasts. Here, we test the hypothesis that PJ protects trophoblasts from hypoxia-induced apoptosis by modulating NDRG1 expression. METHODS: Quantitative rtPCR was used to investigate the effects of PJ treatment on mRNA levels of 22 candidate genes involved in apoptosis, oxidative stress, and differentiation in trophoblasts. Western blotting and immunofluorescence were used to analyze NDRG1 protein levels. siRNA-mediated NDRG1 knockdown was used to investigate the role of NDRG1 in response to PJ in hypoxic BeWo choriocarcinoma cells and hypoxic cultured primary human trophoblasts. RESULTS: The mRNA levels of eight genes were altered, with NDRG1 showing the largest response to PJ and thus, we pursued the role of NDRG1 here. PJ significantly increased NDRG1 protein expression in primary trophoblasts and in BeWo cells. Knockdown of NDRG1 in hypoxic BeWo cells in the presence of PJ yielded increased apoptosis. In contrast, knockdown of NDRG1 in hypoxic primary trophoblasts in the presence of PJ did not increase apoptosis. DISCUSSION: We conclude that the PJ-mediated decrease in cell death in hypoxia is partially mediated by NDRG1 in BeWo cells but not in primary trophoblasts. The disparate effects of NDRG1 between BeWo cells and primary trophoblasts indicate caution is required when extrapolating from results obtained with cell lines to primary trophoblasts.

Reduced apoptosis in term placentas from gestational diabetic pregnancies.

Gestational diabetic mellitus (GDM) pregnancies have an increased risk of macrosomic infants and large placental mass, though the mechanisms explaining each of these is uncertain. We sought to evaluate the contribution of apoptosis to placental size and the expression of glucose transporters (SLC2A) in GDM pregnancies. Maternal age and pre-pregnancy body weight were documented. Newborn weights were recorded after delivery. Placentas 37-40-week gestation from control patients (no pregnancy complication) (n = 5), or with GDM (n = 5) were weighed immediately after delivery. Villous samples (4 mm diameter) were collected and divided into specimens; one was fixed in 4% paraformaldehyde for immunostaining using terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) and activated caspase-3. The other specimen was snap frozen in liquid nitrogen and stored at -80°C for active caspase-3, poly(ADP-ribose) polymerase (PARP), SLC2A1 and SLC2A3 gene expression analysis. Our results showed that maternal age and pre-pregnancy body weight were significantly higher in the GDM group when compared with those from the controls (P < 0.05). The mean neonatal birth weight and placenta weight were significantly higher in the GDM group compared with that from the controls (P < 0.05). The apoptotic index of placentas (0.05 ± 0.01 v. 0.17 ± 0.04, P < 0.04), active caspase-3 polypeptide fragments and PARP protein were significantly decreased in GDM placentas as compared with controls. Further, the level of placental SLC2A1 protein expression was ∼3-fold higher in GDM placentas. Our results suggest that reduced apoptosis in GDM placentas may contribute to increased placental tissue, which together with enhanced SLC2A1 expression, could play a role in fetal macrosomia.

Pericellular oxygen concentration of cultured primary human trophoblasts.

INTRODUCTION: Oxygen is pivotal in placental development and function. In vitro culture of human trophoblasts provides a useful model to study this phenomenon, but a hotly debated issue is whether or not the oxygen tension of the culture conditions mimics in vivo conditions. We tested the hypothesis that ambient oxygen tensions in culture reflect the pericellular oxygen levels. METHODS: We used a microelectrode oxygen sensor to measure the concentration of dissolved oxygen in the culture medium equilibrated with 21%, 8% or <0.5% oxygen. RESULTS: The concentration of oxygen in medium without cells resembled that in the ambient atmosphere. The oxygen concentration present in medium bathing trophoblasts was remarkably dependent on the depth within the medium where sampling occurred, and the oxygen concentration within the overlying atmosphere was not reflected in medium immediately adjacent to the cells. Indeed, the pericellular oxygen concentration was in a range that most would consider severe hypoxia, at ≤0.6% oxygen or about 4.6 mm Hg, when the overlying atmosphere was 21% oxygen. CONCLUSIONS: We conclude that culture conditions of 21% oxygen are unable to replicate the pO(2) of 40-60 mm Hg commonly attributed to the maternal blood in the intervillous space in the second and third trimesters of pregnancy. We further surmise that oxygen atmospheres in culture conditions between 0.5% and 21% provide different oxygen fluxes in the immediate pericellular environment yet can still yield insights into the responses of human trophoblast to different oxygen conditions.

Live-cell imaging shows apoptosis initiates locally and propagates as a wave throughout syncytiotrophoblasts in primary cultures of human placental villous trophoblasts.

Human placental villi are surfaced by the syncytiotrophoblast, a multinucleated, epithelial-cell layer that functions in maternal-fetal exchange. Mononucleated cytotrophoblasts are subjacent to the syncytiotrophoblast. Using confocal fluorescence microscopy of third-trimester villi, we previously found that cytotrophoblasts are often interdigitated into the syncytiotrophoblast, that cytotrophoblasts undergo caspase-mediated apoptosis, and that apoptosis is much lower, and perhaps completely inhibited, in intact syncytiotrophoblast lacking fibrin-type fibrinoid. Previous analysis of primary cultures of human trophoblasts also indicated lower levels of apoptosis in syncytiotrophoblast compared to cytotrophoblasts. Here, using confocal microscopy we find that cultured cytotrophoblasts and syncytiotrophoblasts display complex structural relationships, as in vivo, and that apoptosis of a cytotrophoblast or syncytiotrophoblast does not induce apoptosis of adjacent trophoblasts. Using live-cell imaging of mitochondrial depolarization and nuclear condensation in cultured syncytiotrophoblasts, we show apoptosis initiates in a localized region and propagates radially at ∼5 µm/min with no loss of velocity until the entire syncytium has undergone apoptosis. The rate of propagation is similar in cases of spontaneous apoptosis and in apoptosis that occurs in the presence of cobalt chloride or rotenone, two inducers of apoptosis. We suggest that inhibition of syncytiotrophoblast apoptosis in vivo is important to prevent widespread syncytiotrophoblast death, which would result in placental dysfunction and contribute to poor pregnancy outcomes.

Survival by self-destruction: a role for autophagy in the placenta?

Autophagy is a burgeoning area of research from yeast to humans. Although previously described as a death pathway, autophagy is now considered an important survival phenomenon in response to environmental stressors to which most organs are exposed. Despite an ever expanding literature in non-placental cells, studies of autophagy in the placenta are lagging. We review the regulation of autophagy, summarize available placental studies of autophagy, and highlight potential areas for future research. We believe that such studies will yield novel insights into how placentas protect the survival of the species by "self-eating".

Villous trophoblast apoptosis is elevated and restricted to cytotrophoblasts in pregnancies complicated by preeclampsia, IUGR, or preeclampsia with IUGR.

Human placental villi are surfaced by an outer multinucleated syncytiotrophoblast and underlying mononucleated cytotrophoblasts. Conflicting data have attributed one, or the other, of these villous trophoblast phenotypes to undergo enhanced apoptosis in complicated pregnancies, compared to term, normotensive pregnancies. We use high-resolution confocal microscopy after co-staining for E-cadherin, as a trophoblast plasma membrane marker, and for the cleavage products of cytokeratin 18 and PARP1, as markers for caspase-mediated apoptosis, to distinguish between apoptotic cytotrophoblasts and apoptosis within the syncytiotrophoblast. We test the hypothesis that increased caspase-mediated apoptosis occurs in villi of placentas derived from pregnancies complicated by preeclampsia, intrauterine growth restriction (IUGR), or both. We find significantly elevated apoptosis in villous cytotrophoblasts from women with preeclampsia and/or IUGR, compared to term, normotensive pregnancies. Apoptosis of cytotrophoblasts in villi from complicated pregnancies appears to progress similarly to what we found previously for apoptotic cytotrophoblasts in villi from in term, normotensive pregnancies. Notably, caspase-mediated apoptosis was not detectable in regions with intact syncytiotrophoblast, suggesting strong repression of apoptosis in this trophoblast phenotype in vivo. We suggest that the elevated apoptosis in cytotrophoblasts in preeclampsia contributes to the placental dysfunction characteristic of this disorder. We also propose that repression of apoptosis in the syncytiotrophoblast is important to prevent apoptosis sweeping throughout the syncytium, which would result in widespread death of this essential interface for maternal-fetal exchange.

First-trimester placental protein 13, PAPP-A, uterine artery Doppler and maternal characteristics in the prediction of pre-eclampsia.

OBJECTIVE: To test the hypothesis that a combination of PP13, PAPP-A and first-trimester uterine artery Doppler would improve the prediction of pre-eclampsia. METHODS: This is a prospective cohort study of pregnant women followed from the first-trimester to delivery. PP13 and PAPP-A were determined by immunoassay of maternal serum at 11-14 weeks', when uterine artery Doppler measurements were assessed. Cases identified with any form of pre-eclampsia were compared with a control group without pre-eclampsia. The sensitivity of each marker or their combinations in predicting pre-eclampsia for different fixed false positive rates was calculated from the ROC curves. RESULTS: Forty two women were diagnosed with pre-eclampsia and 410 women with pregnancies not complicated by pre-eclampsia were used as controls. For a fixed false positive rate (FPR) of 20%, PP13, PAPP-A and mean uterine artery pulsatility index identified 49%, 58% and 62% respectively, of women who developed any form of pre-eclampsia. PP13 was best in predicting early onset pre-eclampsia with a sensitivity of 79% at a 20% FPR. Combinations of the three first-trimester assessments did not improve the prediction of pre-eclampsia in later pregnancy. CONCLUSION: First-trimester PP13, PAPP-A and uterine artery PI are reasonable, individual predictors of women at risk to develop pre-eclampsia. Combinations of these assessments do not further improve the prediction of pre-eclampsia.

First-trimester serum analytes, biophysical tests and the association with pathological morphometry in the placenta of pregnancies with preeclampsia and fetal growth restriction.

OBJECTIVE: We test the hypothesis that first-trimester serum analytes, 4-D power Doppler placental vascular indices and uterine artery Doppler (UAD) predicts abnormal placental morphometry in pregnancies with preeclampsia (PE) and fetal growth restriction (FGR). STUDY DESIGN: Maternal serum analytes (PAPP-A, hCG, ADAM12s, and PP13), bilateral UADs, and placental vascular indices were measured at 11-14 weeks in a nested-case control study within a prospective cohort of women followed from the first-trimester to delivery. Vascularization index (VI), flow index (FI), and vascularization flow index (VFI) were obtained from 4-D power Doppler histograms. Serum analytes were measured using immunofluorometric assays and values converted to multiples of the median (MoM) for gestational age. Morphometric analysis was performed on placentas from pregnancies complicated by PE (n = 13), gestational hypertension (HBP, n = 7) and FGR (defined as fetal weight <10th percentile with abnormal umbilical artery Doppler: n = 7); and 20 uncomplicated pregnancies. Two pregnancies had both FGR and PE. Each placenta was weighed and random samples taken, and fixed in formalin within 1 h of delivery. Hematoxylin & Eosin stained slides were analyzed by design-based stereology to quantify linear dimensions, surface areas and volumes of placental components. Paired t-test and ANOVA with adjustments for multiple comparisons were used. RESULTS: The surface areas of terminal and intermediate villi as well as the volume of terminal villi were significantly smaller in placentas from pregnancies complicated by FGR and PE. Compared with the control group the mean PAPP-A (MoM) was lower in the pregnancies with abnormal placenta morphometry (1.1 ± 0.5 versus 0.7 ± 0.5, P = 0.03). The morphometric indices were lower in those pregnancies with low PAPP-A and IUGR compared with preeclampsia. CONCLUSION: First-trimester PAPP-A levels are associated with abnormal placental morphometry at delivery in pregnancies with PE and IUGR. These findings may explain the association between adverse pregnancy outcomes and first-trimester PAPP-A.

Review: Oxygen and trophoblast biology--a source of controversy.

Oxygen is necessary for life yet too much or too little oxygen is toxic to cells. The oxygen tension in the maternal plasma bathing placental villi is <20 mm Hg until 10-12 weeks' gestation, rising to 40-80 mm Hg and remaining in this range throughout the second and third trimesters. Maldevelopment of the maternal spiral arteries in the first trimester predisposes to placental dysfunction and sub-optimal pregnancy outcomes in the second half of pregnancy. Although low oxygen at the site of early placental development is the norm, controversy is intense when investigators interpret how defective transformation of spiral arteries leads to placental dysfunction during the second and third trimesters. Moreover, debate rages as to what oxygen concentrations should be considered normal and abnormal for use in vitro to model villous responses in vivo. The placenta may be injured in the second half of pregnancy by hypoxia, but recent evidence shows that ischemia with reoxygenation and mechanical damage due to high flow contributes to the placental dysfunction of diverse pregnancy disorders. We overview normal and pathologic development of the placenta, consider variables that influence experiments in vitro, and discuss the hotly debated question of what in vitro oxygen percentage reflects the normal and abnormal oxygen concentrations that occur in vivo. We then describe our studies that show cultured villous trophoblasts undergo apoptosis and autophagy with phenotype-related differences in response to hypoxia.

Vitamin D effects on pregnancy and the placenta.

Vitamin D is a pleiotropic secosteroid hormone important for health and disease prevention. The actions of vitamin D are mediated by the vitamin D receptor that binds the active form of vitamin D [1,25(OH)(2)D] to induce both transcriptional and non-genomic responses. Vitamin D has well known classical functions in calcium uptake and bone metabolism, but more recent work highlights the importance of the nonclassical actions of vitamin D in a variety of cell types. These actions include modulation of the innate and adaptive immune systems and regulation of cell proliferation. Adequate vitamin D intake is essential for maternal and fetal health during pregnancy, and epidemiological data indicate that many pregnant women have sub-optimal vitamin D levels. Notably, vitamin D deficiency correlates with preeclampsia, gestational diabetes mellitus, and bacterial vaginosis, and an increased risk for C-section delivery. Recent work emphasizes the importance of nonclassical roles of vitamin D in pregnancy and the placenta. The placenta produces and responds to vitamin D where vitamin D functions as a modulator of implantation, cytokine production and the immune response to infection. We describe vitamin D metabolism and the cellular responses to vitamin D, and then summarize the role of vitamin D in placental trophoblast, pregnancy and the fetus.

The levels of hypoxia-regulated microRNAs in plasma of pregnant women with fetal growth restriction.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the post-transcriptional level. While mostly intracellular, a portion of cellular miRNAs is released to the circulation and their level in the plasma is altered in certain pathological conditions such as cancer, and also during pregnancy. We examined the circulating levels of a set of trophoblastic miRNAs, which we recently found to be regulated by hypoxia, in the plasma of pregnant women with fetal growth restriction (FGR). Pregnancy was associated with increased plasma levels of several placenta-specific miRNAs, compared to non-pregnant controls. Among pregnant women, the overall levels of miRNA species that we analyzed were increased by 1.84-fold (p < or = 0.01) in plasma of women with pregnancies complicated by FGR, but decreased in FGR placentas by 24% (p < or = 0.01) compared to values from uncomplicated pregnancies. Together, our results show that plasma concentration of miRNAs is regulated in pregnancy, and that FGR is associated with increased circulating miRNA levels, highlighting the need to explore plasma miRNAs as potential biomarkers for placental diseases.

IFPA meeting 2008 workshops report.

Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.

The C5b-9 membrane attack complex of complement activation localizes to villous trophoblast injury in vivo and modulates human trophoblast function in vitro.

The complement system plays an important role in normal human pregnancy. Uncontrolled activation of this system has been associated with many disease states. We tested the hypothesis that the C5b-9 membrane attack complex (MAC) localizes to sites of villous injury and modulates trophoblast function. Placental sections from pregnancies with no complications, intrauterine growth restriction, or preeclampsia were immunostained and the surface density for MAC and fibrin was determined by morphometric analysis. Primary cytotrophoblasts from term placentas were cultured in a FiO(2) of <1%, 8% and 20% with 10% human serum containing active MAC or heat-inactivated control serum. Immunofluorescent MAC binding to trophoblast was quantified, and the neoepitopes formed in cytokeratin 18 filaments and poly-ADP-ribose polymerase during apoptosis were used to measure cell death. Trophoblast differentiation was assessed by HCG secretion, formation of syncytia, and expression of syncytin. MAC localized to fibrin deposits in normal placentas, and especially in placentas from IUGR and preeclampsia. MAC binding to cytotrophoblasts was inversely proportional to FiO(2) and enhanced apoptosis. MAC increased markers of differentiation in cultures at 72h (medium HCG, syncytia and syncytin expression). Our findings demonstrate that MAC associates with fibrin deposits at sites of villous injury in vivo. Hypoxia also enhances MAC deposition in cultured trophoblasts and MAC alters trophoblast function in a phenotype specific manner.