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Pericytes are multifunctional cells wrapped around capillary endothelia, essential for vascular health, development, and blood flow regulation, although their role in human placental chorionic villi has not been fully explored. The second half of normal pregnancy is characterized by a progressive decline in placental and fetal oxygen levels which, by term, comprises a substantial degree of hypoxia. We hypothesized this hypoxia would stimulate pericyte regulation of chorionic villous capillary function. This study's objective was to investigate the role of hypoxia on normal term placental pericytes (PLVP) and their signaling to endothelial cells. First, we confirmed fetoplacental hypoxia at term by a new analysis of umbilical arterial blood oxygen tension of 3,010 healthy singleton neonates sampled at caesarean section and before labor. We then measured the release of cytokines, chemokines, and small extracellular vesicles (PLVPsv), from PLVP cultured at 20%, 8% and 1% O 2 . As O 2 levels decreased, secreted cytokines and chemokines [interleukin-6 (IL-6), interleukin-1α (IL-1α) and vascular endothelial growth factor (VEGF)], and small extracellular vesicle markers, (Alix, Syntenin and CD9) increased significantly in the culture supernatants. When primary human umbilical vein endothelial cells (HUVEC) were cultured with PLVPsv, polygon formation, number, and tube formation length was significantly increased compared to cells not treated with PLVPsv, indicating PLVPsv stimulated angiogenesis. We conclude that adding PLVPsv stimulates angiogenesis and vessel stabilization on neighboring endothelial cells in response to hypoxia in term pregnancy compared to no addition of PLVPsv. Our finding that PLVP can release angiogenic molecules via extracellular vesicles in response to hypoxia may apply to other organ systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Designing cities for thermal comfort is an important priority in a warming and urbanizing world. As temperatures in cities continue to break extreme heat records, it is necessary to develop and test new approaches capable of tracking human thermal sensations influenced by microclimate conditions, complex urban geometries, and individual characteristics in dynamic settings. Thermal walks are a promising novel research method to address this gap. During a thermal walk in Phoenix, Arizona, USA, we examined relationships between the built environment, microclimate, and subjective thermal judgments across a downtown city neighborhood slated for redevelopment. Subjects equipped with GPS devices participated in a 1-hour walk on a hot sunny day and recorded their experience in a field guide. Microclimate measurements were simultaneously collected using the mobile human-biometeorological instrument platform MaRTy. Results revealed significant differences in physiologically equivalent temperature (PET) and modified physiologically equivalent temperature (mPET) and between street segments with more than 18 °C (25 °C mPET) between the maximum and minimum values. Wider range of mPET values reflected the inclusion of individual level data into the model. Streets with higher sky view factor (SVF) and east-west orientation showed a higher PET and mPET overall. Furthermore, we showed evidence of thermal alliesthesia, the pleasure resulting from slight changes in microclimate conditions. Participants' sense of pleasure was related to the mean PET of the segment they just walked, with linear regression explaining over 60% of the variability. We also showed that estimated percent shade was significantly correlated with SVF, PET, mPET, and pleasure, indicating that participants could sense minor changes in microclimate and perceived shade as pleasant. Although generalization of results is limited by a low sample size, findings of this study improve the understanding of dynamic thermal comfort in complex urban environments and highlight the value of thermal walks as a robust research method., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Preeclampsia (PE) is associated with an exaggerated maternal systemic inflammatory response. Throughout gestation, the placenta releases extracellular vesicles through the syncytiotrophoblast layer (STB) into the maternal circulation and this is increased in PE. Expression of Siglec-6, a transmembrane receptor of molecular weight 50 KDa, is upregulated in PE placental tissue., Methods: Here we investigated respective abundance of Siglec-6 in PE -and normal pregnancy- (NP) derived placental lysates (PL) and syncytiotrophoblast-derived extracellular vesicles (STBEV). STBEV from PE and NP placentas were isolated through dual-lobe placental perfusion and serial ultracentrifugation. Siglec-6 was characterized by immunohistochemistry, immunoblotting, mass spectrometry (MS), and deglycosylation., Results: Immunoblotting revealed the expected Siglec-6 (50 KDa) band present in both PE and NP PL, however an additional heavier band was observed at 70 KDa only in PE PL, but not in NP. When interrogating STBEV we saw an absence of the expected 50 KDa band but the 70 KDa was present predominantly only in the PE STBEV. Deglycosylation of PL and STBEV from PE showed that the 70 KDa and the 50 KDa bands were reduced to 48 KDa, suggesting glycosylation. Both 48 KDa and 70 KDa bands were subjected to MS, confirming Siglec-6 expression in both., Discussion: Our data shows that the inability to detect Siglec-6 in circulation might be due to the placenta secreting STBEV carrying a modified glycosylated form of Siglec-6 with a 70 KDa molecular weight, significantly and uniquely upregulated in PE STBEV., Competing Interests: Declaration of competing interest None., (Copyright © 2020. Published by Elsevier Inc.)

The placenta releases syncytiotrophoblast-derived extracellular vesicles (STB-EV) into the maternal circulation throughout gestation. STB-EV dependent signalling is believed to contribute to the widespread maternal adaptive physiological changes seen in pregnancy. Transfer RNA (tRNA) halves have been identified in vesicles released from other human and murine organ systems, which alter gene expression in target cells. Here, we characterise tRNA-half expression in STB-EV and demonstrate biological activity of a highly abundant tRNA-half. Short RNA from ex-vivo, dual-lobe placental perfusion STB-EV was sequenced, showing that most (>95%) comprised tRNA species. Whole placental tissue contained <50% tRNA species, suggesting selective packaging and export of tRNA into STB-EV. Most tRNA within STB-EV were 5'-tRNA halves cleaved at 30-32 nucleotides. The pattern of tRNA expression differed depending on the size/origin of the STB-EV; this was confirmed by qPCR. Protein synthesis was suppressed in human fibroblasts when they were cultured with a 5'-tRNA half identified from STB-EV sequencing. This study is the first to evaluate tRNA species in STB-EV. The presence of biologically active 5'-tRNA halves, specific to a vesicular origin, suggests a novel mechanism for maternal-fetal signalling in normal pregnancy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2018 there were nine themed workshops, five of which are summarised in this report. These workshops discussed new perspectives and knowledge in the following areas of research: 1) preeclampsia; 2) abnormally invasive placenta; 3) placental infection; 4) gestational trophoblastic disease; 4) drug delivery to treat placental dysfunction., (Copyright © 2019. Published by Elsevier Ltd.)

Introduction: Acute atherosis (AA) is a lesion affecting uteroplacental spiral arteries during pregnancy, most frequently in preeclampsia but occasionally in normal pregnancy. It is commonly observed in untransformed spiral arteries (intact smooth muscle cell layer, lacking intramural trophoblasts). The mechanism causing lesion development is unknown. AA shares some morphological similarities with atherosclerosis, in which endothelial activation occurs early. Here we histologically characterize decidua basalis spiral arteries with and without AA, focusing on endothelial status and activation., Methods: Formalin-fixed and paraffin-embedded decidua basalis tissue sections from 32 patients (16 normotensive, 5 with AA, 16 preeclampsia, 7 with AA) were stained with H + E, PAS, MSB (Martius Scarlet Blue), desmin, CK7, CD68, CD31, vWF and ICAM-1. We logged remodeling status, presence of AA, endothelial morphology, endothelial CD31 intensity and activation (ICAM-1-positive cells)., Results: We observed fully or partially transformed spiral arteries in most decidua basalis samples, and no untransformed arteries. AA arteries were also observed, characterized by intramural CD68-positive vacuolated cells and fibrinoid necrosis. They lacked a smooth muscle cell layer and intramural trophoblasts. The fibrinoid necrosis in AA lesions stained red with MSB. AA arteries were associated with lower CD31 staining intensity of endothelial cells. More arteries had an abnormal or destroyed endothelium relative to arteries without AA. Endothelial activation was not observed in the majority of AA arteries., Discussion: Our results indicate an altered endothelial phenotype as important in the development of AA, supporting previous observations. The histology of AA differs from that of atherosclerosis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Circulating sFlt-1 increases significantly in pregnancy compared to the non-pregnant state and even more so in the event of preeclampsia. We set out to determine if circulating sFlt-1 is placentally derived in normal pregnancy. Paired uterine and peripheral vein samples were collected at time of caesarean section. A follow up peripheral sample was collected in the postpartum period. There was a significant sFlt-1 gradient between uterine and peripheral veins. sFlt-1 levels dropped significantly when the placenta has been removed, i.e. postnatally. This is in keeping with the placenta being the main site of sFlt-1 production in normal pregnancies., (Copyright © 2019 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

The fullPIERS model is a risk prediction model developed to predict adverse maternal outcomes within 48 h for women admitted with pre-eclampsia. External validation of the model is required before implementation for clinical use. We assessed the temporal and external validity of the fullPIERS model in high income settings using five cohorts collected between 2003 and 2016, from tertiary hospitals in Canada, the United States of America, Finland and the United Kingdom. The cohorts were grouped into three datasets for assessing the primary external, and temporal validity, and broader transportability of the model. The predicted risks of developing an adverse maternal outcome were calculated using the model equation and model performance was evaluated based on discrimination, calibration, and stratification. Our study included a total of 2429 women, with an adverse maternal outcome rate of 6.7%, 6.6%, and 7.0% in the primary external, temporal, and combined (broader) validation cohorts, respectively. The model had good discrimination in all datasets: 0.81 (95%CI 0.75-0.86), 0.82 (95%CI 0.76-0.87), and 0.75 (95%CI 0.71-0.80) for the primary external, temporal, and broader validation datasets, respectively. Calibration was best for the temporal cohort but poor in the broader validation dataset. The likelihood ratios estimated to rule in adverse maternal outcomes were high at a cut-off of ≥30% in all datasets. The fullPIERS model is temporally and externally valid and will be useful in the management of women with pre-eclampsia in high income settings although model recalibration is required to improve performance, specifically in the broader healthcare settings., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Placental syncytiotrophoblast (STB) release extracellular vesicles (STB-EVs) that communicate physiological and pathological placental signals to the maternal organs. STB-EV release also increases in preeclampsia (PE). Here we explored the cargo of PP13 in STB-EVs from PE versus control placentas., Methods: Placentae were harvested following cesarean section deliveries, and dual placental lobe perfusion was used to harvest STB-EV. Maternal side perfusate was centrifuged at 10,000 × g to yield the STB microvesicles, and then at 150,000 × g to yield STB exosomes. Total STB-EVs (tSTB-EVs) were collected using a one step 150,000 × g centrifugation. Placental origin and size distribution were assessed by Western blotting and Nanoparticle Tracking Analysis, respectively. PP13 expression was determined by Western blot and ELISA., Results: Placental alkaline phosphatase (PLAP; a STB specific marker) was present in all preparations. Total tSTB-EVs and STB-EXs also expressed the exosome markers such as the Apoptosis-Linked Gene 2-Interacting Protein X (Alix) and the cluster differentiation protein 9 (CD9). PP13 was localized to the outer surface and intra-vesicular compartments of all fractions. Surface to total PP13 ratios were ∼1:1 for all STB-EV preparations. In contrast to the previously reported higher circulating concentrations of soluble PP13 in PE, significantly lower levels of PP13, normalized to total vesicular protein, were observed in PE samples. PP13 reduction in all STB-EVs' sub-populations may be attributed to differences in gestational age (GA). A simple correction for GA suggested that PE may be an important influence., Conclusions: PP13 is located in and on all types of STB-EVs. Circulating PP13 may therefore be either soluble or associated with extracellular vesicles with different pathophysiological effects in the maternal circulation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aberrant placental ageing is implicated in a high percentage of birth complications, stillbirths and neonatal deaths. Understanding how this complex organ is established and maintained for the 9-10 months of pregnancy and then how and why it undergoes the physiological changes that result in labour at term is therefore of enormous clinical importance. In this review, we assess the evidence that placental ageing results from cellular senescence, a state of terminal proliferation arrest accompanied by characteristic morphological and metabolic changes including a shift to a pro-inflammatory phenotype. We discuss how senescence both contributes to placental formation during cytotrophoblast fusion, and to the changes necessary for labour onset, such as cervical remodelling and increased sterile inflammatory signalling. Based on evidence from human clinical studies and experimental interventions in mice, we assess possible biochemical pathways that may drive senescence, and speculate on how aberrant senescence in the placenta may contribute to pre-eclampsia, pre-term birth and stillbirth., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: miRNAs are small non-coding RNAs important for the regulation of mRNA in many organs including placenta. Adipokines and specifically leptin are known to be dysregulated in preeclampsia, but little is known regarding their regulation by miRNAs during pregnancy., Methods: We performed high-throughput sequencing of small RNAs in placenta from 72 well-defined patients: 23 early-onset preeclampsia (PE), 26 late-onset PE and 23 controls. The regulation of some miRNAs was confirmed on qRT-PCR. Maternal circulating levels and placental mRNA of leptin, resistin and adiponectin were measured using Bio-Plex and qRT-PCR., Results: We found that miR-1301, miR-223 and miR-224 expression was downregulated in early-onset PE, but not in late-onset PE, compared to controls. In silico analysis predicted the leptin gene (LEP) to be a target for all three miRNAs. Indeed, we found significant correlation between maternal circulating levels of leptin and placental LEP expression. In addition, we found a significant inverse correlation between maternal circulating leptin/placental LEP expression and placental miR-1301 expression levels. Interestingly, placental expression of miR-1301 was also correlated with newborn weight percentile and inversely correlated with both maternal systolic and diastolic blood pressure prior to delivery., Discussion: Our results confirm that placenta is a major site of LEP expression during pregnancy. It further suggests that miR-1301 could be involved in the regulation of leptin during pregnancy and may play a role in early-onset PE., Conclusions: miR-1301 is dysregulated in early-onset preeclampsia and could possibly play a role in the regulation of leptin during pregnancy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We would like to know the cause of pre-eclampsia. We would also like to be able to diagnose and treat it quickly and effectively, to be able to predict it accurately with one simple test and prevent it with safe and simple medication. But we cannot, nor on present evidence, will we ever be able to do so. Why is this? There are several reasons, which I will attempt to summarise: the heart of the problem is the complexity and variability of the disorder, which is so daunting that every specific statement about preeclampsia seems to have exceptions - it is truly a disease of exceptions. The cause of pre-eclampsia appears to be the placenta - in that the disorder can occur without a fetus as in hydatidiform mole, without a uterus as in abdominal pregnancy and is resolved by delivery (of the placenta). On the other hand some of the worst presentations are of women who are normal until delivery and then get a major crisis, eclampsia or the HELLP syndrome, immediately afterwards, when the placenta has gone. The placental problem is poor uteroplacental circulation secondary to inadequate remodelling of the spiral arteries that occurs between weeks 8 and week 18 (poor placentation). The maternal symptoms and signs arise from maternal endothelial dysfunction and an associated vascular inflammation. But the placental pathology can occur without features of pre-eclampsia and endothelial dysfunction can arise in mothers without placental disease (maternal pre-eclampsia). This apparent confusion can be resolved in part by recognising that pre-eclampsia is not a disease but a syndrome - encompassed by its defining features of new hypertension and proteinuria that remit after delivery. A syndrome is simply an empirical definition of a clinical presentation that demands action. It tells you nothing about pathogenesis. One syndrome usually encompasses several conditions and this is likely to be true of pre-eclampsia. The key here is to acknowledge that there are many routes to pre-eclampsia; that the final disorder arises from different mixes of environmental and genetic factors, with different contributions from the mother and the placenta. In terms of disease, the unique feature of the latter is that two individuals are involved, mother and baby, each with their different genetic make-ups. This can explain the heterogeneity of pre-eclampsia: for example the difference between early-onset disease with its high preponderance of fetuses that are growth restricted and prominent placental pathology in contrast to the late onset disease, which may be associated with large for gestational age fetuses and routinely reveals little placental pathology. Finally, many very mild features of pre-eclampsia arise towards term in normal healthy women. It is possible that all women are destined to get the disorder. Timing is the key feature, such that late gestation is a race against time: will spontaneous delivery occur before pre-eclampsia or vice versa?, (Copyright © 2014.)

Unlabelled: The diagnosis of preeclampsia can be difficult with considerable uncertainty when the presenting features are borderline. Moreover underlying medical conditions may obscure or simulate the onset of pre-eclampsia. There is a clinical need to be able to diagnose, more accurately, those forms of the disease that are likely to progress to an adverse fetal or maternal outcome. Maternal circulating placental growth factor (PIGF) concentrations are decreased in these women, in correlation with disease severity and gestational age of onset. In this study its diagnostic performance was evaluated with an accurate, point of care test. The PELICAN study was a prospective observational cohort study, in seven maternity units in the British Isles. 625 women presenting between 20+0 and 40+6 weeks gestation with suspected preeclampsia were recruited, excluding those already diagnosed with preeclampsia. PIGF was measured (Alere Triage® PIGF test) in the enrolment sample after all participants had delivered so that clinical decisions were not affected by its results. ISSHP definitions of hypertensive disease were assigned, blinded to PIGF values. Analysis was conducted to assess diagnostic accuracy of the enrolment PIGF level for placental dysfunction requiring delivery within 14days. In women who presented before 35 week's gestation the PIGF test predicted placental dysfunction, that required deliverywithin the next 14days, with the following characteristics: sensitivity 0.95 (0.86-0.98); specificity 0.56 [0.49-0.63); NPV 0.97 (0.92-0.99); PPV0.42 (0.35-0.51)., Conclusion: PIGF is a strong predictor of time-to-delivery and may be an independent and complementary tool to assist in diagnosis and risk stratification of pregnancies complicated by placental dysfunction. These valuable attributes are to be tested in prospecting randomised double-blind controlled trial., (Copyright © 2014.)

For many years pre eclampsia has been considered to be a two-stage disease. The first stage comprises poor placentation. The second stage is the clinical expression of the disease namely new hypertension and new proteinuria. The first stage is preclinical and symptomless, which evolves between weeks 8 and 18 of pregnancy, when the uteroplacental circulation is established by spiral artery remodelling. Its consequence is dysfunctional perfusion of the intervillous space of the placenta with oxidative and haemodynamic stress. The damaged placenta releases excessive pro-inflammatory and antiangiogenic factors into the maternal circulation. With increasing knowledge, this model has become inadequate. First the antecedents of poor placentation have become clearer and are immunological in origin, reflecting the mother's ability to accommodate to the genetic foreignness of her unborn child. They begin, as discussed already in this meeting, with preconceptual tolerisation of the mother to the semen of the prospective father of her child. A lack of tolerisation, arising from a short interval between first coitus and conception increases the likelihood of poor placentation and pre-eclampsia (Stage 1). This is presumed to affect the health and growth of the embryo immediately after implantation but there is little evidence of this at the moment (Stage 2). Placentation begins after week 8 when the uteroplacental circulation, which previously has been closed by trophoblast plugs in the spiral arteries, begins to open. Defective placentation may arise from premature opening, and perfusion of the intervillous space by oxygenised arterial blood before the placenta is equipped to cope with the stress. Placentation extends over about 10 weeks and, when it is defective, constitutes stage 3 of pre-eclampsia. Stages 4-6 all occur in the second half of pregnancy. Stage 4 is associated with excessive or deficient placental derived factors in the mother's blood, secondary to placental damage, before the appearance of clinical signs. When the diagnosis of pre-eclampsia can be made stage 5 has begun. Stage 6 affects less than half of women with pre-eclampsia. It is the superimposition of a second and later spiral artery lesion called acute atherosis, which has some resemblance to atherosclerosis, which is suffered by middle and old-aged, non-pregnant adults. Its importance is that it further reduces uteroplacental perfusion and predisposes to spiral artery thrombosis, which underlies the occurrence of placental infarcts. The evidence for and the mechanisms of these multiple stages will be briefly presented., (Copyright © 2014.)

Incomplete spiral artery remodelling is the first of two stages of pre-eclampsia, typically of early onset. The second stage comprises dysregulated uteroplacental perfusion and placental oxidative stress. Oxidatively stressed syncytiotrophoblast (STB) over-secretes proteins that perturb maternal angiogenic balance and are considered to be pre-eclampsia biomarkers. We propose that, in addition and more fundamentally, these STB-derived proteins are biomarkers of a cellular (STB) stress response, which typically involves up-regulation of some proteins and down-regulation of others (positive and negative stress proteins respectively). Soluble vascular growth factor receptor-1 (sVEGFR-1) and reduced growth factor (PlGF) then exemplify positive and negative STB stress response proteins in the maternal circulation. Uncomplicated term pregnancy is associated with increasing sVEGFR-1 and decreasing PlGF, which can be interpreted as evidence of increasing STB stress. STB pathology, at or after term (for example focal STB necrosis) demonstrates this stress, with or without pre-eclampsia. We review the evidence that when placental growth reaches its limits at term, terminal villi become over-crowded with diminished intervillous pore size impeding intervillous perfusion with increasing intervillous hypoxia and STB stress. This type of STB stress has no antecedent pathology, so the fetuses are well-grown, as typifies late onset pre-eclampsia, and prediction is less effective than for the early onset syndrome because STB stress is a late event. In summary, abnormal placental perfusion and STB stress contribute to the pathogenesis of early and late onset pre-eclampsia. But the former has an extrinsic cause - poor placentation, whereas the latter has an intrinsic cause, 'microvillous overcrowding', as placental growth reaches its functional limits. This model explains important features of late pre-eclampsia and raises questions of how antecedent medical risk factors such as chronic hypertension affect early and late sub-types of the syndrome. It also implies that all pregnant women may be destined to get pre-eclampsia but spontaneous or induced delivery averts this outcome in most instances., (Copyright © 2013 IFPA and Elsevier Ltd. All rights reserved.)

The decidua basalis is one of the frontiers between placenta and mother. Its spiral arteries ensure that the placenta and fetus have adequate access to the maternal circulation, without compromising maternal health. Normally this requires a tightly regulated collaboration between tissues of genetically different individuals. But like all frontiers it can become a battlefield. The decidua is difficult to sample systematically. Some of the problems have been resolved by our vacuum suction method. We review the technique and how it has contributed to what we know of decidual tissue, especially when it becomes a battlefield in preeclampsia, with its increased oxidative stress and inflammation. Acute atherosis is a poorly studied decidual lesion of late pregnancy, which mainly affects the decidual tips of spiral arteries in preeclampsia. It is characterized by lipid-filled foam cells and resembles early atherosclerosis. Poorly remodelled spiral arteries seem to be especially susceptible. The underlying mechanisms are largely unknown, but are likely to be similar to those of atherosclerosis and primarily the consequence of vascular inflammation. Acute atherosis also occurs in other pregnancy complications, even in normal pregnancies. It appears not to be confined to maladapted spiral arteries nor be caused by hypertension. It is important that foam cells result from inflammatory stimulation of macrophages. Hence, we propose that decidual inflammation of multiple causes underlies acute atherosis, with or without preeclampsia. Women suffering from preeclampsia have an augmented risk of cardiovascular disease later in life and of premature death. Acute atherosis may more specifically identify those women at augmented risk for such later cardiovascular disorders, whether or not it is associated with preeclampsia., (Copyright © 2014 IFPA and Elsevier Ltd. All rights reserved.)

Biobanks provide an important repository of samples for research purposes. However, for those samples to reflect the in vivo state, and for experimental reliability and reproducibility, careful attention to collection, processing and storage is essential. This is particularly true for the placenta, which is potentially subjected to stressful conditions during delivery, and sample collection may be delayed owing to routine postpartum inspection by clinical staff. In addition, standardisation of the collection procedure enables samples to be shared among research groups, allowing larger datasets to be established. Here, we provide an evidence-based and experts' review of the factors surrounding collection that may influence data obtained from the human placenta. We outline particular requirements for specific techniques, and propose a protocol for optimal sample collection. We recognise that the relevance of these factors, and of the sample types collected to a particular study will depend on the research questions being addressed. We therefore anticipate that researchers will select from the protocol to meet their needs and resources available. Wherever possible, we encourage researchers to extend their collection to include additional samples that can be shared on an international collaborative basis, with appropriate informed consent, to raise the quality, as well as quantity, of placental research., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Biological stress may affect individual cells, tissues or whole organisms, arising from disturbed homoeostasis of any cause. Stress is rarely localised. Because biological systems are closely integrated, it spreads to involve other systems. Stress responses are highly integrated and work to restore homoeostasis. Different response pathways overlap and interlink. If the responses fail or decompensate, distress ensues, of which the end-stage is death. Pre-eclampsia results from a series of biological stresses, possibly from conception, which become established by abnormal placentation and affect the mother, her foetus and her placenta. The stresses involve dialogue between mother and placenta. Even a normal placenta imposes substantial stress on maternal systems. When placental growth and perfusion is abnormal (poor placentation) then the placenta, particularly its outer trophoblast layer, becomes stressed - loosely denoted hypoxic damage or oxidative stress. Signals from the placenta spread the stress to the mother, who develops signs of pre-eclampsia. Cellular stress sensors initiate stress responses. Different stresses may trigger similar responses in specific cell types. The first cell response is reduced protein synthesis. However some synthetic pathways are spared or activated to produce stress signals. In relation to pre-eclampsia and the placenta, an excessive release of sFlt-1 a soluble decoy receptor for vascular endothelial growth factor (VEGF) is a trophoblast related stress signal. SFlt1 perturbs the angiogenic balance in the maternal circulation and is considered to cause many of the specific features of the maternal syndrome in pre-eclampsia. Three key points will be emphasised. First, multiple stressors, not simply hypoxia, stimulate the release of sFlt-1 from trophoblast. Second, sFlt-1 is only one of the group of stress signals delivered by trophoblast to the mother. Third, sFlt-1 is not the only trophoblast derived factor to perturb the maternal circulation in pre-eclampsia., (Copyright © 2013. Published by Elsevier B.V.)

Introduction: Circulating angiogenic factors are potential markers for preeclampsia, but heterogeneous studies have failed to identify precise predictive/diagnostic properties. The Global CoLaboratory is investigating how to merge published data of angiogenic factors for meta-analysis on an individual sample basis., Objective: To amalgamate pregnancy angiogenic factor studies, investigate diagnostic and predictive properties of these markers in preeclampsia and placenta-related pregnancy complications, and to test if measures from disparate platforms can be standardised. This is the first report using PlGF measures to diagnose preeclampsia., Methods: Data were derived from 15 cohorts, within and outside the CoLaboratory network. Women were classified as either case (confirmed diagnosis of preeclampsia at sampling) or non-case (no preeclampsia at sampling). Individual PlGF measurements from four different analytical platforms were used, along with transformations of the data (e.g. log-transformations, transformations to a baseline platform). Transformed measurements were standardised both for specific platforms and globally, stratifying on gestational age. Different statistical techniques were compared., Results: The database currently contains 1442 cases and 11,512 non-cases, which were used to define an algorithm to merge PlGF measurements from different platforms. Non-case distributions were used to standardise case results. Diagnostic PlGF measurements in relation to preeclampsia will be presented and confirm feasibility., Conclusions: Future studies can extend this approach to other angiogenic factors, prediction as well as diagnosis and to other placenta-related disorders., (Copyright © 2013. Published by Elsevier B.V.)

Preeclampsia is a serious complication of pregnancy, potentially lethal for women and offspring. Affected women have an augmented risk of later cardiovascular disease and premature death and may have risk factors in common with older persons developing cardiovascular disease. In some cases of preeclampsia, lipid-filled foam cells accumulate in the walls of the spiral arteries of the uteroplacental circulation (acute atherosis). These lesions resemble the early stages of atherosclerosis and are thought to regress after delivery. The mechanisms that contribute to acute atherosis are largely unknown, but are related to defective vascular remodeling of the spiral arteries in the first half of pregnancy. Spiral artery lipid deposition may also occur in normal pregnancies, which suggests that it may not be confined exclusively to maladapted spiral arteries or caused by hypertension. Our first hypothesis is that there are several pathways to the development of acute atherosis, which converge at the point of excessive decidual inflammation in the final common pathway. Our second hypothesis is that acute atherosis, evolving during the short time of pregnancy, identifies a subset of women at augmented risk for atherosclerosis and later chronic arterial disease better than the diagnosis of preeclampsia itself. If confirmed, this may enable better preventive management for the affected women., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: Early-onset preeclampsia is associated with a greater risk of cardiovascular disease than late-onset preeclampsia., Objectives: We tested the hypothesis that young women, with previous early-onset preeclampsia, have unique differences in long term cardiovascular phenotype compared to late-onset preeclampsia or normal pregnancy., Methods: 140 women (mean age 40 yrs) were followed up 6-13years following pregnancy. 90 had had preeclampsia (45 early onset (before 34 weeks of gestation), 45 late onset) and 50 had normotensive uncomplicated pregnancies. Women with cardiovascular risk factors present before pregnancy were excluded. Fasting lipids, glucose, insulin and circulating cytokines were measured. Central blood pressure (BP) and arterial stiffness (pulse wave velocity (PWV)/augmentation index (AI)) were assessed by applanation tonometry, common carotid intima media thickness (cIMT) by ultrasound and cutaneous capillary density by intravital microscopy. 46 women returned for assessment of cardiac structure and function by magnetic resonance and echocardiography as well as ambulatory blood pressure monitoring., Results: All women with a previous history of preeclampsia had 5-10mmHg higher peripheral and central BP (P<0.001) as well as elevated total: HDL cholesterol (P<0.003), insulin resistance (P<0.04) and circulating TNFα (P<0.007). They also had increased arterial stiffness (P<0.04) and cIMT (P<0.005). Cardiac size and systolic function were preserved but there was evidence of abnormal diastolic relaxation (E/E' -P<0.04). In contrast early-onset preeclampsia was associated with characteristic differences in peri-pregnancy blood pressure, long term ambulatory measures and microvascular function., Conclusion: Early onset preeclampsia is associated with unique features in long term cardiovascular phenotype. Pregnancy disease characteristics may identify women at greatest potential benefit from monitoring and primary prevention., (Copyright © 2010. Published by Elsevier B.V.)

Introduction: In pre-eclampsia, the consequences of poor placentation lead to the second stage of pre-eclampsia, which involves activation of a maternal systemic inflammatory response (MSIR). Endothelial and other inflammatory cellular dysfunction cause the diverse features which characterise the disorder. We have previously shown that syncytiotrophoblast microvesicles (STBM) are pro-inflammatory and circulate in increased amounts in pre-eclamptic women. We hypothesise that multiple placental "danger signals" are carried by STBM into the maternal circulation in increased amounts in PE with pro-inflammatory, anti-angiogenic and pro-coagulant activity, implicating STBM in the pathophysiology of PE., Objectives: To characterise the proteins carried by STBM from normal and PE placentas. For the first time multi-dimensional protein identification technology (MudPIT) was used to derive the proteome profiles of normal and PE placenta STBM., Methods: STBM were prepared from placentas (normal term: n=9 and PE: n=5) by dual lobe perfusion, isolated by ultracentrifugation and stored at -80°C. Normal and PE derived placenta STBM pools were then subjected to MudPIT analysis., Results: 538 proteins unique to PE STBM, 604 proteins unique to normal STBM and 1421 proteins common to both preparations were found. Preliminary analysis indicates the presence of alarmins (HSP70, and galectin 3), exosomal proteins (CD63,CD9,CD81), immunoregulatory molecules (CD26,CD200,CD47,Galectin 1), complement and complement regulatory molecules (C1q,C3,CD55,CD59 and vitronectin), amino acid transporters (CD98) and anti-angiogenic molecules (endoglin). Our analysis also reveals that proteins known to be elevated in blood before, or at, the time of pre-eclampsia are elevated or unique in STBM from PE placentas, including Fetuin A, Inter-alpha (globulin) inhibitor H4, Serum amyloid P component, Apolipoprotein H (or B2GP1) and Apolipoprotein AII. Thus, as predicted, a large number of circulating molecules are associated with STBM. The inter-relationships between proteins that are unique to either PE or normal pregnancy and the processes in which they are involved are being determined by Ingenuity Pathways Analysis software. In terms of biofunctions, preliminary analysis shows that proteins unique to PE STBM have a highly significant association (p<10(-11)) with 6 disease pathways including inflammatory, immunological, cardiovascular and reproductive system diseases and organ injury, whereas for proteins unique to normal STBM only protein synthesis was significant at the same level., Conclusion: STBM contain a heterogeneous population of vesicles that convey a large repertoire of placental proteins into the maternal circulation. The profound differences between PE and normal STBM indicate their pro-inflammatory potential., (Copyright © 2012. Published by Elsevier B.V.)

A variety of 'debris' is shed from the syncytial surface of the human placenta ranging from large deported multinuclear fragments to sub-cellular components. It is increasingly clear that at least some of this material has signalling functions. Many categories of circulating debris are increased in pre-eclampsia, and exhibit proteins that are pro-inflammatory and could contribute to the systemic inflammatory response in normal pregnancy, which is exaggerated in pre-eclampsia. It is now evident that there is a large 'hidden' population of microvesicles and nanovesicles (including exosomes) which are hard to investigate because of their size. We have used a new technology, nanoparticle tracking analysis, to measure the size and concentration of syncytiotrophoblast vesicles prepared by placental perfusion. The vesicles range in size from 50 nm to 1 μm with the majority being <500 nm (which includes both exosomes and microvesicles). We speculate whether changes not only in the numbers, but also in the size (beneficial syncytiotrophoblast exosomes and harmful microvesicles) might be important in the maternal syndrome of pre-eclampsia., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Micronutrient status is increasingly recognized to play an important role in the health and well-being of pregnant women and in the development and long-term health of the offspring. On 26th - 28th February 2009, The Child Health Foundation invited leading experts in this area to a scientific workshop at Obergurgl, Austria to review and critically discuss current knowledge, to identify issues that may need to be addressed in future recommendations, and to highlight priorities and opportunities for future research. This report summarizes updated key conclusions of the workshop with regards to micronutrients' intake and physiological role related to mother, placenta and fetus, as well as relevance for adverse pregnancy and long-term outcomes., (2011 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

The Pregenesys Consensus Meeting held in Cambridge on 13 July 2009 was organized by the Pregenesys Consortium to review and critically discuss current knowledge regarding early markers of preeclampsia, to identify priorities and opportunities for future research, to consider issues that may need to be addressed in future recommendations and to highlight key issues in cost effectiveness and national policies concerning prediction and early screening for the risk of developing preeclampsia. This report summarizes the outcome of the Consensus Meeting and draws attention to issues for further investigation with specific regard to single versus multiple markers, early versus late risk identification, and the long-term effects on both maternal and perinatal health and the need to include these in any future cost-benefit assessment., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

It is 40 years since I started researching pre-eclampsia. Much has changed but some old problems persist. These include the debate of how to define a syndrome, the inheritance and genetics of pre-eclampsia, why primiparae are so susceptible and is primipaternity important? If it is, in a multiparous pregnancy (after changing partners), the old hypothesis that pre-eclampsia is the outcome of failed maternal immunoregulation to accommodate nature's transplant - the fetus - must be confronted. These points are briefly reviewed and a four stage model of pre-eclampsia derived., (Copyright © 2010 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

In pre-eclampsia, poor placentation causes both oxidative and endoplasmic reticulum stress of the placenta. It is believed placental hypoxia stimulates excessive production of soluble fms-like tyrosine kinase 1 (sFlt-1), which binds and deactivates circulating vascular endothelial growth factor (VEGF). When maternal endothelium is deprived of VEGF it becomes dysfunctional hence leading to the clinical syndrome of the mother. In this paper the previous claim that poor placentation may predispose more to placental oxidative stress than hypoxia is reiterated. We show why pre-eclampsia is not only an endothelial disease, but also a disorder of systemic inflammation. We question that hypoxia is the only or indeed the main stimulus to release of sFlt-1; and emphasise the role of inflammatory mechanisms. Hypoxia cannot be assumed simply because hypoxia-inducible transcription factors (HIF) are upregulated. Concurrent assessments of nuclear factor-kappaB (NF-kappaB), a transcription factor for inflammatory responses are desirable to obtain a more complete picture. We point out that the pre-eclampsia placenta is the source of bioactive circulating factors other than sFlt-1 in concentrations that are much higher than in normal pregnancy. These may also contribute to the final inflammatory syndrome. We propose a modified version of the two-stage model for pre-eclampsia.

The clinical features of the maternal syndrome of pre-eclampsia can be explained by generalised maternal endothelial cell dysfunction, which is a part of a more global maternal systemic inflammatory response. There is growing evidence that these effects are associated with the shedding of cellular debris, including syncytiotrophoblast microparticles (STBM), cell-free DNA and mRNA, from the surface of the placenta (syncytiotrophoblast) into the maternal circulation. The increased shedding of this debris seen in pre-eclampsia is believed to be caused by placental ischaemia, reperfusion and oxidative stress. This study was carried out to determine whether uterine contractions during labour and subsequent placental separation lead to an acute increase in the release of placental debris into the maternal circulation. To assess the effects of labour, samples were taken from 10 normal pregnant (NP) and 10 pre-eclamptic (PE) women at varied time points. Similarly to assess the effects of placental delivery, plasma samples were taken from 10 NP and 10 PE women undergoing elective caesarean section. There was a significant increase in the shedding of STBM in pre-eclampsia which was not seen in normal pregnancy and there was a small rise in STBM levels at placental separation in both normal pregnant and pre-eclamptic women undergoing caesarean section, but the differences were not significant. However, levels of placental cell-free corticotrophin releasing hormone mRNA were significantly increased in labour in both normal pregnancy and pre-eclampsia and were still high 24 h after delivery in the pre-eclamptic women. There was no significant increase in fetal or total DNA in labour, but the overall levels of total DNA (maternal and fetal) was increased in labour in pre-eclampsia compared to normal labour. The enhanced shedding of STBM and CRH mRNA in pre-eclampsia labour may have a role in cases of postpartum worsening of pre-eclampsia.

Preeclampsia can be lethal to both mother and baby. The prominent symptoms of this syndrome are hypertension, proteinuria and oedema, resulting from an exaggerated aseptic systemic inflammatory response, triggered by placental factors shed into the maternal circulation. Syncytiotrophoblast microparticles (STBM) are one possible factor, shed when the placenta is exposed to stressors such as hypoxia/reperfusion. These can disrupt mitochondria, triggering apoptosis and necrosis, placental pathologies which are increased in preeclampsia. We tested the effects of antioxidant vitamins C (50 microM) and E (50 microM) on trophoblast in culture, using term villous cytotrophoblast preparations. Following Percoll gradient centrifugation and MHC class I expressing cell depletion of placenta digests, syncytial fragments were removed using anti-placental alkaline phosphatase antibody. This yielded cytotrophoblasts of consistently high purity. EGF (10 ng/ml) stimulated syncytialisation and hCG and progesterone production. However, mitochondrial induced apoptosis (MIA) was evident 96h post-isolation, as mitochondrial membrane potential loss and caspase 9 and caspase 3 activation. ROCK-1 cleavage and syncytiotrophoblast particle shedding increased concurrently with apoptosis induction. Vitamins blocked MIA and syncytiotrophoblast particle shedding and significantly increased hCG (p<0.005) and progesterone (p<0.02) concentrations in culture supernatants, reflecting the increased survival rates. Although more cells survived in culture, syncytialisation rate (%) was significantly reduced (p<0.005). We conclude that vitamins C and E can significantly reduce mitochondrial damage generated following syncytialisation in vitro. However, further work is required to determine whether antioxidant vitamins interfere with normal fusion processes.

Cellular particles may be larger shed microparticles (>or=100 nm, MPs) that are the products of cell activation or necrosis. There are also smaller endocytic nanoparticles (<100 nm), called exosomes, which are internal vesicles of late endosomes or multivesicular bodies and are released into the extracellular milieu upon fusion of the multivesicular body with the cell surface. Both MPs and exosomes can be detected in the circulations of non-pregnant and pregnant women. In the former MPs are increased in conditions associated with systemic inflammation such as sepsis or metabolic syndrome. During normal pregnancy MPs are increased and they increase further with pre-eclampsia. They include not only MPs derived from platelets, endothelium and various leukocytes but also syncytiotrophoblast derived MPs (often called STBMs). STBMs interact with both immune and endothelial cells and may contribute to the systemic inflammation of both normal and pre-eclamptic pregnancies. However inhibitory activity has also been ascribed to trophoblast derived exosomes. In vitro, they down-regulate T cell activity, a T cell change that has been repeatedly observed, ex vivo, during normal pregnancy.

Objectives: Vitamin D is vitally important in maintaining skeletal health. A low plasma vitamin D concentration is associated with increased parathyroid hormone secretion, increased bone turnover, osteomalacia, and osteoporosis. As a result, vitamin D deficiency is associated with a higher incidence of hip and other fractures. Although Vitamin D deficiency has been reported in long-term care facilities, optimal methods of replenishment have not been defined. The objective of the present study was to identify the pattern of calcium and vitamin D supplementation in nursing home residents and to identify vitamin D deficiency in residents already on supplement therapy., Design: Retrospective chart review., Setting: Five academic nursing homes staffed by faculty from the University of Arkansas for Medical Sciences., Participants: Elderly residents aged 65 and older receiving calcium and vitamin D supplements., Measurements: Data on dose, frequency, and levels of calcium and vitamin D were collected. The medication list and creatinine levels were also recorded., Results: Forty-four (40%) residents were receiving 1000 mg, 48 (44%) were receiving 1200 mg, and 9 (8.2%) were receiving 1500 mg of calcium carbonate. Similarly, 79 (72%) residents were on 400 IU, 13 (12%) were on 600 IU, and only 8 (7%) were on 800 IU of vitamin D3 (cholecalciferol). Low levels of Vitamin D 25 (OH) D (values <30 ng/mL) were identified in 49.4% of residents; 16% were found to have deficiency (<20 ng/mL)., Conclusion: Despite clear benefit, nursing home residents were not supplemented adequately with calcium and vitamin D.

Lipid rafts are detergent-insoluble, low-density membrane domains that are rich in cholesterol and sphingolipids; caveolae are a subdomain of the biochemically defined glycolipid raft whose expression is associated with the protein caveolin-1. This protein associates with numerous signalling molecules, regulating their activity by holding them inactive. Human villous cytotrophoblasts contain caveolin-1, but levels reduce greatly during their differentiation into syncytiotrophoblast. Since caveolin-1 is a known regulator of apoptosis and trophoblast syncytialisation involves the apoptotic cascade, we hypothesised that cytotrophoblast caveolin-1 may also play a role in regulating fusion events involved in syncytium formation. The BeWo choriocarcinoma cell line has previously proved valuable for studying trophoblast syncytialisation, hence the present work was carried out to determine whether BeWo cells could be used as a model for the exploration of caveolin-1's role in regulating the syncytialisation process. Undifferentiated BeWo cells were found to express caveolin-1 in similar amounts to villous cytotrophoblasts isolated from term placenta. Lipid raft fractions prepared from these BeWo cells at confluence contained the raft-associated proteins caveolin-1 and -2, flotillin-1 and -2, stomatin and the heterotrimeric G protein, Galphaq. Confocal immunofluorescence studies revealed that caveolin-1 is internalized to the mitochondria, but not to the Golgi or endoplasmic reticulum, in subconfluent BeWo and that the protein relocates to the plasma membrane upon confluence, an observation confirmed by caveolin-1 and cytochrome c Western blotting of lipid raft fractions and mitochondria purified from confluent and subconfluent cells. Western blotting and immunofluorescence experiments comparing undifferentiated cells and those induced to differentiate using the cAMP analogue, dibutyryl cAMP, showed that BeWo syncytialisation was accompanied by a reduction in caveolin-1 levels, similar to the situation in primary villous cytotrophoblasts. Confluent, undifferentiated BeWo cultures were then used to investigate the cellular localisation of Rock-1, a protein which promotes cytoskeletal re-organisation important for syncytialisation and apoptosis. Its association with caveolin-1 was evidenced by the demonstration that the 160kDa proenzyme form of Rock-1 co-immunoprecipitates with caveolin-1 and vice versa, as well as by the co-localisation of the two proteins at the plasma membrane, as shown in immunofluorescence studies. A proportion of the total cell Rock-1 content was found in BeWo lipid raft fractions, confirming its membrane presence in confluent cells. This close association of plasmalemmal caveolin-1 with Rock-1 protein raises the possibility that caveolin-1 may regulate Rock-1 in these trophoblasts. We conclude that cell-cell contact is required for BeWo trophoblast to exhibit plasmalemmal caveolin-1; BeWo cells at confluence offer a useful model for the study of trophoblast raft behaviour during syncytialisation and for the exploration of the potential Rock-1-regulating role of caveolin-1 in this process.

Pre-eclampsia is a common and potentially dangerous disorder of human pregnancy. The maternal syndrome of hypertension, proteinuria and oedema is part of a severe systemic inflammatory response that includes leukocyte and endothelial cell activation. Although the origins of pre-eclampsia remain unclear, a major cause is the failure to develop an adequate blood supply to the placenta, leading to placental oxidative stress. This results in the excess release of placental factors, such as syncytiotrophoblast debris or soluble fms-like tyrosine kinase-1 (sFlt-1), the soluble receptor for vascular endothelial growth factor (VEGF), into the maternal circulation, where they trigger an inflammatory response and endothelial dysfunction. Alternatively, pre-eclampsia can develop in the presence of a normal placenta in women that are susceptible to systemic inflammation, such as with chronic cardiovascular disease or diabetes. While clinical management of pre-eclampsia does not currently include anti-inflammatory agents, current research is focusing on ways to reduce inflammation and oxidative stress.

Rationale: Syncytiotrophoblast microparticles (STBM) are shed into the maternal circulation in higher amounts in pre-eclampsia compared to normal pregnancy and are believed to be the stimulus for the systemic inflammatory response and endothelial cell damage which characterises the maternal syndrome. The excess shedding of STBM may be caused by hypoxia as a result of poor placentation, which is often a feature of pre-eclampsia. Similar placental pathology occurs in some cases of normotensive intrauterine growth restriction (nIUGR), but in the absence of maternal disease., Objective: To examine whether the shedding of STBM in nIUGR occurs to the same extent as in pre-eclampsia., Methods: A prospective case-control study in a tertiary referral centre of: 1) women with early-onset pre-eclampsia (EOPET < 34 week), 2) women with late-onset pre-eclampsia (LOPET > or = 34 week), 3) women with nIUGR), 4) matched normal pregnant women (NPC), and 5) non-pregnant women. An ELISA using the antitrophoblast antibody NDOG2 was used to measure STBM levels in peripheral venous plasma. Non-parametric analyses were conducted with statistical significance set at p < 0.05., Results: STBM levels rise during normal pregnancy. EOPET was associated with increased STBM levels (EOPET (median): 41 ng/ml, n = 15) compared with matched normal pregnancy (16 ng/ml, n = 15; Wilcoxon p = 0.005). LOPET (50 ng/ml, n = 10) and nIUGR (18 ng/ml, n = 8) STBM levels did not differ from matched normal pregnancy (36 ng/ml, n = 15, and 36 ng/ml, n = 8, respectively). Background levels in non-pregnant plasma were 0.49 ng/ml, n = 10., Conclusions: Increased STBM levels were found in EOPET but not in nIUGR providing further evidence for their role in the pathogenesis of the maternal syndrome.

Placental trophoblast syncytialization is a unique biological process. We have studied the time course of this process using DNA microarray in a cell model of syncytialization (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin). Total RNA was extracted from BeWo cells and labelled-cRNA target was then hybridized to a specific oligonucleotide probe set containing probes to over 12?000 human transcripts. Detectable levels of signal were found on average for 44 per cent of the total number of genes assayed. The correlation coefficient for the level of expression of independent replicates was #10878;0.99. The mRNA expression profile of specific genes analysed by microarray correlated quantitatively well with that analysed by reverse transcription-polymerase chain reaction and with protein secretion. In the absence of forskolin there are relatively few changes in gene expression (reaching a threshold of two fold); in the presence of forskolin there are a substantial number of changes. By clustering the patterns of altered gene expression at least ten groups could be extracted. Seven of these clusters involved increased gene expression and three decreased expression. Each cluster has been categorized by gene ontology (confining the analysis to genes with 'known' function). Among the genes with increased expression following forskolin treatment were many required for cellular communication (such as placental specific peptide hormones) and metabolism (such as cholesterol side chain cleavage enzyme). Several genes known to be involved in cell adhesion and fusion have markedly changed expression levels very early following forskolin exposure, thus preceding morphological fusion of BeWo cells. Further analysis of this data and expression profiling in general will be able to contribute to understanding the functional basis for the formation of the placental syncytiotrophoblast.

Caveolae are flask-shaped invaginations of the plasma membrane found in many cell types, particularly endothelium. A major structural component is the membrane protein caveolin-1 which associates with numerous signalling molecules, including endothelial nitric oxide (eNOS). Caveolin-1, which co-immunoprecipitates with eNOS in preparations from endothelial cells, regulates eNOS activity, holding it inactive. Controversy now exists regarding the presence of caveolae and caveolin-1 in trophoblasts, hence this study was carried out to examine whether the high levels of eNOS expressed in human syncytiotrophoblast are associated with caveolin-1, and to find out if caveolae are present in villous cytotrophoblasts and syncytiotrophoblast. Immunohistochemistry of term placentae revealed only weak labelling for caveolin-1 in the syncytiotrophoblast although the endothelium of the terminal villus vessels stained strongly. By electron microscopy, numerous caveolae were identified in the villus capillary endothelium but were extremely rare in the syncytium. Caveolin-1 staining was extensive in purified, isolated term villous cytotrophoblasts, with the purity of these cytokeratin positive cells confirmed by cytospin analysis and flow cytometry. Caveolae were clearly demonstrated in ultrastructural sections of the purified cytotrophoblasts. The time course of expression of caveolin-1 and eNOS during differentiation of villous cytotrophoblast into syncytiotrophoblast in culture was studied. Western analysis showed that caveolin-1 expression evident in day 1 whole cell lysates decreased at day 3 when the cells had syncytialized and declined further by day 6, while the levels of actin (control) remained high. eNOS expression in the same samples followed a different pattern, with the low levels in day 1 cells increasing substantially by 3 days in culture, subsiding again by day 6. eNOS association with caveolin-1 in day 1 and day 3 trophoblast cultures was evidenced by the demonstration that eNOS co-immunoprecipitates with caveolin-1 and vice versa. We conclude that human villous cytotrophoblasts express caveolin-1, which assembles into caveolae. Differentiation into syncytium results in a decrease, but not disappearance, of expression of caveolin-1 and a marked reduction of the caveolae.