Senescent Syncytiotrophoblast Secretion During Early Onset Preeclampsia.

BACKGROUND: Preeclampsia is a severe hypertensive disorder in pregnancy that causes preterm delivery, maternal and fetal morbidity, mortality, and life-long sequelae. Understanding the pathogenesis of preeclampsia is a critical first step toward protecting mother and child from this syndrome and increased risk of cardiovascular disease later in life. However, effective early predictive tests and therapies for preeclampsia are scarce. METHODS: To identify novel markers and signaling pathways for early onset preeclampsia, we profiled human maternal-fetal interface units (fetal villi and maternal decidua) from early onset preeclampsia and healthy controls using single-nucleus RNA sequencing combined with spatial transcriptomics. The placental syncytiotrophoblast is in direct contact with maternal blood and forms the barrier between fetal and maternal circulation. RESULTS: We identified different transcriptomic states of the endocrine syncytiotrophoblast nuclei with patterns of dysregulation associated with a senescence-associated secretory phenotype and a spatial dysregulation of senescence in the placental trophoblast layer. Elevated senescence markers were validated in placental tissues of clinical multicenter cohorts. Importantly, several secreted senescence-associated secretory phenotype factors were elevated in maternal blood already in the first trimester. We verified the secreted senescence markers, PAI-1 (plasminogen activator inhibitor 1) and activin A, as identified in our single-nucleus RNA sequencing model as predictive markers before clinical preeclampsia diagnosis. CONCLUSIONS: This indicates that increased syncytiotrophoblast senescence appears weeks before clinical manifestation of early onset preeclampsia, suggesting that the dysregulated preeclamptic placenta starts with higher cell maturation resulting in premature and increased senescence-associated secretory phenotype release. These senescence-associated secretory phenotype markers may serve as an additional early diagnostic tool for this syndrome.

Trophoblast Side-Population Markers are Dysregulated in Preeclampsia and Fetal Growth Restriction.

Dysregulated progenitor cell populations may contribute to poor placental development and placental insufficiency pathogenesis. Side-population cells possess progenitor properties. Recent human trophoblast side-population isolation identified enrichment of 8 specific genes (CXCL8, ELL2, GATA6, HK2, HLA-DPB1, INTS6, SERPINE3 and UPP1) (Gamage et al. 2020, Stem Cell Rev Rep). We characterised these trophoblast side-population markers in human placenta and in placental insufficiency disorders: preeclampsia and fetal growth restriction (FGR). Trophoblast side-population markers localised to mononuclear trophoblasts lining the placental villous basement membrane in preterm control, preeclamptic and FGR placental sections (n = 3, panel of 3 markers/serial section). Analysis of single-cell transcriptomics of an organoid human trophoblast stem cell (hTSC) to extravillous trophoblast (EVT) differentiation model (Shannon et al. 2022, Development) identified that all side-population genes were enriched in mononuclear trophoblast and trophoblasts committed to differentiation under hTSC culture conditions. In vitro validation via 96 h time course hTSC differentiation to EVTs or syncytiotrophoblasts (n = 5) demonstrated ELL2 and HK2 increased with differentiation (p < 0.0024, p < 0.0039 respectively). CXCL8 and HLA-DPB1 were downregulated (p < 0.030, p < 0.011 respectively). GATA6 and INTS6 increased with EVT differentiation only, and UPP1 reduced with syncytialisation. SERPINE3 was undetectable. Trophoblast side-population marker mRNA was measured in human placentas (< 34-weeks' gestation; n = 78 preeclampsia, n = 30 FGR, and n = 18 gestation-matched controls). ELL2, HK2 and CXCL8 were elevated in preeclamptic (p = 0.0006, p < 0.0001, p = 0.0335 respectively) and FGR placentas (p = 0.0065, p < 0.0001, p = 0.0001 respectively) versus controls. Placental GATA6 was reduced in pregnancies with preeclampsia and FGR (p = 0.0014, p = 0.0146 respectively). Placental INTS6 was reduced with FGR only (p < 0.0001). This study identified the localisation of a unique trophoblast subset enriched for side-population markers. Aberrant expression of some side-population markers may indicate disruptions to unique trophoblast subtypes in placental insufficiency.

Fluid shear stress induces a shift from glycolytic to amino acid pathway in human trophoblasts.

BACKGROUND: The human placenta, a tissue with a lifespan limited to the period of pregnancy, is exposed to varying shear rates by maternal blood perfusion depending on the stage of development. In this study, we aimed to investigate the effects of fluidic shear stress on the human trophoblast transcriptome and metabolism. RESULTS: Based on a trophoblast cell line cultured in a fluidic flow system, changes caused by shear stress were analyzed and compared to static conditions. RNA sequencing and bioinformatics analysis revealed an altered transcriptome and enriched gene ontology terms associated with amino acid and mitochondrial metabolism. A decreased GLUT1 expression and reduced glucose uptake, together with downregulated expression of key glycolytic rate-limiting enzymes, hexokinase 2 and phosphofructokinase 1 was observed. Altered mitochondrial ATP levels and mass spectrometry data, suggested a shift in energy production from glycolysis towards mitochondrial oxidative phosphorylation. This shift in energy production could be supported by increased expression of glutamic-oxaloacetic transaminase variants in response to shear stress as well as under low glucose availability or after silencing of GLUT1. The shift towards amino acid metabolic pathways could be supported by significantly altered amino acid levels, like glutamic acid, cysteine and serine. Downregulation of GLUT1 and glycolytic rate-limiting enzymes, with concomitant upregulation of glutamic-oxaloacetic transaminase 2 was confirmed in first trimester placental explants cultured under fluidic flow. In contrast, high fluid shear stress decreased glutamic-oxaloacetic transaminase 2 expression in term placental explants when compared to low flow rates. Placental tissue from pregnancies with intrauterine growth restriction are exposed to high shear rates and showed also decreased glutamic-oxaloacetic transaminase 2, while GLUT1 was unchanged and glycolytic rate-limiting enzymes showed a trend to be upregulated. The results were generated by using qPCR, immunoblots, quantification of immunofluorescent pictures, padlock probe hybridization, mass spectrometry and FRET-based measurement. CONCLUSION: Our study suggests that onset of uteroplacental blood flow is accompanied by a shift from a predominant glycolytic- to an alternative amino acid converting metabolism in the villous trophoblast. Rheological changes with excessive fluidic shear stress at the placental surface, may disrupt this alternative amino acid pathway in the syncytiotrophoblast and could contribute to intrauterine growth restriction.

Maternal COVID-19 causing intrauterine foetal demise with microthrombotic placental insufficiency: a case report.

BACKGROUND: Pregnant women have an increased risk of getting infected with SARS-CoV-2 and are more prone to severe illness. Data on foetal demise in affected pregnancies and its underlying aetiology is scarce and pathomechanisms remain largely unclear. CASE: Herein we present the case of a pregnant woman with COVID-19 and intrauterine foetal demise. She had no previous obstetric or gynaecological history, and presented with mild symptoms at 34 + 3 weeks and no signs of foetal distress. At 35 + 6 weeks intrauterine foetal death was diagnosed. In the placental histopathology evaluation, we found inter- and perivillous fibrin depositions including viral particles in areas of degraded placental anatomy without presence of viral entry receptors and SARS-CoV-2 infection of the placenta. CONCLUSION: This case demonstrates that maternal SARS-CoV-2 infection in the third trimester may lead to an unfavourable outcome for the foetus due to placental fibrin deposition in maternal COVID-19 disease possibly via a thrombogenic microenvironment, even when the foetus itself is not infected.

Can single-cell and spatial omics unravel the pathophysiology of pre-eclampsia?

Pre-eclampsia is a leading cause of maternal and fetal morbidity and mortality. Characterised by the onset of hypertension and proteinuria in the second half of pregnancy, it can lead to maternal end-organ injury such as cerebral ischemia and oedema, pulmonary oedema and renal failure, and potentially fatal outcomes for both mother and fetus. The causes of the different maternal end-organ phenotypes of pre-eclampsia and why some women develop pre-eclampsia condition early in pregnancy have yet to be elucidated. Omics methods include proteomics, genomics, metabolomics, transcriptomics. These omics techniques, previously mostly used on bulk tissue and individually, are increasingly available at a single cellular level and can be combined with each other. Multi-omics techniques on a single-cell or spatial level provide us with a powerful tool to understand the pathophysiology of pre-eclampsia. This review will explore the status of omics methods and how they can and could contribute to understanding the pathophysiology of pre-eclampsia.

CD39 abrogates platelet-derived factors induced IL-1ß expression in the human placenta.

Tissue insults in response to inflammation, hypoxia and ischemia are accompanied by the release of ATP into the extracellular space. There, ATP modulates several pathological processes, including chemotaxis, inflammasome induction and platelet activation. ATP hydrolysis is significantly enhanced in human pregnancy, suggesting that increased conversion of extracellular ATP is an important anti-inflammatory process in preventing exaggerated inflammation, platelet activation and hemostasis in gestation. Extracellular ATP is converted into AMP, and subsequently into adenosine by the two major nucleotide-metabolizing enzymes CD39 and CD73. Here, we aimed to elucidate developmental changes of placental CD39 and CD73 over gestation, compared their expression in placental tissue from patients with preeclampsia and healthy controls, and analyzed their regulation in response to platelet-derived factors and different oxygen conditions in placental explants as well as the trophoblast cell line BeWo. Linear regression analysis showed a significant increase in placental CD39 expression, while at the same time CD73 levels declined at term of pregnancy. Neither maternal smoking during first trimester, fetal sex, maternal age, nor maternal BMI revealed any effects on placental CD39 and CD73 expression. Immunohistochemistry detected both, CD39 and CD73, predominantly in the syncytiotrophoblast layer. Placental CD39 and CD73 expression were significantly increased in pregnancies complicated with preeclampsia, when compared to controls. Cultivation of placental explants under different oxygen conditions had no effect on the ectonucleotidases, whereas presence of platelet releasate from pregnant women led to deregulated CD39 expression. Overexpression of recombinant human CD39 in BeWo cells decreased extracellular ATP levels after culture in presence of platelet-derived factors. Moreover, platelet-derived factors-induced upregulation of the pro-inflammatory cytokine, interleukin-1ß, was abolished by CD39 overexpression. Our study shows that placental CD39 is upregulated in preeclampsia, suggesting an increasing demand for extracellular ATP hydrolysis at the utero-placental interface. Increased placental CD39 in response to platelet-derived factors may lead to enhanced conversion of extracellular ATP levels, which in turn could represent an important anti-coagulant defense mechanism of the placenta.

Platelet-derived factors dysregulate placental sphingosine-1-phosphate receptor 2 in human trophoblasts.

RESEARCH QUESTION: Sphingosine-1-phosphate (S1P) is an essential and bioactive sphingolipid with various functions, which acts through five different G-protein-coupled receptors (S1PR1-5). What is the localization of S1PR1-S1PR3 in the human placenta and what is the effect of different flow rates, various oxygen concentrations and platelet-derived factors on the expression profile of S1PR in trophoblasts? DESIGN: Expression dynamics of placental S1PR1-S1PR3 were determined in human first trimester (n = 10), pre-term (n = 9) and term (n = 10) cases. Furthermore, the study investigated the expression of these receptors in different primary cell types isolated from human placenta, verified the findings with publicly available single-cell RNA-Seq data from first trimester and immunostaining of human first trimester and term placentas. The study also tested whether the placental S1PR subtypes are dysregulated in differentiated BeWo cells under different flow rates, different oxygen concentrations or in the presence of platelet-derived factors. RESULTS: Quantitative polymerase chain reaction revealed that S1PR2 is the predominant placental S1PR in the first trimester and reduces towards term (P < 0.0001). S1PR1 and S1PR3 increased from first trimester towards term (P < 0.0001). S1PR1 was localized in endothelial cells, whereas S1PR2 and S1PR3 were predominantly found in villous trophoblasts. Furthermore, S1PR2 was found to be significantly down-regulated in BeWo cells when co-incubated with platelet-derived factors (P = 0.0055). CONCLUSION: This study suggests that the placental S1PR repertoire is differentially expressed across gestation. S1PR2 expression in villous trophoblasts is negatively influenced by platelet-derived factors, which could contribute to down-regulation of placental S1PR2 over time of gestation as platelet presence and activation in the intervillous space increases from the middle of the first trimester onwards.

Candesartan Does Not Activate PPARγ and Its Target Genes in Early Gestation Trophoblasts.

Angiotensin II receptor 1 blockers are commonly used to treat hypertension in women of childbearing age. While the fetotoxic effects of these drugs in the second and third trimesters of pregnancy are well documented, their possible impacts on placenta development in early gestation are unknown. Candesartan, a member of this group, also acts as a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, a key regulator shown to be important for placental development. We have previously shown that trophoblasts do not express the candesartan target-receptor angiotensin II type 1 receptor AGTR1. This study investigated the possible role of candesartan on trophoblastic PPARγ and its hallmark target genes in early gestation. Candesartan did not affect the PPARγ protein expression or nuclear translocation of PPARγ. To mimic extravillous trophoblasts (EVTs) and cytotrophoblast/syncytiotrophoblast (CTB/SCT) responses to candesartan, we used trophoblast cell models BeWo (for CTB/SCT) and SGHPL-4 (EVT) cells as well as placental explants. In vitro, the RT-qPCR analysis showed no effect of candesartan treatment on PPARγ target genes in BeWo or SGHPL-4 cells. Treatment with positive control rosiglitazone, another PPARγ agonist, led to decreased expressions of LEP and PPARG1 in BeWo cells and an increased expression of PPARG1 in SGHPL-4 cells. Our previous data showed early gestation-placental AGTR1 expression in fetal myofibroblasts only. In a CAM assay, AGTR1 was stimulated with angiotensin II and showed increased on-plant vessel outgrowth. These results suggest candesartan does not negatively affect PPARγ or its target genes in human trophoblasts. More likely, candesartan from maternal serum may first act on fetal-placental AGTR1 and influence angiogenesis in the placenta, warranting further research.

Automated Quantitative Image Evaluation of Antigen Retrieval Methods for 17 Antibodies in Placentation and Implantation Diagnostic and Research.

Immunostaining in clinical routine and research highly depends on standardized staining methods and quantitative image analyses. We qualitatively and quantitatively compared antigen retrieval methods (no pretreatment, pretreatment with pepsin, and heat-induced pretreatment with pH 6 or pH 9) for 17 antibodies relevant for placenta and implantation diagnostics and research. Using our newly established, comprehensive automated quantitative image analysis approach, fluorescent signal intensities were evaluated. Automated quantitative image analysis found that 9 out of 17 antibodies needed antigen retrieval to show positive staining. Heat induction proved to be the most efficient form of antigen retrieval. Eight markers stained positive after pepsin digestion, with ß-hCG and vWF showing enhanced staining intensities. To avoid the misinterpretation of quantitative image data, the qualitative aspect should always be considered. Results from native placental tissue were compared with sections of a placental invasion model based on thermo-sensitive scaffolds. Immunostaining on placentas in vitro leads to new insights into fetal development and maternal pathophysiological pathways, as pregnant women are justifiably excluded from clinical studies. Thus, there is a clear need for the assessment of reliable immunofluorescent staining and pretreatment methods. Our evaluation offers a powerful tool for antibody and pretreatment selection in placental research providing objective and precise results.

Molecular characteristics of established trophoblast-derived cell lines.

INTRODUCTION: Research on human placental development and function lacks a conclusive in vivo model. To investigate the intracellular molecular mechanisms in trophoblast cells, different cell lines have been established during the last decades. So far, none of these accomplishes all features of primary trophoblast, thus their suitability as well as the transferability of the results has been discussed. The aim of this study is to assess molecular markers and features matching different trophoblast subpopulations in trophoblastic cell lines to provide orientation on their suitability and relevance for distinct research questions. METHODS: The commonly used trophoblastic cell lines, BeWo, JEG-3, HTR-8/SVneo, AC1-M59, AC1-M32, ACH-3P and Swan71 were selected. qPCR and immunoblotting were used to determine expression of characteristic molecular markers. C14MC, C19MC and miR-371-3 miRNA expression were investigated by real time PCR. Proliferation, migration and network stabilization assays were performed. Hormone secretion was determined by chemiluminescent-immunoassays. DNA profiles were obtained by Short Tandem Repeat (STR)-genotyping. RESULTS: Immortalized cell lines differ from choriocarcinoma-derived ones in the expression of HLA-G, E-cadherin, N-cadherin, VE-cadherin, cadherin-11, cytokeratin 7, vimentin, ADAM12 and PRG2. Compared to choriocarcinoma-derived cell lines, expression of C19MC and hormone secretion were almost absent in immortalized cell lines. Conversely, they express C14MC and exhibit higher migration and network stabilization. DISCUSSION: The data presented will help justify the use of a cell line to evaluate distinct features of trophoblast biology and pathology. In general, characteristics and markers of choriocarcinoma derived cell lines seem to be more similar to in vivo trophoblast than immortalized cell lines and thus might be regarded as more suitable models.

Maternal Angiotensin Increases Placental Leptin in Early Gestation via an Alternative Renin-Angiotensin System Pathway: Suggesting a Link to Preeclampsia.

[Figure: see text].

Maternal platelets pass interstices of trophoblast columns and are not activated by HLA-G in early human pregnancy.

In early human gestation, maternal arterial blood flow into the intervillous space of the developing placenta is obstructed by invaded trophoblasts, which form cellular plugs in uterine spiral arteries. These trophoblast plugs have recently been described to be loosely cohesive with clear capillary-sized channels into the intervillous space by 7 weeks of gestation. Here, we analysed localisation of maternal platelets at the maternal-foetal interface of human first trimester pregnancy, and tested the hypothesis whether HLA-G, which is primarily expressed by extravillous trophoblasts, affects aggregation and adhesion of isolated platelets. Immunohistochemistry of first trimester placental sections localised maternal platelets in vessel-like channels and adjacent intercellular gaps of extravillous trophoblasts in distal parts of columns. Furthermore, this localisation was confirmed by transmission electron microscopy. Neither co-incubation of HLA-G overexpressing JAR cells with isolated platelets, nor incubation with cell-derived soluble HLA-G or recombinant HLA-G affected platelet adhesion and aggregation. Our study suggests that maternal platelets flow through vessel-like channels of distal trophoblast columns and spread into adjacent lateral intercellular gaps, where platelet-derived factors could contribute to trophoblast differentiation into the invasive phenotype.

Platelet-derived factors impair placental chorionic gonadotropin beta-subunit synthesis.

During histiotrophic nutrition of the embryo, maternal platelets may be the first circulating maternal cells that find their way into the placental intervillous space through narrow intertrophoblastic gaps within the plugs of spiral arteries. Activation of platelets at the maternal-fetal interface can influence trophoblast behavior and has been implicated in serious pregnancy pathologies. Here, we show that platelet-derived factors impaired expression and secretion of the human chorionic gonadotropin beta-subunit (ßhCG) in human first trimester placental explants and the trophoblast cell line BeWo. Impaired ßhCG synthesis was not the consequence of hampered morphological differentiation, as assessed by analysis of differentiation-associated genes and electron microscopy. Platelet-derived factors did not affect intracellular cAMP levels and phosphorylation of CREB, but activated Smad3 and its downstream-target plasminogen activator inhibitor (PAI)-1 in forskolin-induced BeWo cell differentiation. While TGF-ß type I receptor inhibitor SB431542 did not restore impaired ßhCG production in response to platelet-derived factors, Smad3 inhibitor SIS3 interfered with CREB activation, suggesting an interaction of cAMP/CREB and Smad3 signaling. Sequestration of transcription co-activators CBP/p300, known to bind both CREB and Smad3, may limit ßhCG production, since CBP/p300 inhibitor C646 significantly restricted its forskolin-induced upregulation. In conclusion, our study suggests that degranulation of maternal platelets at the early maternal-fetal interface can impair placental ßhCG production, without substantially affecting morphological and biochemical differentiation of villous trophoblasts. KEY MESSAGES: Maternal platelets can be detected on the surface of the placental villi and in intercellular gaps of trophoblast cell columns from gestational week 5 onwards. Platelet-derived factors impair hCG synthesis in human first trimester placenta. Platelet-derived factors activate Smad3 in trophoblasts. Smad3 inhibitor SIS3 interferes with forskolin-induced CREB signaling. Sequestration of CBP/p300 by activated Smad3 may limit placental hCG production.

Disturbed Cardiorespiratory Adaptation in Preeclampsia: Return to Normal Stress Regulation Shortly after Delivery?

Women with pregnancies complicated by preeclampsia appear to be at increased risk of metabolic and vascular diseases in later life. Previous research has also indicated disturbed cardiorespiratory adaptation during pregnancy. The aim of this study was to follow up on the physiological stress response in preeclampsia several weeks postpartum. A standardized laboratory test was used to illustrate potential deviations in the physiological stress responding to mildly stressful events of the kind and intensity in which they regularly occur in further everyday life after pregnancy. Fifteen to seventeen weeks postpartum, 35 women previously affected by preeclampsia (19 mild, 16 severe preeclampsia), 38 women after uncomplicated pregnancies, and 51 age-matched healthy controls were exposed to a self-relevant stressor in a standardized stress-reactivity protocol. Reactivity of blood pressure, heart rate, stroke index, and systemic vascular resistance index as well as baroreceptor sensitivity were analyzed. In addition, the mutual adjustment of blood pressure, heart rate, and respiration, partitioned for influences of the sympathetic and the parasympathetic branches of the autonomic nervous system, were quantified by determining their phase synchronization. Findings indicated moderately elevated blood pressure levels in the nonpathological range, reduced stroke volume, and elevated systemic vascular resistance in women previously affected by preeclampsia. Despite these moderate abnormalities, at the time of testing, women with previous preeclampsia did not differ from the other groups in their physiological response patterns to acute stress. Furthermore, no differences between early, preterm, and term preeclampsia or mild and severe preeclampsia were observed at the time of testing. The findings suggest that the overall cardiovascular responses to moderate stressors return to normal in women who experience a pregnancy with preeclampsia a few weeks after delivery, while the operating point of the arterial baroreflex is readjusted to a higher pressure. Yet, their regulation mechanisms may remain different.

Placental CX3CL1 is Deregulated by Angiotensin II and Contributes to a Pro-Inflammatory Trophoblast-Monocyte Interaction.

CX3CL1, which is a chemokine involved in many aspects of human pregnancy, is a membrane-bound chemokine shed into circulation as a soluble isoform. Placental CX3CL1 is induced by inflammatory cytokines and is upregulated in severe early-onset preeclampsia. In this study, the hypothesis was addressed whether angiotensin II can deregulate placental CX3CL1 expression, and whether CX3CL1 can promote a pro-inflammatory status of monocytes. qPCR analysis of human placenta samples (n = 45) showed stable expression of CX3CL1 and the angiotensin II receptor AGTR1 throughout the first trimester, but did not show a correlation between both or any influence of maternal age, BMI, and gestational age. Angiotensin II incubation of placental explants transiently deregulated CX3CL1 expression, while the angiotensin II receptor antagonist candesartan reversed this effect. Overexpression of recombinant human CX3CL1 in SGHPL-4 trophoblasts increased adhesion of THP-1 monocytes and significantly increased IL8, CCL19, and CCL13 in co-cultures with human primary monocytes. Incubation of primary monocytes with CX3CL1 and subsequent global transcriptome analysis of CD16⁺ subsets revealed 81 upregulated genes, including clusterin, lipocalin-2, and the leptin receptor. Aldosterone synthase, osteopontin, and cortisone reductase were some of the 66 downregulated genes present. These data suggest that maternal angiotensin II levels influence placental CX3CL1 expression, which, in turn, can affect monocyte to trophoblast adhesion. Release of placental CX3CL1 could promote the pro-inflammatory status of the CD16⁺ subset of maternal monocytes.