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.

Various studies found an association of different renin-angiotensin system (RAS) components with gestational duration and preterm birth, as well as with preeclampsia. Approximately 25% of first-time pregnant women develop a mild to severe hypertension in pregnancy or even preeclampsia. Based on recently published single-cell RNA-sequencing, we hypothesized an alternative RAS function in placenta and furthermore, an implication in hypertensive disorders in pregnancy. Placental RAS expression and localization was analyzed via quantitative polymerase chain reaction and in situ mRNA padlock probes. Tissue was collected from first-trimester elective termination (n=198), from healthy third-trimester controls (n=54), from early-onset preeclamptic (n=54) and age-matched controls (n=29), as well as first-trimester placentae from women with a high uterine artery resistance index (high-risk for preeclampsia, n=9) and controls (n=8). Serum levels of Ang (angiotensin) I to IV from women before and after conception were measured via mass spectrometry (n=10). Placental explants were cultured in 2.5% oxygen with Ang II, candesartan, and leptin. Seahorse XF96 MitoStress assays assessed trophoblast metabolism. Here, we show that maternal angiotensin acts on placental LNPEP (leucine aminopeptidase), that is, angiotensin IV-receptor and fetal angiotensin on placental AGTR1 (angiotensin II receptor type 1). Maternal circulating RAS shifts towards Ang IV in pregnancy. Ang IV decreases trophoblastic mitochondrial respiration and increases placental leptin via placental LNPEP. Lower placental LNPEP in preeclampsia and in first-trimester patients at high-risk for preeclampsia suggests a new alternative route in maternal RAS signaling and may contribute to hypertension and disease in pregnancy. The study shows how hypertensive disorders in pregnancy may be connected metabolic alterations that finally seem to contribute to the multifactorial disease in pregnancy, preeclampsia.

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.

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.

Summary: Pre-eclampsia (PE) is a syndrome that affects multiple organ systems and is the most severe hypertensive disorder in pregnancy. It frequently leads to preterm delivery, maternal and fetal morbidity and mortality and life-long complications ¹ . We currently lack efficient screening tools ^{2, 3} and early therapies ^{4, 5} to address PE. To investigate the early stages of early onset PE, and identify candidate markers and pathways, we performed spatio-temporal multi-omics profiling of human PE placentae and healthy controls and validated targets in early gestation in a longitudinal clinical cohort. We used a single-nuclei RNA-seq approach combined with spatial proteo- and transcriptomics and mechanistic in vitro signalling analyses to bridge the gap from late pregnancy disease to early pregnancy pathomechanisms. We discovered a key disruption in villous trophoblast differentiation, which is driven by the increase of transcriptional coactivator p300, that ultimately ends with a senescence-associated secretory phenotype (SASP) of trophoblasts. We found a significant increase in the senescence marker activin A in preeclamptic maternal serum in early gestation, before the development of clinical symptoms, indicating a translation of the placental syndrome to the maternal side. Our work describes a new disease progression, starting with a disturbed transition in villous trophoblast differentiation. Our study identifies potential pathophysiology-relevant biomarkers for the early diagnosis of the disease as well as possible targets for interventions, which would be crucial steps toward protecting the mother and child from gestational mortality and morbidity and an increased risk of cardiovascular disease later in life.