OBJECTIVE:Maternal physical activity (PA), sedentary time (ST) and body-mass index (BMI) might influence placental development. We examined associations between PA, ST during pregnancy and pre-pregnancy BMI, and placental structural outcomes as well as mRNA expression levels of vascular growth factors. METHODS:The FitMum randomized controlled trial included 220 healthy, pregnant women. PA and ST were objectively measured 24/7 from inclusion before gestational week 15 + 0 until delivery. This secondary analysis of FitMum includes all participants with an available placenta sample (n = 118). Placental sections were stained with immunofluorescence and H&E. Villous density as well as vessel area were assessed with Visiopharm software. Placental samples with sufficient quality (n = 107) were bulk RNA-sequenced and genes relevant for placental vascularization were selected for analysis. Associations of moderate-to-vigorous intensity PA (MVPA), ST and pre-pregnancy BMI, with villous density, vessel surface area, placental weight, and mRNA-counts, were analyzed with multiple regressions. RESULTS:Increased MVPA was associated with increased placental weight (p = 0.005), while higher pre-pregnancy BMI was associated with lower area of medium-sized and large blood vessels in the placenta (p = 0.002). No associations were observed between MVPA or ST and histologic outcomes or mRNA expression levels. CONCLUSION:PA during pregnancy was not associated with vessel per villous area or villous density nor mRNA expression levels of vascular growth factors, but with increased placental weight. High pre-pregnancy BMI was associated with lower area of medium-sized and large blood vessels in the placenta, suggesting metabolic status before pregnancy may play a key role in placental vascular development.

The 3D visualization of the maternal-fetal interface is essential for understanding human placental development, yet remains technically challenging due to tissue opacity and structural complexity. Here, we describe a robust light-sheet fluorescence microscopy workflow optimized for imaging first-trimester placental villi and decidua, which were cleared, multiplex immunofluorescence labeled, and imaged by light-sheet microscopy. Pan-cytokeratin, CD31 or CD34, and alpha smooth muscle actin staining enabled 3D reconstruction of the vascular architecture in placental villi and decidua parietalis. This workflow may aid in elevating previous 2D observations into a 3D context, offering a more comprehensive view of tissue organization at the early maternal-fetal interface.

STUDY QUESTION:Are maternal levels of moderate-to-vigorous physical activity (MVPA) and sedentary time (ST) in obese pregnant women associated with placental structural adaptations for facilitating oxygen delivery to the fetus? SUMMARY ANSWER:Higher maternal MVPA and ST are associated with a higher density of villi, a proxy measure of placental surface area for oxygen delivery to the fetus, without further added placental vessels. WHAT IS KNOWN ALREADY:Physical activity during pregnancy intermittently reduces uterine blood flow, potentially limiting placental and fetal oxygen supply. The placenta can mount several adaptive responses, including enlargement of the surface area of villi and/or feto-placental vessels to accommodate fetal needs. Early research on the morphology and growth of the placenta with exercise interventions has shown inconsistencies and is lacking, particularly in non-lean pregnant women. STUDY DESIGN, SIZE, DURATION:This study is a secondary longitudinal analysis of the vitamin D and lifestyle intervention for gestational diabetes prevention (DALI) randomized controlled trial. The prospective study was conducted between 2012 and 2015 in nine European countries at 11 different sites. In this analysis, 92 pregnant women with a BMI ≥ 29 kg/m2 were combined into one cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS:MVPA and percentage of time spent sedentary (% ST) were measured with accelerometers during gestation. Placental sections were immunostained for endothelial cell-specific CD34. Artificial intelligence (AI)-based stereology assessed villous density, number, and cross-sectional area of vessels on whole-slide images and in selected regions comprising peripheral villi only, where the majority of vascular adaptations occur. Expression of pro- and anti-angiogenic factors was quantified using molecular counting analysis. MAIN RESULTS AND THE ROLE OF CHANCE:In multivariable regression, higher levels of maternal MVPA (min/day) were associated with a higher density of villi in both whole-slide images (beta 0.12; 95% CI 0.05, 0.2) and selected regions (0.17; CI 0.07, 0.26). Unexpectedly, ST was also positively associated with density of villi (0.23; CI 0.04, 0.43). MVPA and ST were not associated with vessel count/mm2 villous area, vessel area, or pro- and anti-angiogenic factor mRNA expression. All estimates and statistical significance of the sensitivity analyses excluding smokers, women who developed gestational diabetes or pre-eclampsia and/or pregnancy-induced hypertension were similar in the main analysis. LIMITATIONS, REASONS FOR CAUTION:The placenta is a complex organ undergoing dynamic changes. While various adjustments were made to account for different maternal contributing factors, in addition to the outcome measures, various other factors could impact oxygen delivery to the fetus. WIDER IMPLICATIONS OF THE FINDINGS:For the first time, we evaluated the association between placental structures quantified using an AI-based approach with objectively measured physical activity and ST at multiple time points in pregnant women with obesity. The observed adaptations contribute to the advancement of our understanding of the hemodynamics and adaptations of the placental unit in response to MVPA and ST. However, our results might not be generalizable to lean pregnant women. STUDY FUNDING/COMPETING INTEREST(S):The DALI project has received funding from the European Community's 7th Framework Program (FP7/2007-2013) under grant agreement no. 242187. The funders had no role in study design, collection of data, analyses, writing of the article, or the decision to submit it for publication. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER:ISRCTN70595832.

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This setting maintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces new possibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This setting maintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces new possibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

The etiology of the pregnancy syndrome preeclampsia is still unclear, while most hypotheses center on the placenta as the major contributor of the syndrome. Especially changes of the placental metabolism, including the use of glucose to produce energy, are important features. As an example, inositol phosphoglycan P-type molecules, second messengers involved in the glucose metabolism of all cells, can be retrieved from maternal urine of preeclamptic women, even before the onset of clinical symptoms. Alterations in the placental metabolism may subsequently lead to negative effects on the plasma membrane of the placental syncytiotrophoblast. This in turn may have deleterious effects on the glycocalyx of this layer and a disruption of this layer in all types of preeclampsia. The interruption of the glycocalyx in preeclampsia may result in changes of inositol phosphoglycan P-type signaling pathways and the release of these molecules as well as the release of soluble receptors such as sFlt-1 and sEndoglin. The release of placental factors later affects the maternal endothelium and disrupts the endothelial glycocalyx as well. This in turn may pave the way for edema, endothelial dysfunction, coagulation, all typical symptoms of preeclampsia.

Introduction: CD24 is a mucin-like glycoprotein expressed at the surface of hematopoietic and tumor cells and was recently shown to be expressed in the first trimester placenta. As it was postulated as an immune suppressor, CD24 may contribute to maternal immune tolerance to the growing fetus. Preeclampsia (PE), a major pregnancy complication, is linked to reduced immune tolerance. Here, we explored the expression of CD24 in PE placenta in preterm and term cases. Methods: Placentas were derived from first and early second trimester social terminations (N = 43), and third trimester normal term delivery (N = 67), preterm PE (N = 18), and preterm delivery (PTD) (N = 6). CD24 expression was determined by quantitative polymerase chain reaction (qPCR) and Western blotting. A smaller cohort included 3–5 subjects each of term and early PE, and term and preterm delivery controls analyzed by immunohistochemistry. Results: A higher expression (2.27-fold) of CD24 mRNA was determined in the normal term delivery compared to first and early second trimester cases. The mRNA of preterm PE cases was only higher by 1.31-fold compared to first and early second trimester, while in the age-matched PTD group had a fold increase of 5.72, four times higher compared to preterm PE. The delta cycle threshold (ΔCt) of CD24 mRNA expression in the preterm PE group was inversely correlated with gestational age (r = 0.737) and fetal size (r = 0.623), while correlation of any other group with these parameters was negligible. Western blot analysis revealed that the presence of CD24 protein in placental lysate of preterm PE was significantly reduced compared to term delivery controls (p = 0.026). In immunohistochemistry, there was a reduction of CD24 staining in villous trophoblast in preterm PE cases compared to gestational age-matched PTD cases (p = 0.042). Staining of PE cases at term was approximately twice higher compared to preterm PE cases (p = 0.025) but not different from normal term delivery controls. Conclusion: While higher CD24 mRNA expression levels were determined for normal term delivery compared to earlier pregnancy stages, this expression level was found to be lower in preterm PE cases, and could be said to be linked to reduced immune tolerance in preeclampsia.

Human umbilical vein and artery endothelial cells (HUVEC; HUAEC), placental endothelial cells (fpAEC), and endothelial colony-forming cells (ECFC) from cord blood are a widely used model for researching placental vascular development, fetal and placental endothelial function, and the effect of adverse conditions in pregnancy thereon. However, placental vascular development and angiogenesis start in the first weeks of gestation, and adverse conditions in pregnancy may also affect endothelial function before term, suggesting that endothelial cells from early pregnancy may respond differently. Thus, we established a novel, gentle flow-through method to isolate pure human umbilical endothelial cells from first trimester (FTUEC). FTUEC were characterized and their phenotype was compared to the umbilical endothelium in situ as well as to other fetal endothelial cell models from term of gestation, i.e. HUVEC, fpAEC, ECFC. FTUEC possess a CD34-positive, juvenile endothelial phenotype, and can be expanded and passaged. We regard FTUEC as a valuable tool to study developmental processes as well as the effect of adverse insults in pregnancy in vitro.

During pregnancy, freely floating placental villi are adapted to fluid shear stress due to placental perfusion with maternal plasma and blood. In vitro culture of placental villous explants is widely performed under static conditions, hoping the conditions may represent the in utero environment. However, static placental villous explant culture dramatically differs from the in vivo situation. Thus, we established a flow culture system for placental villous explants and compared commonly used static cultured tissue to flow cultured tissue using transmission and scanning electron microscopy, immunohistochemistry, and lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) measurements. The data revealed a better structural and biochemical integrity of flow cultured tissue compared to static cultured tissue. Thus, this new flow system can be used to simulate the blood flow from the mother to the placenta and back in the most native-like in vitro system so far and thus can enable novel study designs.

The human placenta is a unique organ serving as the lung, gut, liver, and kidney of the fetus, mediating the exchange of different endogenous as well as exogenous substances and gases between the mother and fetus during pregnancy. Additionally, the placental barrier protects the fetus from a range of environmental toxins, bacterial and viral infections, since any contaminant bridging the placenta may have unforeseeable effects on embryonal and fetal development. A more recent concern in placenta research, however, involves the ability of engineered nanoparticles to cross the placental barrier and/or affect its barrier function. To advance nanoparticle risk assessment at the human placental barrier, we have developed as proof-of-principle a highly integrated placenta-on-a-chip system containing embedded membrane-bound impedance microsensor arrays capable of non-invasively monitoring placental barrier integrity. Barrier integrity is continuously and label-free evaluated using porous membrane-based interdigitated electrode structures located on top of a porous PET membrane supporting a barrier of trophoblast-derived BeWo cell barrier in the absence and presence of standardized silicon dioxide (SiO2), titanium dioxide (TiO2), and zinc oxide (ZnO) nanomaterials.

The placental barrier is crucial for the supply of nutrients and oxygen to the developing fetus and is maintained by differentiation and fusion of mononucleated cytotrophoblasts into the syncytiotrophoblast, a process only partially understood. Here transcriptome and pathway analyses during differentiation and fusion of cultured trophoblasts yielded p53 signaling as negative upstream regulator and indicated an upregulation of autophagy-related genes. We further showed p53 mRNA and protein levels decreased during trophoblast differentiation. Reciprocally, autophagic flux increased and cytoplasmic LC3B-GFP puncta became more abundant, indicating enhanced autophagic activity. In line, in human first trimester placenta p53 protein mainly localized to the cytotrophoblast, while autophagy marker LC3B as well as late autophagic compartments were predominantly detectable in the syncytiotrophoblast. Importantly, ectopic overexpression of p53 reduced levels of LC3B-II, supporting a negative regulatory role on autophagy in differentiating trophoblasts. This was also shown in primary trophoblasts and human first trimester placental explants, where pharmacological stabilization of p53 decreased LC3B-II levels. In summary our data suggest that differentiation-dependent downregulation of p53 is a prerequisite for activating autophagy in the syncytiotrophoblast.

OBJECTIVE:To investigate whether differences between early preeclampsia and early fetal growth restriction can be explained by differential placental expression patterns of sFlt-1, Flt-1, and PlGF.METHODS:Placental tissues and maternal blood samples from six cases of preeclampsia, seven IUGR, and six age-matched controls were studied for mRNA and protein levels as well as protein localization and expression intensity.RESULTS:Neither placental PlGF mRNA and protein expression nor placental villous trophoblast expression intensity of PlGF was altered by placental dysfunction.CONCLUSION:High sFlt-1 concentrations may account for diminished maternal serum PlGF levels.

The human placenta plays a crucial role as the interface between mother and fetus. It represents a unique tissue that undergoes morphological as well as functional changes on the cellular and tissue level throughout pregnancy. To better understand how the placenta works, a variety of techniques has been developed to re-create this complex physiological barrier in vitro . However, due to the low availability of freshly isolated primary cells, choriocarcinoma cell lines remain the usual suspects as in vitro models for placental research. Here, we present a comparative study on the functional aspects of the choriocarcinoma cell lines BeWo, JAR and Jeg-3, as well as the first trimester trophoblast cell line ACH-3P as placental in vitro barrier models for endocrine and transport studies. Functional assays including tight junction immunostaining, sodium fluorescein retardation, trans epithelial resistance, glucose transport, hormone secretion as well as size-dependent polystyrene nanoparticle transport were performed using the four cell types to evaluate key functional parameters of each cell line to act a relevant in vitro placental barrier model.

During the first trimester of pregnancy, extravillous trophoblasts (EVTs) invade into the decidual interstitium to the first third of the myometrium, thereby anchoring the placenta to the uterus. They also follow the endovascular and endoglandular route of invasion; plug, line and remodel spiral arteries, thus being responsible for the establishment of hemotrophic nutrition with the beginning of the second trimester and invade and open uterine glands toward the intervillous space for a histiotrophic nutrition during the first trimester. The aim of this study was to provide proof that uterine veins are invaded by EVTs similar to uterine arteries and glands in first trimester of pregnancy. Therefore, serial sections from in situ first trimester placenta were immuno-single- and immuno-double-stained to distinguish in a first step between arteries and veins and secondly between invaded and non-invaded vessels. Subsequently, invasion of EVTs into uterine vessels was quantified. Our data show that uterine veins are significantly more invaded by EVTs than uterine arteries (29.2 ± 15.7 %) during early pregnancy. Counted vessel cross sections revealed significantly higher EVT invasion into veins (59.5 ± 7.9 %) compared to arteries (29.2 ± 15.7 %). In the lumen of veins, single EVTs were repeatedly found, beside detached glandular epithelial cells or syncytial fragments. This study allows the expansion of our hitherto postulated concept of EVT invasion during first trimester of pregnancy. We suggest that invasion of EVTs into uterine veins is responsible the draining of waste and blood plasma from the intervillous space during the first trimester of pregnancy.

During pregnancy, the fetal–maternal interface establishes immune tolerance between the fetus and the mother. CD24, a mucin-like glycoprotein expressed at the surface of hematopoietic cells and diverse tumor cells, is known to interact with the sialic acid-binding immunoglobulin-type lectins (Siglecs). This interaction was assessed as a candidate complex for the immune suppression response in the placenta. CD24 was affinity purified from term placenta and characterized by SDS-PAGE, Western blot and ELISA. Binding of recombinant Siglecs to placental CD24 was evaluated by ELISA. The expression of CD24 and Siglec-10 in first trimester placental tissues was investigated by immunohistochemistry and immunofluorescence. Placental CD24 had an apparent molecular weight of 30–70 kDa consistent with its high degree of N- and O-linked glycosylation. EDTA-sensitive CD24–Siglec-10 interaction via the terminal sialic acid glycan residues of CD24 was observed. CD24 did not interact with Siglec-3 or Siglec-5. During the first trimester, and already in gestational week (GA) 8, CD24 showed high expression in villous and extravillous cytotrophoblasts. There was also a mild expression in stromal cells, while syncytiotrophoblasts were negative. Co-localization of CD24 with Siglec-10 was observed in endometrial glands and in first trimester decidual cells in close vicinity to extracellular trophoblasts. This study is the first to demonstrate the early presence of CD24 in the placenta cytotrophoblast layers, placental bed and maternal uterine glands. The presence of the CD24–Siglec-10 in these regions of fetal–maternal interactions suggests a possible role in mediating immune tolerance at the fetal–maternal interface.