Background: The placenta is an important organ for fetal and maternal health during pregnancy and impacts offspring health late in life. Defects in placental vasculature and trophoblast have been identified in several pregnancy complications. Thus, the detailed molecular profile and heterogeneity of endothelial cells and trophoblasts in placentas will aid us in better understanding placental behaviors and improving pregnancy outcomes. Methods: Single-cell RNA sequencing (scRNA-seq) was performed to profile the transcriptomics of human placental villous tissues from eleven patients with normal pregnancies in the first and second trimesters (6–16 weeks of gestation). Results: The transcriptomic landscape of 52,179 single cells was obtained, and the cells were classified as trophoblasts, fibroblasts, endothelial cells, erythroid cells, Hofbauer cells, and macrophages. Our analysis further revealed the three subtypes of placental endothelial cells, with distinct metabolic signatures and transcription factor regulatory networks. We also determined the transcriptomic features of the trophoblast subpopulations and characterized two distinct populations of progenitor cells in cytotrophoblasts, which were capable of differentiating to extravillous trophoblasts and syncytiotrophoblasts, respectively. Conclusions: Our study provided a high-resolution molecular profile of the human placenta between 6 and 16 weeks of gestation. Our data revealed the placental cell complexity and demonstrated the transcriptional networks and signaling involved in placental endothelial and trophoblast differentiation during early pregnancy, which will be a resource for future studies of the human placental development.

Background: Preeclampsia is a unique multisystem disorder that affects 5–8% of pregnancies. A high level of soluble fms-like tyrosine kinase-1 (sFlt-1) is a hallmark of preeclampsia that causes endothelial dysfunction. Exosomes derived from mesenchymal stem cells (MSCs) have been indicated to improve endothelial performances by transporting signals to target cells. We hypothesized that exosomes derived from MSCs have potential effects against preeclampsia. Methods: We collected human umbilical cord MSC-derived exosomes (HUCMSC-exos) by ultracentrifugation. The size and morphology of the exosomes were examined using a transmission electron microscope and nanoparticle tracking analysis. Pregnant mice were injected with murine sFlt-1 adenovirus to build the preeclampsia-like mouse model and then treated with HUCMSC-exos. Human umbilical vein endothelial cells (HUVECs) were infected with lentiviruses expressing tet-on-sFlt-1 to obtain cells overexpressing sFlt-1. Cell proliferation and migration assays were used to measure the endothelial functions. The exosomes enriched proteins underlying mechanisms were explored by proteomic analysis. Results: In the current study, we successfully collected the cup-shaped HUCMSC-exos with diameters of 30–150 nm. In the sFlt-1-induced preeclampsia mouse model, HUCMSC-exos exhibited beneficial effects on adverse birth events by decreasing blood pressure and improving fetal birth weight. In addition, preeclamptic dams that were injected with HUCMSC-exos had rebuilt dense placental vascular networks. Furthermore, we observed that HUCMSC-exos partially rescued sFlt-1-induced HUVECs dysfunction in vitro. Proteomics analysis of HUCMSC-exos displayed functional enrichment in biological processes related to vesicle-mediated transport, cell communication, cell migration, and angiogenesis. Conclusion: We propose that exosomes derived from HUCMSCs contain abundant Versican and play beneficial roles in the birth outcomes of sFlt-1-induced preeclamptic mice by promoting angiogenesis. Graphical

Although preterm birth (PTB) is one of the major causes of perinatal mortality and neonatal morbidity, little is known about its complex etiology. An abnormal cervicovaginal microbiome during pregnancy is associated with an increased risk of PTB. The cervicovaginal microbiota and its active metabolites, such as short-chain fatty acids (SCFAs), might be effectively used to predict and diagnose PTB. However, the roles of these proteins and the underlying mechanisms involved remain elusive. We conducted 16S rRNA gene sequencing and used a targeted metabolomics approach to study cervicovaginal swabs obtained from 51 singleton pregnancies and 52 twin pregnancies in the second trimester. Next, functional in vitro experiments were performed to investigate the roles and mechanisms of SCFAs in placental trophoblast cells (HTR8/SVneo cells). Significant cervicovaginal microbiome dysbiosis, characterized by a substantial reduction in the abundance of lactobacilli and overgrowth of anaerobes, was revealed in the second trimester and was strongly associated with subsequent PTB ( P = 0.036). Among the paired samples ( n = 103), acetic acid was significantly greater in the preterm group than in the term group ( P = 0.047). Data obtained from integrated gas chromatography‒mass spectrometry and 16S RNA studies revealed metabolites that were distinctly associated with particular microbial communities. Gardnerella vaginalis was the species most positively associated with acetic acid content. In addition, we identified a marker set consisting of the pregnancy type, acetic acid concentration, and community state type to accurately diagnose PTB. Acetate was associated with increased interleukin (IL)-8 and IL-6 levels and extravillous trophoblast cell migration and invasion through the activation of the extracellular signal-regulated kinase 1/2 signaling pathway in HTR8/SVneo cells. Cervicovaginal microbiota dysbiosis is an important etiological factor of PTB. The cervicovaginal microbiota and its active metabolites can be efficiently used to predict and diagnose PTB. Our findings enrich the microbiota–placenta axis theory and contribute to the development of microecological products for pregnancy. IMPORTANCE: Preterm birth (PTB) is a leading cause of infant mortality and long-term health issues, affecting millions of families worldwide. Despite its prevalence, the exact causes of PTB remain unclear. Our study reveals that certain bacteria and their metabolic byproducts in the cervicovaginal environment, specifically short-chain fatty acids (SCFAs), are linked to the risk of preterm birth. By analyzing samples from pregnant women, we found that an imbalance in the vaginal microbiota and increased levels of SCFAs are associated with changes in cells that can lead to early labor. This research provides new insights into how the microbiome influences pregnancy outcomes and highlights potential biomarkers for predicting preterm birth. Understanding these microbial influences could lead to innovative strategies for early diagnosis and prevention, ultimately improving maternal and infant health.

Preeclampsia (PE) is a pregnancy-specific, multisystemic disorder that affects 2–8% pregnancies worldwide and is a leading cause of maternal and perinatal mortality. At present, there is no cure for PE apart from delivery the placenta. Therefore, it is important and urgent to possess a suitable animal model to study the pathology and treatment of PE. When exogenous soluble fms-like tyrosine kinase-1 (sFlt-1) is administered, pregnant animals develop a PE-like phenotype. However, there is no report on the comparison between different methods of constructing PE mouse models using sFlt-1. In this study, the adenovirus carrying sFlt-1(ADV-Flt-1) and recombinant murine sFlt-1 protein (RM Flt-1) are two different methods were used to induce and compare PE-like mouse models. Pregnancy outcomes were examined on E14.5 and E17.5. Our data showed that on E14.5, the adenovirus carrying sFlt-1 induced PE-like phenotype, whereas recombinant murine sFlt-1 protein not. On E17.5, both the two methods induced PE-like phenotype including hypertension, proteinuria, fetal growth restriction, placental and glomerular endotheliosis. Importantly, in the adenoviral-mediated sFlt-1 group, the circulating concentration of sFlt-1 were higher than in the recombinant sFlt-1 group, leading to earlier and more severe symptoms of PE. The ADV-Flt-1 group is easy to operate, quickly effective and efficient. The RM Flt-1 group is safer and more stable, with good repeatability, but slower to take effect. We proposed that the adenoviral-mediated sFlt-1 model can better simulate early-onset and severe PE.