Sandeep Raha

Introduction: Preeclampsia (PE) is a hypertensive pregnancy disorder in which placental dysfunction is thought to play a central role. Although hypoxia is widely used to model PE-associated syncytiotrophoblast (ST) dysfunction in vitro, considerable variability in oxygen control conditions and hypoxic exposure paradigms has limited standardization across studies. This study aimed to establish physiologically relevant oxygen control conditions and identify a hypoxic exposure paradigm that consistently recapitulates characteristic features of PE-associated ST dysfunction. Methods: Human blastocyst-derived trophoblast stem cells were differentiated into STs and exposed to physiological (8% O2), atmospheric (21% O2), or hypoxic (1% O2) conditions for 24 or 48 h. Angiogenic and apoptotic marker expression, cell death, and mitochondrial stress were assessed. Results: Compared with atmospheric oxygen, 8% O2 reduced cell death and more closely reflected physiological ST conditions. Acute hypoxia (24 h) induced a robust PE-like phenotype in STs characterized by induction of anti-angiogenic markers, sFlt-1 and sEng, reduced expression of the pro-angiogenic factor PlGF, and increased cell death, apoptotic signalling and mitochondrial stress. Notably, induction of several of these markers was attenuated or reversed following prolonged hypoxic exposure. Conclusions: These findings identify 8% O2 as a physiologically relevant control condition for modelling PE-associated ST dysfunction relative to 21% O2, and demonstrate that 24 h of severe hypoxia more consistently reproduces key molecular and cellular features of PE than prolonged hypoxia. This work establishes a framework for modelling hypoxia-induced ST dysfunction while highlighting the importance of oxygen control conditions and hypoxic exposure duration when modeling PE in vitro.

Background: Prenatal cannabis use is increasing, yet the effects of inhaled cannabis smoke on placental molecular function remain poorly defined. Objective: To characterize placental transcriptomic alterations following prenatal cannabis smoke exposure using a physiologically relevant murine inhalation model. Methods: Timed-pregnant CD-1 mice were exposed to cannabis cigarette smoke or room air from embryonic day (E)6.5 to 18.5. Placentae were collected at E18.5 for microarray-based transcriptomic profiling. Differential gene expression was assessed using fetal sex adjusted linear models and false discovery rate correction. Gene set variation and ranked pathway enrichment were performed to evaluate compartment-associated and biological transcriptional programs. Selected genes were validated by quantitative PCR. Results: Large-scale transcriptomic profiles revealed no clear visual clustering by exposure; however, 23 genes were differentially expressed (FDR 0.5). Labyrinth-associated transcriptional signatures were reduced, whereas junctional- associated signatures were increased in exposed placentae. Pathway analyses revealed negative enrichment of fatty acid metabolism, bioenergetic, and transmembrane transport pathways, alongside positive enrichment of RNA processing and transcriptional-regulatory pathways. Quantitative PCR confirmed altered expression of multiple transport-related genes. Electron transport chain-associated gene expression was altered despite preserved placental respiratory measurements. Conclusions: Prenatal cannabis smoke exposure is associated with subtle yet coordinated placental transcriptional alterations involving metabolic, transport-associated, and gene-regulatory pathways, alongside compartment-associated transcriptional shifts. These findings provide insight into placental responses to whole cannabis smoke exposure and identify pathways that may contribute to altered placental function during pregnancy. Graphical

Successful pregnancy outcomes largely depend on the functional integrity of the placental barrier, which serves as a crucial interface between the maternal and fetal bloodstreams. Accurate modeling of the placental barrier is essential for understanding its physiological processes, developing therapeutics, and conducting transport studies for drug discovery and development that could have averted clinical disasters such as thalidomide-induced teratogenesis. Current models often fail to mimic the placental barrier's complex multilayer structure and consistently form nonleaky barriers due to traditional cell-seeding techniques and the inability to replicate the intricate architecture of the placental barrier. To our knowledge, a true 3D model that recapitulates the multilayer structure, mimics the syncytiotrophoblast layer with high confluency, facilitates cell-extracellular matrix interactions, and supports transport studies under dynamic conditions has not been previously reported. Here, we utilize the self-assembly process of BeWo placental trophoblastic cells and collagen I, a major component of the placental extracellular matrix, to create 3D tubular constructs. These constructs represent a significant advancement in the fabrication of physiologically relevant placental barrier models, enabling the modeling of the syncytiotrophoblast layer with underlying supportive cytotrophoblast layers and allowing for transport studies under dynamic conditions. Fluorescence imaging confirmed syncytial fusion and the formation of a continuous barrier. Permeability tests demonstrated that our model closely mirrors the in vivo transport properties of the placental barrier, highlighting the dominant role of the syncytiotrophoblast layer in governing the placental barrier transport. These models can be utilized for further transport studies, drug development and delivery, and placental barrier tissue engineering.

Cannabis use during pregnancy continues to increase with smoking remaining the most common mode of consumption. While clinical studies highlight an association between prenatal cannabis use and adverse pregnancy outcomes, less is known about placental outcomes, even though many of the reported pregnancy outcomes are thought to be mediated via placental dysfunction. Here, we established a mouse model of gestational cannabis smoke exposure to investigate the impacts on fetal outcomes and placental structure and function. Pregnant CD1 mice were exposed daily to Δ9-tetrahydrocannabinol (THC)-dominant cannabis smoke (12–14% THC, 0–2% CBD) or filtered air from embryonic day (E)6.5 to E18.5 or parturition. Cannabinoid analyses in cannabis smoke-exposed, paired maternal and fetal livers revealed total THC and 11-Nor-9-carboxy-THC (THCA) concentrations of 135.95 ± 13.60 ng/g and 30.84 ± 4.68 ng/g, respectively. Moreover, Cyp1a1 , a smoke-inducible enzyme, was induced by 4-fold in cannabis smoke-exposed placentae. No changes in offspring body weights were observed; however, there was a marked decrease in the brain-to-body weight ratio of exposed postnatal day 1 (PND1) offspring. Placentae from exposed dams were significantly reduced in size, with altered zonation marked by a significantly decreased junctional zone and increased labyrinth zone. Key trophoblast differentiation markers ( Tfap2c , Tpbpa , Pcdh12 ) and placental endocrine regulators ( Pl2 , Igf1r ) were significantly downregulated following cannabis smoke exposure in placentas. Furthermore, transcript levels of placental nutrient and vascularization markers, Glut1 , Vegfa and Pparg were significantly decreased in cannabis smoke-exposed placentas. By employing a physiologically relevant platform of prenatal cannabis exposure in vivo we demonstrate the adverse effects of prenatal cannabis smoke exposure on placental structure and function as well as on fetal brain growth.

Smoking cannabis remains the most common mode of consumption amongst pregnant people, yet the effects on placentation remain poorly understood. While prior studies have focused on exposure to single components of cannabis (i.e., Δ9-THC and CBD), this approach overlooks the complex toxicology and pharmacology of cannabis smoke exposure. In this study, we used an in vitro model of human trophoblast differentiation to investigate the impact of CaSE (cannabis smoke extract) compared to Δ9-THC. We show that CaSE, but not Δ9-THC induces CYP1A1 expression, a marker of exposure to combustion by-products. CaSE reduced hCG protein levels and syncytin-1 gene expression, suggesting impaired syncytialization. Lower concentrations of CaSE (1%, 2.5%) elevated reactive oxygen species without impacting membrane potential, whereas higher concentrations (5%, 10%) disrupted mitochondrial respiration, indicating dose-dependent bioenergetic dysfunction. Antioxidant genes, superoxide dismutase 1 and 2, were distinctly altered indicating the divergence in oxidative stress responses. Interestingly, CB1R antagonism rescued the effects of Δ9-THC exposure, but not CaSE-mediated effects on differentiation markers. Since most cannabis users consume cannabis by smoking, and smoke exposure differs from single components (Δ9-THC), it is important that preclinical models consider smoking when evaluating the impacts of cannabis use during pregnancy.

The placenta is a metabolically active organ whose mitochondrial activity is tightly linked to fetal growth, oxygenation, and nutrient transport, mediating fetal susceptibility to environmental exposures. Accordingly, aberrant mitochondrial function has been implicated in the progression of placental dysfunction. However, existing respirometry platforms require primarily fresh or cryopreserved placental tissue and offer limited throughput, rendering these platforms impractical in the context of large-scale placental dissections. Here, we describe and validate a Seahorse XF approach for measuring mitochondrial respiration in previously frozen placentae, enabling the functional interrogation of placental mitochondria in prenatal studies. Our protocol fundamentally relies on the restoration of matrix substrates that are depleted due to increased mitochondrial membrane permeability following freeze-thaw cycles. We provide a strategy to assess complex I and II-associated respiration adapted for the Seahorse XFe24 Analyzer and further demonstrate comparable oxygen consumption readouts between fresh and frozen placentae. We further demonstrate distinct differences in the magnitude of oxygen consumption between fresh and frozen placentae in the absence of exogenous NADH. Taken together, we present a simplified and convenient protocol for the assessment of respiratory enzyme complex-associated respiration from archived placental tissue.

Cannabis use during pregnancy continues to increase with smoking remaining the most common mode of consumption. While clinical studies highlight an association between prenatal cannabis use and adverse pregnancy outcomes, less is known about placental outcomes, even though many of the reported pregnancy outcomes are thought to be mediated via placental dysfunction. Here, we established a mouse model of gestational cannabis smoke exposure to investigate the impacts on fetal outcomes and placental structure and function. Pregnant CD1 mice were exposed daily to Δ9-tetrahydrocannabinol (THC)-dominant cannabis smoke (12-14% THC, 0-2% CBD) or filtered air from embryonic day (E)6.5 to E18.5 or parturition. Cannabinoid analyses in cannabis smoke-exposed, paired maternal and fetal livers revealed total THC and 11-Nor-9-carboxy-THC (THCA) concentrations of 135.95 ± 13.60 ng/g and 30.84 ± 4.68 ng/g, respectively. Moreover, Cyp1a1, a smoke-inducible enzyme, was induced by 4-fold in cannabis smoke-exposed placentae. No changes in offspring body weights were observed; however, there was a marked decrease in the brain-to-body weight ratio of exposed postnatal day 1 (PND1) offspring. Placentae from exposed dams were significantly reduced in size, with altered zonation marked by a significantly decreased junctional zone and increased labyrinth zone. Key trophoblast differentiation markers (Tfap2c, Tpbpa, Pcdh12) and placental endocrine regulators (Pl2, Igf1r) were significantly downregulated following cannabis smoke exposure in placentas. Furthermore, transcript levels of placental nutrient and vascularization markers, Glut1, Vegfa and Pparg were significantly decreased in cannabis smoke-exposed placentas. By employing a physiologically relevant platform of prenatal cannabis exposure in vivo we demonstrate the adverse effects of prenatal cannabis smoke exposure on placental structure and function as well as on fetal brain growth.

Trophoblast differentiation is a crucial process in the formation of the placenta where cytotrophoblasts (CTs) differentiate and fuse to form the syncytiotrophoblast (ST). The bioactive components of cannabis, such as Δ⁹-THC, are known to disrupt trophoblast differentiation and fusion, as well as mitochondrial dynamics and respiration. However, less is known about the impact of cannabidiol (CBD) on trophoblast differentiation. Due to the central role of mitochondria in stem cell differentiation, we evaluated the impact of CBD on trophoblast mitochondrial function and differentiation. Using BeWo b30 cells, we observed decreased levels of mRNA for markers of syncytialization (GCM1, ERVW1, hCG) following 20 µM CBD treatment during differentiation. In CTs, CBD elevated transcript levels for the mitochondrial and cellular stress markers HSP60 and HSP70, respectively. Furthermore, CBD treatment also increased the lipid peroxidation and oxidative damage marker 4-hydroxynonenal. Mitochondrial membrane potential, basal respiration and ATP production were diminished with the 20 µM CBD treatment in both sub-lineages. mRNA levels for endocannabinoid system (ECS) components (FAAH, NAPEPLD, TRPV1, CB1, CB2, PPARγ) were altered differentially by CBD in CTs and STs. Overall, we demonstrate that CBD impairs trophoblast differentiation and fusion, as well as mitochondrial bioenergetics and redox homeostasis.