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Background: Organoids are stem-cell derived, self-organised, three-dimensional cultures that improve in vitro recapitulation of tissue structure. The generation of trophoblast organoids using primary placental villous digests (containing cytotrophoblasts and trophoblast stem cells (TSC)) improved high-throughput assessment of early trophoblast differentiation. However, the relative contributions of cytotrophoblasts and TSCs to trophoblast organoid growth and differentiation remain unclear, with implications for model interpretation. Here we sought to generate organoids from side-population trophoblasts (SpTSCs) to better understand the contribution of TSC to trophoblast organoid formation., Methods: Methods were adapted from Haider et al., 2018 to generate organoids from Okae TSCs (OkTSCs) or SpTSCs. Organoid growth was compared with primary villous trophoblast organoids and cellular composition interrogated by immunohistochemistry., Results: Organoids can be derived from first-trimester SpTSCs that exhibit similar architecture to those from primary villous trophoblast. However, organoids established from pure TSC populations (OkTSC or SpTSC) have different growth dynamics to primary placental villous digest-derived organoids - with OkTSCs developing faster and spontaneously generating migratory cells, whilst SpTSC organoids grow more slowly. Importantly, depletion of SpTSC from first-trimester villous digests ablates organoid formation. Finally, the capacity of the side-population technique to isolate late-gestation TSC enabled the generation of trophoblast organoids from term placentae, although these were significantly smaller than their first-trimester SpTSC counterparts., Discussion: Together, this work highlights the requirement of TSC for organoid formation, and the functional distinction between TSC and cytotrophoblasts. Proof-of-principle data demonstrating organoid generation from late gestation TSC isolated directly from the placenta lays the groundwork for future disease models., Competing Interests: Declaration of competing interest The authors have no competing conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: The complex arborization of the feto-placental vasculature is crucial for optimal fetal nutrition, waste exchange and ultimately, development. Ethical and experimental limitations constrain research into the human placenta, hence experimental animal models such as mice and rats, are crucial to understand placental function. It is unclear how well the mouse and rat feto-placental vascular structure emulates human. Moreover, the implications of differences in vascular structure, especially in arborization, for placental function remain unclear., Methods: We use micro-computed tomography imaging, high frequency Doppler ultrasound and computational fluid dynamics to characterize feto-placental vasculature structure and haemodynamics in mice, rats, and human., Results: Our data suggest that despite structural differences between rat and mouse placenta, haemodynamics are similar and that both hold applicability to investigating feto-placental structure and function. We show that human cotyledons demonstrate vascularity-dependent haemodynamic behaviour (including flow deceleration and oxygen exchange) similar to rodents and can be analysed in the same spectrum as rodents. Finally, we show strong structure-function relationships when interspecies datasets are combined; notably, we demonstrate that surrogate measures such as vascularity, can be used to estimate placental oxygen exchange function., Discussion: Pre-clinical placental research utilising rat and mouse placentae to understand the impact of feto-placental arborization on placental function and fetal development can inform the human context., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The opening session of the 2023 International Federation of Placenta Associations meeting included a powerful patient perspective from invited guest Dawn Mark about her experience with preeclampsia and stillbirth. As part of this issue of Trophoblast Research we invited Dawn to provide this in written form, to more widely share her important message. Her children's names have been changed to protect privacy., Competing Interests: Declaration of competing interest I have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Career trajectories in science are often unpredictable, with many early and mid-career researchers working multiple successive fixed-term contracts, and physically relocating to take up employment opportunities. Whilst this can provide exciting opportunities to change research direction, acquire new skills, and see the world, the precarity of this scenario is also a significant cause of anxiety for many, and can have a negative impact on their ability to maintain career momentum and trajectory, access institutional financial benefits, or make long term career or financial plans. Here, we build on a pair of workshops held at the 2021 International Federation of Placenta Associations annual conference to discuss two key areas important to help early career researchers navigate their careers - building an academic profile, and the financial ramifications of academic careers., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Early placental development lays the foundation of a healthy pregnancy, and numerous tightly regulated processes must occur for the placenta to meet the increasing nutrient and oxygen exchange requirements of the growing fetus later in gestation. Inadequacies in early placental development can result in disorders such as fetal growth restriction that do not present clinically until the second half of gestation. Indeed, growth restricted placentae exhibit impaired placental development and function, including reduced overall placental size, decreased branching of villi and the blood vessels within them, altered trophoblast function, and impaired uterine vascular remodelling, which together combine to reduce placental exchange capacity. This review explores the importance of early placental development across multiple anatomical aspects of placentation, from the stem cells and lineage hierarchies from which villous core cells and trophoblasts arise, through extravillous trophoblast invasion and spiral artery remodelling, and finally remodelling of the larger uterine vessels., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Adequate development of the feto-placental circulation is critical for placental exchange function and healthy fetal growth. Understanding the structure of this circulation and how it informs fetal outcomes is important both in the human placenta, and the rodent, a purported comparative experimental model. Vascular casting and micro-CT imaging approaches enable detailed quantification of the complex vascular relationships in the feto-circulation, and provide detailed data to parameterise in silico models. Here, to assist researchers to apply these technically challenging methods we provide detailed approaches to cast and image; 1) human placentas at the cotyledon-level, and 2) whole rodent placentas., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Placental structures at the nano-, micro-, and macro scale each play important roles in contributing to its function. As such, quantifying the dynamic way in which placental structure evolves during pregnancy is critical to both clinical diagnosis of pregnancy disorders, and mechanistic understanding of their pathophysiology. Imaging the placenta, both exvivo and invivo, can provide a wealth of structural and/or functional information. This review outlines how imaging across modalities and spatial scales can ultimately come together to improve our understanding of normal and pathological pregnancies. We discuss how imaging technologies are evolving to provide new insights into placental physiology across disciplines, and how advanced computational algorithms can be used alongside state-of-the-art imaging to obtain a holistic view of placental structure and its associated functions to improve our understanding of placental function in health and disease., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: Derivation of pure fetal placental mesenchymal stem/stromal cells (pMSCs) is key to understand their role in placental development. However, isolated pMSCs are often contaminated by maternal-derived decidual MSCs (dMSCs). EGM-2 medium promotes the derivation of term fetal pMSCs, but the extent of first-trimester maternal pMSC contamination remains unclear. Culture media can also affect MSC phenotype. Here, we examined the effects of culture media on maternal pMSC contamination and fetal pMSC phenotype across gestation., Methods: pMSCs were derived from first-trimester or term placentae in advanced-DMEM/F12 medium or EGM-2 medium. Proportions of maternal (XX) and fetal (XY) cells in male pMSC cultures were determined by fluorescence in-situ hybridization. pMSC phenotype was analysed by flow cytometry, immunohistochemistry and Alamar blue proliferation assays., Results: When derived in advanced-DMEM/F12, all first trimester pMSC isolates exhibited maternal contamination (>72% XX cells, n = 5), whilst 7/9 term pMSC isolates were >98% fetal. When derived in EGM-2, all first trimester (n = 4) and term (n = 9) pMSC isolates contained 95-100% fetal cells. Fetal pMSCs in EGM-2 proliferated 2-fold (first-trimester) or 4-fold (term) faster than those in advanced-DMEM/F12 (p < 0.05, n = 3). Fetal pMSCs in both media expressed the generic MSC marker profile (CD90 + , CD105+, CD73 + , CD31-, CD34-, CD144-). However, pMSCs transferred from EGM-2 to advanced-DMEM/F12 increased expression of smooth muscle cell markers calponin and α-smooth muscle actin, and decreased expression of the vascular cell marker VEGFR2 (n = 3)., Conclusions: Deriving first-trimester pMSC in EGM-2 dramatically reduces maternal dMSC contamination. Media affects fetal pMSC phenotype, and careful consideration should be given to application specific culture conditions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The placenta is essential for the efficient delivery of nutrients and oxygen from mother to fetus to maintain normal fetal growth. Dysfunctional placental development underpins many pregnancy complications, including fetal growth restriction (FGR) a condition in which the fetus does not reach its growth potential. The FGR placenta is smaller than normal placentae throughout gestation and displays maldevelopment of both the placental villi and the fetal vasculature within these villi. Specialized epithelial cells called trophoblasts exhibit abnormal function and development in FGR placentae. This includes an altered balance between proliferation and apoptotic death, premature cellular senescence, and reduced colonisation of the maternal decidual tissue. Thus, the placenta undergoes aberrant changes at the macroscopic to cellular level in FGR, which can limit exchange capacity and downstream fetal growth. This review aims to compile stereological, in vitro, and imaging data to create a holistic overview of the FGR placenta and its pathophysiology, with a focus on the contribution of trophoblasts., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: Uterine artery (UtA) Doppler indices are one of the most commonly employed screening tests for pre-eclampsia worldwide. Abnormal indices appear to result from increased uterine vascular resistance, but anatomical complexity and lack of appropriate animal models mean that little is known about the relative contribution of each of the components of the uterine vasculature to the overall UtA Doppler waveform. Previous computational models suggested that trophoblast-mediated spiral artery remodeling has a dominant effect on the UtA Doppler waveform. However, these models did not incorporate the myometrial arterio-venous anastomoses, which have significant potential to affect utero-placental haemodynamics., Methods: We present a more anatomically complete computational model, explicitly incorporating a structural description of each component of the uterine vasculature, and crucially including myometrial arterio-venous anastomoses as parallel pathways for blood-flow away from the placental bed. Wave transmission theory was applied to the network to predict UtA waveforms., Results: Our model shows that high UtA resistance indices, combined with notching, reflect an abnormal remodeling of the entire uterine vasculature. Incomplete spiral artery remodeling alone is unlikely to cause abnormal UtA Doppler waveforms as increased resistance in these arteries can be 'buffered' by upstream anastomoses. Critically, our results indicate that the radial arteries, may have a more important effect on utero-placental flow dynamics, and the UtA Doppler waveform than previously thought., Conclusions: This model suggests that to appropriately interpret UtA Doppler waveforms they must be considered to be reflecting changes in the entire system, rather than just the spiral arteries., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Necrotic but not apoptotic trophoblastic debris can induce endothelial cell activation but the mechanism by which endothelial cells distinguish apoptotic from necrotic debris is unclear. The NALP3 inflammasome is a pattern recognition receptor that macrophages employ to recognise "danger signals" in necrotic cell corpses. In this study, we hypothesized that endothelial cells can identify and respond to necrotic trophoblastic debris via the NALP3 inflammasome., Methods: The effect of trophoblastic debris on endothelial expression of NALP3 inflammasome components was investigated using qRT-PCR, immunoassays and fluorescent caspase 1 activity assay. IL-1β in was quantified by ELISA. Endothelial cell activation was measured by cell surface ICAM expression and monocytes adhesion assay., Results: The NALP3 inflammasome was expressed in resting vascular endothelial cells and is involved in endothelial response to danger signals. However, exposure to necrotic trophoblastic debris did not significantly alter the expression of any of the three components of the NALP3 inflammasome at the mRNA level, nor was caspase-1 activation increased. Conditioned media from endothelial cells exposed to necrotic trophoblastic debris contained elevated levels of IL-1β which was derived from the necrotic debris and which contributed to endothelial cell activation., Discussion: Necrotic trophoblastic debris induced endothelial cell activation through the IL-1β/IL-1R pathway. However, the NALP3 inflammasome in endothelial cells was not involved in this process., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The transformation of cytotrophoblast (CTB) to extravillous trophoblast (EVT) is an essential process for placental implantation. EVT generated at the tips of the anchoring villi migrate away from the placenta and invade the endometrium and maternal spiral arteries, where they modulate maternal immune responses and remodel the arteries into high-volume conduits to facilitate uteroplacental blood flow. The process of EVT differentiation has several factors in common with the epithelial-to-mesenchymal transition (EMT) observed in embryonic development, wound healing and cancer metastasis. We hypothesized that the generation of invasive EVT from CTB was a form of EMT. We isolated paired CTB and EVT from first trimester placentae, and compared their gene expression using a PCR array comprising probes for genes involved in EMT. Out of 84 genes, 24 were down-regulated in EVT compared to CTB, including epithelial markers such as E-cadherin (-11-fold) and occludin (-75-fold). Another 30 genes were up-regulated in EVT compared to CTB including mesenchymal markers such as vimentin (235-fold) and fibronectin (107-fold) as well as the matrix metalloproteinases, MMP2 and MMP9 (357-fold, 129-fold). These alterations also included major increases in the ZEB2 (zinc finger E-box binding homeobox 2, 198-fold) and TCF4 (transcription factor 4, 18-fold) transcription factors, suggesting possible stimulatory mechanisms. There was substantial up-regulation of the genes encoding TGFβ1 and TGFβ2 (48-fold, 115-fold), which may contribute to the maintenance of the mesenchymal-like phenotype. We conclude that transformation of CTB to EVT is consistent with an EMT, although the differences with other types of EMT suggest this may be a unique form., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In pregnancy disorders such as pre-eclampsia, intrauterine growth restriction (IUGR) and recurrent miscarriage a poorly functioning placenta is thought to be a major component of the disease process. However, despite their prevalence, we currently have no way to fix dysfunctional placentae or directly treat these disorders. Over the past two decades our understanding of the role that stem cells play in organ development and regeneration has expanded rapidly, and over the past 5 years the therapeutic use of stem cells to both regenerate damaged tissues, and act as potent modulators of diseased microenvironments, has become a reality in many organs including the heart, kidney, liver, skin and eye. Over its short lifespan the placenta undergoes rapid and continuous growth and differentiation, meaning that placental 'organogenesis' only truly ends at delivery, and thus stem cells are likely to play important roles in placental function for the duration of pregnancy. Two populations of stem cells exist in the placenta that contribute to this on-going growth and differentiation: trophoblast stem cells and mesenchymal stem cells. This review will address our current understanding of how each of these stem cell populations contributes to successful placental function, how epithelial and mesenchymal stem cell populations are being translated to the clinic in other fields, and whether these advances can teach us anything about how placental stem cells could be used to fix faulty placentae in the future., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: The local actions of glucocorticoids in the placenta can be modulated by 11β-hydroxysteroid dehydrogenase (11βHSD) enzymes, which catalyse inter-conversion of cortisol with its inert metabolite, cortisone, and are known to be expressed in the term placenta and decidua. However, the expression and activity of these enzymes have not been well characterised in the first trimester placenta. The aim of this study was to compare 11βHSD2 expression and activity in first trimester placental tissue from pregnancies at either relatively low or high risk of developing pre-eclampsia as determined by Doppler ultrasound., Methods: Enzyme expression was assessed by western blot analysis and immunohistochemistry while 11βHSD enzyme activities were quantified using radiometric conversion of [3H]-cortisol in the presence of NADP(+) or NAD(+)., Results: 11βHSD2 was expressed in syncytiotrophoblast of first trimester placenta, and there was no difference in the level of expression of placental 11βHSD2 protein between 9 high pre-eclampsia risk and 14 low pre-eclampsia risk pregnancies. NAD(+)-dependent cortisol oxidation was elevated 3-fold in placental tissue from pregnancies at higher risk of pre-eclampsia than in normal pregnancies (50.9 ± 15.9 versus 18.3 ± 1.9 pmol cortisone/mg protein.10 min, n = 11 & 12, respectively; P < 0.05)., Conclusions: Expression of 11βHSD2 is thought to protect the fetus from exposure to maternal cortisol. While other studies have suggested that 11βHSD2 is down regulated in term pre-eclamptic placentae, our study suggests that there is increased cortisol inactivation in first trimester placenta prior to week 10 of gestation, from pregnancies at higher risk of developing pre-eclampsia., (Copyright © 2011 Elsevier Ltd. All rights reserved.)