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Background: Mesenchymal stem cells (MSCs) are increasingly recognized for their regenerative potential. However, their clinical application is hindered by their inherent variability, which is influenced by various factors, such as the tissue source, culture conditions, and passage number., Methods: MSCs were sourced from clinically relevant tissues, including adipose tissue-derived MSCs (ADMSCs, n = 2), chorionic villi-derived MSCs (CMMSCs, n = 2), amniotic membrane-derived MSCs (AMMSCs, n = 3), and umbilical cord-derived MSCs (UCMSCs, n = 3). Passages included the umbilical cord at P0 (UCMSCP0, n = 2), P3 (UCMSCP3, n = 2), and P5 (UCMSCP5, n = 2) as well as the umbilical cord at P5 cultured under low-oxygen conditions (UCMSCP5L, n = 2)., Results: We observed that MSCs from different tissue origins clustered into six distinct functional subpopulations, each with varying proportions. Notably, ADMSCs exhibited a higher proportion of subpopulations associated with vascular regeneration, suggesting that they are beneficial for applications in vascular regeneration. Additionally, CMMSCs had a high proportion of subpopulations associated with reproductive processes. UCMSCP5 and UCMSCP5L had higher proportions of subpopulations related to female reproductive function than those for earlier passages. Furthermore, UCMSCP5L, cultured under low-oxygen (hypoxic) conditions, had a high proportion of subpopulations associated with pro-angiogenic characteristics, with implications for optimizing vascular regeneration., Conclusions: This study revealed variation in the distribution of MSC subpopulations among different tissue sources, passages, and culture conditions, including differences in functions related to vascular and reproductive system regeneration. These findings hold promise for personalized regenerative medicine and may lead to more effective clinical treatments across a spectrum of medical conditions., Competing Interests: The authors declare that they have no competing interests. Chunbing Zheng, Shiping Li, Cheng Wang, Wenjiao Jiang, Yun Liu, Yuan Yang, Tenglong Yan and Zhigang Xue are employed by Yuanpin Cell Technology Co. Ltd., (© 2024 Yi et al.)

Introduction: Knowledge of the size of surfaces available for transport is important for assessing the amount of nutrients that can be transmitted to the fetus for its normal growth and development., Aim: The aim of our study, was to determine the stereological structural parameters of the parenchymal part of placenta, ratio of birth weight and placental weight, and to determine their correlation with the body length and head circumference of the newborns of adolescent pregnant women., Methods: The study was conducted on a total of 60 human placentas of term pregnancy, divided into two groups according to the age of pregnant women. The experimental group consisted of 30 placenta of pregnant women aged 13-19. The control group consisted of 30 placenta of pregnant women aged 20-35. Computer assisted morphological analysis of images of histological preparations using stereological methods was performed., Results: Surface density of terminal villi of adolescent placentas is significant higher than the control group (t = 14,179, df = 29, p <0,0001). The T-test (t = -5,868, df = 29, p <0,0001) showed statistically significant difference in the surface density of fibrinoid in two compared groups. T-test (t = 6.438, df = 29, p <0.0001) found that total surface of terminal villi was significantly higher in adolescent placentas. The T-test (t = -6,747, df = 29, p <0,0001) found that total surface of fibrinoid was significantly lower in adolescent group. The T-test (t = 4.203, df = 29, p <0.0001) found that the ratio of birth weight of newborn and adolescent placental weight was significantly higher in relation to the control group., Conclusion: Adolescent placentas was more efficient in increasing the weight of newborns, compared to the control group placentas., Competing Interests: There are no conflicts of interest., (© 2019 Sergije Markovic, Zlata Zigic, Anis Cerovac, Suad Kunosic, Melisa Lelic, Fejzo Dzafic.)

Background: Preeclampsia (PE) is a pregnancy-related hypertensive disorder, which may stem from impair placentation. Renin-angiotensin system is one of the mediators of decidualization and trophoblastic proliferation. In the present study women with PE were studied in a comparison of normotensive controls to determine whether Angiotensin-converting enzyme (ACE) gene I/D polymorphism affect the placental villi and umbilical cord formation with the assessment of biochemical and clinical risk factors., Methods: Total 400 blood (PE/controls = 200), 400 urine (PE/controls = 200), 90 tissue samples of UC (PE = 50, controls = 40) and 90 placental tissue samples (PE = 50, controls = 40) were recruited. Histomorphological and Histomorphometric analysis were done for placental and umbilical cord tissues. Blood and serum parameters were analyzed, samples were genotyped for I/D polymorphism. Data were statistically analyzed by Independent sample t-test, Chi-square test and the odds ratio., Results: Histological study revealed significant increase (p < 0.001) in distance from Wharton jelly (in both artery and vein) and outer layer thickness of vein; significant reduction (p < 0.01 and p < 0.05) in the lumen area of artery and vein. Abnormal villi, more syncytial knots (SK) and a significant decrease in elongated and large villi in PE placentas. Analysis of ACE gene determined that genotypic frequencies were statistically significant (p < 0.02) among both groups and DD genotype was predominant in the PE group., Conclusion: Present study reveals that ACE I/D polymorphism might affect the normal placental villi and umbilical cord formation in women with PE. In addition, histological studies and genetic evaluation can provide useful information in the determination of various reasons and mechanisms involved in the pathogenesis of PE in Pakistan., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Early Embryonic Arrest (EEA) is one of the major causes of female infertility. Genetic factors including specific genes and miRNAs may play pivotal roles on EEA. However, it is not well defined what genes and micro RNAs participate the pathophysiological alterations of EEA. In this work, we compared the Transcriptome -Seq and microRNA profiles from three pairs of villi (three EEA patients and three normal pregnancy, NP). We first confirmed the array data by qPCR with ten randomly selected differentially expressed genes and ten differentially expressed miRNAs in villi from 20 EEA and 20 NP controls. We next applied Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis and found that these differentially expressed genes enriched in the PI3K-Akt signaling pathway, Jak-STAT signaling pathway, MAPK signaling pathway, Complement and coagulation cascades, Hypertrophic cardiomyopathy (HCM), Dilated cardiomyopathy (DCM). Interestingly, hsa-miR-6515-5p and its target genes NLRP3, UGP2 may regulate the Immune system and carbohydrate metabolism. Hsa-miRNA 518 and its target gene EGR1 may regulate cell proliferation, angiogenesis, and cell apoptosis to impact early embryonic development. Moreover, novel-m0045-5p and its target gene RMDN3 may regulate microtubule formation on the development of EEA. Our research provides novel biomarkers for EEA and establishes a foundation for further study of the mechanism of EEA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: To assess the potential association between polycystic ovary syndrome (PCOS) and chromosomally aberrant miscarriage during treatment with assisted reproductive technology (ART)., Design: A retrospective, single-center study., Setting: University-affiliated reproductive center., Patient(s): A total of 328 patients sent their first trimester miscarried chorionic villus for genetic examination after ART in our center from January 2013 to September 2016, of which 119 cases were women with PCOS and 209 were non-PCOS controls. No known definite miscarriage-related concomitants existed in any study subject., Intervention(s): Single nucleotide polymorphism array analysis was performed on all collected samples., Main Outcome Measure(s): Frequency of aberrant karyotype of miscarried conceptus and the correlation between PCOS and chromosomally aberrant miscarriage., Result(s): A total of 173 (52.7% of 328) conceptuses were identified as chromosomally aberrant by single nucleotide polymorphism array. Chromosomal aberrations were more frequent in conceptuses from PCOS patients compared with controls (61.3% vs. 47.8%). Furthermore, both univariate and multivariable analysis identified PCOS as a risk factor for an embryo/fetus to be chromosomally abnormal, with odds ratios of 1.957 (95% confidence interval, 1.067-3.590) and 2.008 (95% confidence interval, 1.038-3.883), respectively., Conclusion(s): Women with PCOS were at an increased risk of miscarrying a chromosomally aberrant embryo/fetus compared with non-PCOS controls during ART. Mechanisms require further investigation. Preimplantation genetic screening might be an effective approach to decrease the risk of spontaneous miscarriage for women with PCOS., (Copyright © 2019. Published by Elsevier Inc.)

Placental sexual dimorphism is of special interest in prenatal programming. Various postnatal diseases with gender dependent incidence, especially neuropsychiatric disorders like schizophrenia and autism spectrum disorders, have prenatal risk factors established. However, the functional relevance of placental microarchitecture in prenatal programming is poorly investigated, mainly due to a lack of statistically efficient methods. We hypothesized that the recently established 3D microscopic analysis of villous trees would be able to identify microscopic structural correlates of human placental sexual dimorphism. We analyzed the density of cell nuclei of villous trophoblast, i.e. the materno-fetal exchange barrier, in placentas from term pregnancies. The cell nuclei were grouped into proliferative and non-proliferative nuclei by detection of a proliferation marker (PCNA). Normal female placentas showed a higher density of non-proliferating nuclei (PCNA-negative) in villous trophoblast than normal male placentas. The density of PCNA-negative cell nuclei was higher in placentas of pregnancies with intrauterine growth retardation (IUGR) than in control placentas. The data of the present study shows that the density of non-proliferative cell nuclei in the syncytial layer of villous trophoblast is influenced by fetal sex and by IUGR, while proliferation remains unchanged. A novel concept of post-fusion regulation of syncytial structure and function is proposed.

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies., Competing Interests: The authors have declared that no competing interests exist.

Placental oxygen transport takes place at the final branches of the villous tree and is dictated by the relative arrangement of the maternal and fetal circulations. Modeling techniques have failed to accurately assess the structure-function relationship in the terminal villi due to the geometrical complexity. Three-dimensional blood flow and oxygen transport was modeled in four terminal villi reconstructed from confocal image stacks. The blood flow was analyzed along the center lines of capillary segments and the effect of the variability in capillary diameter, tortuosity and branching was investigated. Additionally, a validation study was performed to corroborate the simulation results. The results show how capillary variations impact motion of the fetal blood, and how their bends and dilatations can decelerate the flow by up to 80%. Vortical flow is also demonstrated not to develop in the fetal capillaries. The different geometries are shown to dictate the transport of gases with differences of over 100% in the oxygen flux between samples. Capillary variations are key for efficient oxygen uptake by the fetus; they allow the blood to decelerate where the villous membrane is thinnest allowing for a better oxygenation, but also by reducing the vessel diameter they carry the oxygenated blood away fast. The methodology employed herein could become a platform to simulate complicated in-vivo and in-vitro scenarios of pregnancy complications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We evaluate, in routine H&E histology slides, villus quantity in a given area (villous packing density, VPD) and the pattern or "gappiness" of villous distribution (lacunarity), and test for correlations with a proxy for fetoplacental metabolic rate, β calculated as (ln (placental weight)/ln (birthweight)) from Kleiber's law [1]. Three ∼4.3 mm 2 images each were obtained from 88 term placentas. Ranges of VPD and lacunarity were each correlated with β (r = 0.31, p = 0.003, r = 0.23, p = 0.03 and respectively). The relationship between β and within-placenta variation in VPD and lacunarity highlights the need to study not merely the mean but the variance of villous geometries and spatial distributions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

During human pregnancy, the fetal circulation is separated from maternal blood in the placenta by two cell layers - the fetal capillary endothelium and placental trophoblast. This placental barrier plays an essential role in fetal development and health by tightly regulating the exchange of endogenous and exogenous materials between the mother and the fetus. Here we present a microengineered device that provides a novel platform to mimic the structural and functional complexity of this specialized tissue in vitro. Our model is created in a multilayered microfluidic system that enables co-culture of human trophoblast cells and human fetal endothelial cells in a physiologically relevant spatial arrangement to replicate the characteristic architecture of the human placental barrier. We have engineered this co-culture model to induce progressive fusion of trophoblast cells and to form a syncytialized epithelium that resembles the syncytiotrophoblast in vivo. Our system also allows the cultured trophoblasts to form dense microvilli under dynamic flow conditions and to reconstitute expression and physiological localization of membrane transport proteins, such as glucose transporters (GLUTs), critical to the barrier function of the placenta. To provide a proof-of-principle for using this microdevice to recapitulate native function of the placental barrier, we demonstrated physiological transport of glucose across the microengineered maternal-fetal interface. Importantly, the rate of maternal-to-fetal glucose transfer in this system closely approximated that measured in ex vivo perfused human placentas. Our "placenta-on-a-chip" platform represents an important advance in the development of new technologies to model and study the physiological complexity of the human placenta for a wide variety of applications.

Pregnancy-associated plasma protein-A 2 (PAPPA2) is a placental-enriched gene that is important for normal human placentation and defects in the gene can cause complications in pregnancy. Yet the exact expression pattern and role of PAPPA2 in the human fetomaternal interface are not clear. In this study, in situ hybridization (ISH) and immunohistochemistry (IHC) were employed to examine the spatial and temporal expression of PAPPA2 in the human fetomaternal interface. IHC results exhibited wide expression of PAPPA2 in the fetomaternal interface, with placental syncytiatrophoblast (STB) and extravillous trophoblast (EVT) showing strong expression and the cytotrophoblast (CTB) showing weak expression of PAPPA2. These results were confirmed by ISH. Quantitative reverse transcription-polymerase chain reaction and western blot showed the elevation of PAPPA2 in first trimester EVT differentiation and term CTB spontaneous syncytialization. PAPPA2-siRNA transfection significantly depressed the invasion and migration ability of a trophoblast cell line (HTR8/SVneo) in a transwell migration and Matrigel invasion model compared to a negative control siRNA (P < 0.05), also revealing that matrix metalloproteinase 9 (MMP9) secretion is downregulated. This was confirmed using a human first trimester placental villi explant culture model. Our results reveal the spatial and temporal expression of PAPPA2 in the human fetomaternal interface and show the positive regulatory role of PAPPA2 in human trophoblast invasion and migration through the secretion of MMP9., Competing Interests: The authors declare no conflict of interest.

The placenta exchanges nutrients between the mother and the fetus and requires a constant abundant energy supply. Adiponectin (a cytokine produced primarily by adipose tissue) controls glucose and lipid homeostasis. It is well-known that maternal serum adiponectin levels are inversely related to birth weight, suggesting that adiponectin has a negative effect on fetal growth. This effect appears to be related to the control of nutrient transporters in human placenta. However, the underlying molecular mechanisms have not yet been characterized. In the present work, we studied adiponectin's direct effect on human primary cytotrophoblasts from first-trimester placenta. Our result showed that in placental cells, adiponectin 1) inhibits the expression of the major glucose transporters (GLUT1 and GLUT12) and sodium-coupled neutral amino acid transporters (SNAT1, SNAT2, and SNAT4), 2) enhances total ATP production but decreases lactate production, 3) inhibits mitochondrial biogenesis and function, and 4) stimulates cell death by enhancing the expression of the pro-apoptotic B-cell lymphoma-2 (BCL-2)-associated X protein (BAX) and tumor protein P53 (TP53) gene expression and inducing the caspase activity. Small-interfering RNA mediating the down-regulation of adiponectin receptors (ADIPOR1 and ADIPOR2) was used to demonstrate that adiponectin effects on placental nutrient transport and apoptosis seemed to be essentially mediated by these specific receptors. Taken as a whole, these results strongly suggest that adiponectin regulates human placental function by limiting nutrient transporter expression and inducing apoptosis. These findings may help us to better understand adiponectin's role in placental pathologies such as intrauterine growth restriction, which is characterized by fetal weight loss and drastic apoptosis of placental cells., (© 2016 by the Society for the Study of Reproduction, Inc.)

Aberrant expressions of some miRNAs were reported in the placenta from compromised pregnancies, suggesting that the miRNA profiling of chorionic villi could be related with early recurrent miscarriage (RM). This study was designed to investigate the miRNA expression profile, and to predict their potential target genes and signal pathways. The chorionic villi tissues were collected from 15 early RM cases and 15 controls. We focused on investigating the biological pathways and their potential functions in order to explore what is the likely association and how they are possibly involved in the development of early RM. The chorionic villi tissues were collected from 15 early RM cases and 15 controls. A total of 155 differentially expressed miRNAs over 2-fold changes in expression levels (P<0.05), including 98 up-regulated and 57 down-regulated expressions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the target genes of those miRNAs were mainly involved in the pathways of MAPK, B cell receptor, T cell receptor and tumor-associated signaling in the development of early RM. Those target genes of differentially expressed miRNAs, coupled with multiple biological processes and pathways, would be beneficial to understand the etiology and pathophysiological mechanism of early RM., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Paternally expressed gene 10 (PEG10) is an imprinted and monoallelic expressed gene. Previous study using a knockout mouse model revealed a crucial role of PEG10 in placental development, yet the exact function of PEG10 during placentation remains to be elucidated. In this study, denuded chorionic villi were prepared from first trimester human placentas, and transduced with PEG10 small interference RNA (siRNA) or non-targeting control sequence by lentiviral infection. Immunohistochemical staining revealed that silencing of PEG10 in the chorionic villous explants resulted in reduced immune-reactivity to CK7, Ki67 and integrin α5, implying that silencing of PEG10 impaired the proliferation of villous trophoblasts and may interfere with the activity of extravillous trophoblasts. We further investigated the role of PEG10 in the proliferation, migration and invasion of JEG-3 trophoblast cell line and the primary chorionic villous cells. PEG10-silenced JEG-3 cells and primary chorionic villous cells displayed a reduced proliferation rate and impaired invasiveness in vitro. Silencing of PEG10 in trophoblast cells led to upregulated expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as downregulated expression of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, knockdown of TIMP-1 reversed the suppressed invasiveness of PEG10 siRNA-transduced JEG-3 cells. In conclusion, our study demonstrates that PEG10 plays an important role in trophoblast proliferation and promotes trophoblast invasion through TIMP-1.

All near-equilibrium systems under linear regime evolve to stationary states in which there is constant entropy production rate. In an open chemical system that exchanges matter and energy with the exterior, we can identify both the energy and entropy flows associated with the exchange of matter and energy. This can be achieved by applying statistical mechanics (SM), which links the microscopic properties of a system to its bulk properties. In the case of contractile tissues such as human placenta, Huxley's equations offer a phenomenological formalism for applying SM. SM was investigated in human placental stem villi (PSV) (n = 40). PSV were stimulated by means of KCl exposure (n = 20) and tetanic electrical stimulation (n = 20). This made it possible to determine statistical entropy (S), internal energy (E), affinity (A), thermodynamic force (A / T) (T: temperature), thermodynamic flow (v) and entropy production rate (A / T x v). We found that PSV operated near equilibrium, i.e., A ≺≺ 2500 J/mol and in a stationary linear regime, i.e., (A / T) varied linearly with v. As v was dramatically low, entropy production rate which quantified irreversibility of chemical processes appeared to be the lowest ever observed in any contractile system.

Purpose: Inflammation in chorionic villi is involved in the development of recurrent pregnancy loss (RPL). High mobility group box 1 protein (HMGB1) plays critical roles in inflammation and expression of the protein can be found in chorionic villi. The purpose of the study was to investigate the association between HMGB1 genetic polymorphisms and susceptibility to RPL and to examine the mechanism underlying this correlation., Methods: Two HMGB1 polymorphisms, rs2249825C/G and rs1412125T/C, were examined in 112 RPL patients and 118 healthy controls by the polymerase chain reaction-restriction fragment length polymorphism assay., Results: Percentage of rs2249825GG was significantly increased in patients than in controls (Odd ratio [OR] =2.33, 95 % confidence interval [CI]: 1.18-4.58, P = 0.013). Also, prevalence of rs2249825G allele was significantly higher in RPL cases (OR = 1.77, 95 % CI: 1.20-2.62, P = 0.004). Function analysis of rs2249825C/G revealed that the polymorphism did not affect serum level of HMGB1. Interestingly, we found significantly increased level of HMGB1 in chorionic villi from RPL patients. Moreover, patients with rs2249825GG genotype presented significantly elevated level of HMGB1 in chorionic villi compared to those with CG or CC genotypes., Conclusions: These results suggest that HMGB1 rs2249825C/G polymorphism is associated with increased risk of RPL and can elevate gene expression in chorionic villi.

Long term propagation of human fetal mesenchymal stromal cells (MSC) in vitro has proven elusive due to limited availability of fetal tissue sources and lack of appropriate methodologies. Here, we have demonstrated the presence of fetal and maternal cells within the tips of terminal chorionic villi (TCV) of normal human term placenta, and we have exploited inherent differences in the adhesive and migratory properties of maternal vs. fetal cells, to establish pure MSC cultures of both cell types. The origin and purity of each culture was confirmed by X-Y chromosome-specific fluorescence in situ hybridization (FISH) and short tandem repeat (STR) genotyping. This is the first demonstration of fetal and maternal cells in the TCV of human term placenta and also of deriving pure fetal MSC cultures from them. The concomitant availability of pure cultures of adult and fetal MSC from one tissue provides a good system to compare genetic and epigenetic differences between adult and fetal MSCs; and also to generate new models of cell based therapies in regenerative medicine.

Study Question: What is the in situ localization and function of hyperglycosylated hCG (hCG-H) in first trimester pregnancy tissues?, Summary Answer: HCG-H localizes to the syncytiotrophoblast, cytotrophoblast and invasive extravillous trophoblast within the maternal decidua and promotes invasion during the first trimester of pregnancy., What Is Known Already: Serum levels of hCG-H decline dramatically throughout the first trimester of pregnancy. As hCG-H is produced by choriocarcinoma cells, it is proposed to regulate trophoblast invasion., Study Design, Size, Duration: Tissues were collected from elective first trimester pregnancy terminations. Placental villous and decidua basalis were collected from Week 6 to Week 12 of gestation (n = 49)., Participants/materials, Setting, Methods: Tissues were collected from elective first trimester surgical pregnancy terminations to determine localization, abundance and function of hCG-H. Placental villous outgrowth studies determined the impact of neutralizing endogenous hCG-H on trophoblast function. Real-time proliferation, migration and invasion assays using JEG-3 choriocarcinoma cells further elucidated the role of hCG-H in trophoblast function., Main Results and the Role of Chance: HCG-H localized to syncytiotrophoblast layer of the placental villous from gestational weeks 6-9; thereafter hCG-H localized as a discrete layer between syncytio- and cyto-trophoblast layers. Immunoreactive hCG-H was also observed within the cytotrophoblast layer in Week 7-8 of gestation. HCG-H abundance decreased within placental villous from Weeks 6-12 of gestation (n = 3 placentas per gestational weeks 6-12). HCG-H also localized to anchoring villi within maternal decidua, extravillous trophoblasts invading into the maternal decidua and endovascular trophoblasts remodeling maternal blood vessels. Treatment of primary first trimester villous explants with hCG-H neutralizing antibody reduced trophoblast outgrowth (n = 3 placentas, P < 0.05). Treatment of a trophoblast cell line with neutralizing antibody reduced trophoblast invasion (n = 4, P < 0.05) but did not affect migration or proliferation., Limitations, Reasons for Caution: Functional invasion and migration assays performed using cell lines. Not possible to perform such assays with primary human material., Wider Implications of the Findings: HCG-H is an important autocrine factor facilitating trophoblast invasion in the first trimester of pregnancy. Targeting hCG-H may prove useful in the treatment of pathologic pregnancies, such as ectopic pregnancies, or pregnancy complications including pre-eclampsia and gestational trophoblast diseases., Study Funding/competing Interests: This work was supported by the Victorian Government Operational Infrastructure Support Program. J.E. is supported by NHMRC project grant #1047756, L.A.S. and E.D. by NHMRC Fellowships #1002018 and #550905 respectively and E.M. by an NHMRC Early Career Fellowship #611827. The authors have no conflicts of interest relating to this work., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We present a stream-tube model of oxygen exchange inside a human placenta functional unit (a placentone). The effect of villi density on oxygen transfer efficiency is assessed by numerically solving the diffusion-convection equation in a 2D+1D geometry for a wide range of villi densities. For each set of physiological parameters, we observe the existence of an optimal villi density providing a maximal oxygen uptake as a trade-off between the incoming oxygen flow and the absorbing villus surface. The predicted optimal villi density 0.47±0.06 is compatible to previous experimental measurements. Several other ways to experimentally validate the model are also proposed. The proposed stream-tube model can serve as a basis for analyzing the efficiency of human placentas, detecting possible pathologies and diagnosing placental health risks for newborns by using routine histology sections collected after birth., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

How the turnover of villous trophoblast is regulated is important for understanding normal and complicated pregnancies. There is considerable accord that syncytiotrophoblast (STB) grows and is refreshed by recruiting post-mitotic cells from the deeper cytotrophoblast (CTB). Nuclei in STB exhibit a spectrum of morphologies and packing densities and, until recently, there seemed to be a consensus that this variation reflected a transition from an early undifferentiated CTB-like phenotype to a long pre-apoptotic and brief apoptotic phase. In these later phases, nuclei are sequestered in clusters (syncytial knots) prior to extrusion as part of normal epithelial turnover. Early in gestation, nuclear clustering and formation of protrusions (syncytial sprouts) also occurs as a preliminary to villous sprouting. Nuclei in these clusters have a CTB-like phenotype and some sprouts may also detach from STB and pass into the uteroplacental circulation. However, this apparent consensus has been challenged and new interpretations of events in the proliferative (CTB), terminal differentiation (STB) and deportation compartments have emerged. Several different types of STB fragment are deported in normal pregnancy: larger multinucleate STB fragments, smaller uninucleate elements with CTB-like morphology, anucleate cytoplasmic fragments, microparticles and nanovesicles. This review identifies points of agreement and disagreement and offers possible avenues of future research. An obvious need is to standardise best practice in several areas including choosing appropriate references for cell cycle phase labelling indices and combining immunolabeling of cell cycle and apoptosis markers (at LM or TEM levels) with design-based stereological estimates of absolute numbers of cells and nuclei in different compartments throughout normal gestation. This would also provide a surer foundation for interpreting results from different research groups and changes in normal and complicated pregnancies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Extravillous trophoblast (EVT) cells are of pivotal importance in human embryo implantation and homeostasis of the maternal fetal interface. Invasion of the endometrium by EVT contributes to placental anchorage, spiral artery remodeling, immunological defense, tolerogenic responses, and several collaborative cross talks involved in establishing and maintaining a successful pregnancy. We report here an improved protocol for the isolation of fully differentiated EVT cells from the basal plate of the human term placenta., Methods: The basal plate was carefully dissected from the villous tissue and the amniochorion membrane prior to enzymatic digestion. Term basal EVT cells were isolated using a 30 and 60% Percoll gradient. A panel of markers and characteristics of the isolated cells were used to confirm the specificity and efficiency of the method so that their potential as an investigative tool for placental research could be ascertained., Results: Isolated cells were immunoreactive for cytokeratin-7 (CK-7), placental growth factor, placental alkaline phosphatase, human leukocyte antigen G1 (HLA-G1), and α1 and α5 integrins, similarly to the EVT markers from first trimester placental villi. Around 95% of the isolated cells labeled positively for CK-7 and 82% for HLA-G1. No significant change in viability was observed during 48 h of EVT culture as indicated by propidium iodide incorporation and trypan blue test exclusion. Genes for metalloproteinases MMP-2 and MMP9 (positive regulators of trophoblast invasiveness) were expressed up to 48 h of culturing, as also the gelatinolytic activity of the isolated cells. Transforming growth factor (TGF)-beta, which inhibits proliferation, migration, and invasiveness of first-trimester EVT cells, also reduced invasion of isolated term EVT cells in transwell assays, whereas epidermal growth factor was a positive modulator., Conclusions: Term basal plate may be a viable source of functional EVT cells that is an alternative to villous explant-derived EVT cells and cell lines. Isolated term EVT cells may be particularly useful in investigation of the role of trophoblast cells in pathological gestations, in which the precise regulation and interactive ability of extravillous trophoblast has been impaired.

Introduction: Circulating fetal cells in maternal blood provide a tool for risk-free, non-invasive prenatal diagnosis. However, fetal cells in the maternal circulation are scarce, and to effectively isolate enough of them for reliable diagnostics, it is crucial to know which fetal cell type(s) should be targeted., Materials and Methods: Fetal cells were enriched from maternal blood by magnetic-activated cell sorting using the endothelial cell marker CD105 and identified by XY fluorescence in situ hybridization. Expression pattern was compared between fetal cells and maternal blood cells using stem cell microarray analysis., Results: 39 genes were identified as candidates for unique fetal cell markers. More than half of these are genes known to be expressed in the placenta, especially in extravillous trophoblasts (EVTs). Immunohistochemical staining of placental tissue confirmed CD105 staining in EVTs and 76% of fetal cells enriched by CD105 were found to be cytokeratin-positive., Discussion: The unique combination of mesodermal (CD105) and ectodermal (cytokeratin) markers in EVTs could be a potential marker set for cell enrichment of this cell type in maternal blood and could be the basis for future cell-based non-invasive prenatal diagnosis., (© 2013 S. Karger AG, Basel.)

The placenta is an indispensable organ for intrauterine protection, development and growth of the embryo and fetus. It provides tight contact between mother and conceptus, enabling the exchange of gas, nutrients and waste products. The human placenta is discoidal in shape, and bears a hemo-monochorial interface as well as villous materno-fetal interdigitations. Since Peter Medawar's astonishment to the paradoxical nature of the mother-fetus relationship in 1953, substantial knowledge in the domain of placental physiology has been gathered. In the present essay, an attempt has been made to build an integrated understanding of morphological dynamics, cell biology, and functional aspects of genomic and proteomic expression of human early placental villous trophoblast cells followed by a commentary on the future directions of research in this field., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: Human placental stem villi (PSV) present contractile properties. We studied the role of actin-myosin cross bridges (CBs) and the effects of NO-cGMP pathway modulators in the PSV contraction and relaxation., Methods: In vitro contractile properties were investigated in 71 PSV from term human placentas studied according to their long axis. Contraction was induced by both KCl and electrical tetanic stimulation. Relaxation was induced by inhibiting the CB cycle with either 2,3-butanedione monoxime (BDM) or blebbistatin (BLE) and by activating the NO-cGMP pathway with isosorbide dinitrate (ISDN), sildenafil (SIL) or ISDN + SIL., Results: PSV tension slowly increased by 140% of the basal tone after KCl exposure and by 85% after tetanus. The addition of BDM, BLE, ISDN, SIL and ISDN + SIL induced a relaxation of PSV, the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). These relaxation kinetics were particularly slow. Other relaxation parametres, i.e., maximum lengthening, -peak dT/dt, and resting tension, did not differ between these 5 subgroups., Discussion and Conclusion: Isolated human PSV were able to contract after both KCl exposure and tetanus. This increase in contractility was reversed by inhibiting the CB cycle with BDM or BLE and by stimulating the NO-cGMP pathway with ISDN or SIL. The association ISDN + SIL did not potentiate the relaxing processes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

To determine placental microRNA (miRNA) expression at different gestational age, total RNA from six first and six third trimester placentas was isolated. miRNA expression was analyzed by Affymetrix miRNA microarray, and miRNA clusters were identified by web-based programs MirClust and miRGen Cluster. qRT-PCR was carried out to validate miRNA expression, and in situ hybridization (ISH) was performed to determine compartmental localization of miRNAs within villous tissue. A total of 208 miRNA transcripts, which represent 191 mature miRNAs, were found differently expressed between first and third trimester placentas. miRNAs within the miR-17-92 cluster, C14MC, miR-371 cluster, and C19MC were significantly upregulated in the first trimester placentas. In contrast, miRNAs of the let-7 family, miR-34 family, miR-29a cluster, miR-195 cluster, and miR-181c cluster were significantly upregulated in the third trimester placentas. Increased miR-371-5p, miR-17-3p, and miR-708-5p expression and decreased miR-125b-5p and miR-139-5p expression in the first trimester placentas were confirmed by qRT-PCR. Different expression pattern for miR-371-5p and miR-125b-5p within villous tissue was demonstrated by ISH. Distinct miRNA cluster expression profiles between the first and third trimester placentas were identified. miRNAs that regulate innate/adaptive immune responses are strongly expressed in both first and third trimester placentas. miRNAs that exert oncogenic, angiogenic, and antiapoptotic properties are dominantly expressed in the first trimester placentas, whereas miRNAs that promote cell differentiation and function as tumor suppressors are strongly expressed in the third trimester placentas. These results indicate that miRNAs play critical roles in placental development.

Introduction: The first step in human implantation is the attraction of the blastocyst to the endometrium. We aimed to study attraction of the human blastocyst to the endometrium, and how this process is accomplished by chemokines secreted by the endometrium., Materials and Methods: Blastocyst trophectoderm cells and other trophoblast lineage cells were subjected to attraction assays by IP-10 and other chemokines using transwell migration and chemotaxis assays. Chemokine expression and secretion were investigated using immunohistochemistry, ELISA, FACS analysis, and RT-PCR on material from flushing of the uterine cavity in endometrial biopsies. Chemokine receptor expression by blastocyst trophectoderm following PGD biopsy, trophectoderm derived from hES, placental villi, and other trophoblast lineage cells were characterized by the same methods., Results: IP-10 dramatically attracted trophectoderm derived from hES cells and other lineages by interaction with CXCR3 chemokine receptors, as shown by both chemotaxis and transwell migration. High levels of IP-10 were detected throughout the menstrual cycle at flushing of the uterine cavity. Immunohistochemistry, FACS analysis, and RT-PCR of endometrial biopsy detected IP-10 in glandular and stromal cells of the endometrium. High levels of IP-10 were detected in condition medium of the endometrial stromal and glandular cells. Of all of the chemokine/chemokine receptor combinations examined, the IP-10/CXCR3 interaction was the only cytokine that was significantly elevated., Discussion: While they await the wandering blastocyst, IP-10 is produced by many cells of the endometrium, but not by endometrial natural killer cells., Conclusion: Endometrial IP-10 may specifically attract human blastocyst trophectoderm cells early in implantation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Bone marrow derived mesenchymal stem cells (BM-MSCs) are used extensively in transplantation but their use is associated with many problems including low abundance in BM, low overall number, decreased differentiation potential with age and the invasive isolation procedures needed to obtain BM. We report a novel method of isolating placental MSCs (pMSCs) from chorionic villi, which exhibit the phenotypic and functional characteristics that will make them an attractive source of MSCs for cell-based therapy., Methods: A novel explant approach was used to isolate pMSCs from chorionic villi of human placentae. These pMSCs were characterized by flow cytometry and were differentiated into adipocytes, osteocytes and chondrocytes using differentiation medium as demonstrated by cytochemical staining. The gene and protein expression profiles of pMSCs were also characterized using real time polymerase chain reaction (PCR) and flow cytometry, respectively. In addition, cytokine secretion by pMSCs was also analysed using sandwich enzyme-linked immunosorbent assay (ELISA) technique. Moreover, the migration and proliferation potentials of pMSCs were also determined., Results: pMSCs were isolated from fetal part of the chorionic villi and these pMSCs expressed CD44, CD90, CD105, CD146, CD166 and HLA-ABC but not CD14, CD19, CD40, CD45, CD80, CD83, CD86 and HLA-DR. In addition, these pMSCs differentiated into osteocytes, chondrocytes and adipocytes and they also expressed several adhesion molecules, chemokines/receptors, growth factor receptors and cytokines/receptors. Moreover, they secreted many cytokines (IL-1Ra, IL6, IL8, IL10, IL11 and IL15) and they were able to proliferate. Furthermore, they migrated in response to chemotactic factors including stromal cell-derived factor-1 (SDF-1), platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), and monocyte chemotactic protein-1 (MCP-1)., Conclusions: We devised a novel explant method of isolating pMSCs that expressed many biological factors responsible for mediating cellular processes such as migration/homing, immune modulation and angiogenesis. Therefore, we suggest that pMSCs prepared from human term placental chorionic villous explants are an attractive source of MSCs for cell therapy.

In humans, very little is known about the factors that regulate trophoblast (TB) specification, expansion of the initial TB population, and formation of the cytotrophoblast (CTB) populations that populate the chorionic villi. The absence of human trophoblast progenitor cell (hTPC) lines that can be propagated in vitro has been a limiting factor. Because attempts to derive TB stem cells from the trophectoderm of the human blastocyst have so far failed, investigators use alternative systems as cell culture models including TBs derived from human embryonic stem cells (hESCs), immortalized CTBs, and cell lines established from TB tumors. Additionally, the characteristics of mature TBs have been extensively studied using primary cultures of CTBs and explants of placental chorionic villi. However, none of these models can be used to study TB progenitor self-renewal and differentiation. Furthermore, the propagation of human TB progenitors from villous CTBs (vCTBs) has not been achieved. The downregulation of key markers of cell cycle progression in vCTBs by the end of the first trimester of pregnancy may indicate that these cells are not a source of human TB progenitors later in pregnancy. In contrast, mesenchymal cells of the villi and chorion continue to proliferate until the end of pregnancy. We recently reported isolation of continuously self-renewing hTPCs from chorionic mesenchyme and showed that they differentiated into the mature TB cell types of the villi, evidence that they can function as TB progenitors. This new cell culture model enables a molecular analysis of the seminal steps in human TB differentiation that have yet to be studied in humans. In turn, this information can be used to trace the origins of pregnancy complications that are associated with faulty TB growth and differentiation., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Objective: Trisomy in pregnancy increases risks of miscarriage, fetal anomalies, and perinatal complications, with trisomy 16 the most common trisomy in human conceptions. The pathogenesis and protein expression profiles in trisomic pregnancy have not been well elucidated. Our objective was to profile protein kinase expression in trisomic and chromosomally normal miscarriage., Methods: Placental chorionic villus cultures were set up from first-trimester miscarriages (n = 73). Protein kinases (n = 75) were profiled using a 2D Western blot in cultures from trisomic miscarriages (n = 6) (trisomy 16 or trisomy 15) and compared to chromosomally normal (euploid) miscarriages (n = 4)., Results: Distinct patterns of protein kinase expression were seen in the two trisomic groups, including dosage-dependent over-expression of the chromosome 16-encoded ERK1 in trisomy 16. This supports a role for chromosome-specific effects in the pathogenesis of trisomy (gene dosage hypothesis). In addition, both trisomic groups had increased inter-individual variation in protein kinase expression, which supports a role for amplified sensitivity to environmental and genetic variation in the pathogenesis of trisomy (amplified instability hypothesis)., Conclusion: Both gene dosage effects and amplified instability operate simultaneously on the expression of protein kinases in trisomic pregnancies ending in miscarriage.

This study investigated the effect of epigenetic modification of maspin on extravillous trophoblastic function. The mRNA expression of maspin in placentae from normotensive and preeclamptic pregnant women was detected by RT-PCR. TEV-1 cells, a human first-trimester extravillous trophoblast cell line, were cultured and treated with CoCl(2) (300 μmol/L) to induce chemical hypoxia and with 5-aza (500 nmol/L) to induce demethylation. The mRNA expression of maspin in TEV-1 cells subjected to different treatments was determined by RT-PCR, and the proliferative and migratory abilities of TEV-1 cells were assessed by cell counting kit-8 (CCK-8) and Transwell assays. Our results showed that the maspin mRNA expression level in placentae from preeclamptic women was much higher than that from normotensive women. CoCl(2) or 5-aza could up-regulate the mRNA expression of maspin and significantly suppress the proliferation and migration of TEV-1 cells. It was concluded that the epigenetic modification in promoter region of maspin contributes to incomplete trophoblast invasion, which offers a novel approach for predicting and treating placental dysfunction.

Objective: As human blastocyst-derived extravillous trophoblasts (EVTs) invade the early decidua, they are positioned to interact with immune cells and resident decidual cells, and remodel spiral arteries into high capacity vessels that increase blood flow to the developing fetal-placental unit. Shallow EVT invasion elicits incomplete vascular transformation and reduces uteroplacental blood flow that presages adverse pregnancy outcomes. Excess macrophages in the decidua induce EVT apoptosis via tumor necrosis factor-alpha (TNF-α) secretion. Our previous observation that pro-inflammatory cytokines enhance neutrophil and macrophage activator granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in first trimester decidual cells is now extended to include: (1) the specific macrophage activator M-CSF; (2) macrophage activation and subsequent enhancement of EVT apoptosis by both GM-CSF and M-CSF., Study Design: Quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay assessed M-CSF expression in first trimester decidual cells incubated with interleukin-1 beta (IL-1β) or TNF-α. Peripheral monocyte-derived macrophages pre-incubated with conditioned media from decidual cell cultures were co-cultured with a first trimester EVT cell line, HTR-8/SVneo cells. Macrophage activation was examined and EVT apoptosis evaluated by DNA fragmentation, caspase activation and cell membrane asymmetry., Results: IL-1β or TNF-α significantly enhanced M-CSF expression in first trimester decidual cells. The conditioned media from these cultures activates macrophages, which promote caspase 3/7-dependent EVT apoptosis with antibodies against GM-CSF or M-CSF blocking this effect., Conclusions: Pro-inflammatory cytokines increases synthesis of M-CSF in first trimester decidual cells. Both GM-CSF and M-CSF activate macrophages, which initiate caspase-dependent EVT apoptosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The placenta is a highly vascularized organ thus angiogenesis is a key process in placental development. The contribution that different cells in the villous stroma play in placental angiogenesis is largely unknown. In this study we identified a novel stromal cell type in sections of term placenta which is morphologically fibroblastic and expressing the fibroblast marker TE-7 but also positive for the monocytic markers CD115 and CD14 and designated these cells as fibrocyte-like cells. Populations of fibrocyte-like cells from the placenta were isolated by two methods: culture of adherence-selected placental cells and, for higher purity, by CD45 fluorescence activated cell sorting (FACS). Fibrocyte-like cell conditioned medium increased endothelial tubule-like structure formation 2-fold versus control medium. Both pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) and the anti-angiogenic factor soluble-Flt were found in the conditioned medium. Neutralizing antibodies against VEGF and b-FGF reduced endothelial cell tubule-like structures to control levels. These data suggests that fibrocyte-like cells, a previously unidentified cell of the villous stroma, may play an important role in the regulation of placental angiogenesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

During early human pregnancy, extravillous trophoblast (EVT) cells from the placenta invade the uterine decidual spiral arterioles and mediate the remodelling of these vessels such that a low pressure, high blood flow can be supplied to the placenta. This is essential to facilitate normal growth and development of the foetus. Defects in remodelling can manifest as the serious pregnancy complication pre-eclampsia. During the period of vessel remodelling three key pregnancy-associated hormones, human chorionic gonadotrophin (hCG), progesterone (P(4)) and oestradiol (E(2)), are found in high concentrations at the maternal-foetal interface. Potentially these hormones may control EVT movement and thus act as regulators of vessel remodelling. This review will discuss what is known about how these hormones affect EVT proliferation, migration and invasion during vascular remodelling and the potential relationship between hCG, P(4), E(2) and the development of pre-eclampsia., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The effect of axon guidance factors ephrin-A1/EphA2 on the invasion of trophoblastic cells and the possible mechanism were investigated in this study. The expression of EphA2 in vascular endothelial cells was detected by immunohistochemistry. The proliferation and invasion of TEV-1 cells (an extravillous trophoblastic cell line) in first trimester were determined by cell counting kit-8 (CCK-8) and Transwell invasion assay. Real-time PCR was used to detect the expression of ephrin-A1 in TEV-1 cells treated with EphA2 at different concentrations (10, 50, 100, 500, 1000 and 5000 μg/L). The results showed: (1) EphA2 was expressed in the vascular endothelial cells; (2) EphA2 could promote the proliferation of TEV-1 cells. The proliferative capacity reached a peak in TEV-1 cells treated with 100 μg/L EphA2 (P<0.05); (3) EphA2 could increase the invasion of TEV-1 cells. The invasive ability was the greatest in TEV-1 cells treated with 500 μg/L EphA2 (P<0.05); (4) in the presence of EphA2 (0-500 μg/L), the expression of ephrin-A1 was increased concentration-dependently (P<0.05), but when the concentration of EphA2 was over 500 μg/L, the expression of ephrin-A1 ceased to increase (P>0.05). It was concluded that EphA2 can promote the invasion and proliferation of the human extravillous trophoblastic cells probably by regulating the ephrin-A1 ligand.

Pregnancy-associated glycoproteins (PAGs) are abundant embryo-originated products expressed in the pre-placental trophoblast and later in the post-implantational chorionic epithelium of some ungulate species. This paper describes the cellular immunolocalization of the chorionic PAG family in the epitheliochorial placenta type of the alpaca (Lama pacos-Lp), in which the PAGs were named 'LpPAGs'. Placental Lp sections (5 μm) of different females near mid-pregnancy (150 days post coitum; dpc), advanced pregnancy (244-263 dpc) and late pregnancy (347 dpc) were used for cross-species (heterologous-ht) double fluorescent immunohistochemistry (htdF-IHC). The htdF-IHC was performed with primary rabbit polyvalent anti-porcine PAG polyclonals. The LpPAG immuno-complexes were visualized with secondary goat anti-rabbit immunoglobulins-conjugated with Alexa 488 fluorophore (green), among all nuclei of placental cells stained with propidium iodide (red). This is the first study reporting the immunolocalization of the LpPAG family identified by htdF-IHC at the feto/maternal interface during different pregnancy stages of the alpaca. The most dominant and strongest immune-positive LpPAG signals were found in the well-developed chorionic cell layer. Our htdF-IHC indicated relatively high epitope resemblance to that of the PAGs in camelids and pigs. These data increase our general knowledge of chorionic PAG localization during pregnancy-stage dependent development of the epitheliochorial diffuse placenta type in the alpaca., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Cyclic 3',5'-adenosine monophosphate (cAMP) is a critical second messenger for human trophoblasts and regulates the expression of numerous genes. It is known to stimulate in vitro the fusion and differentiation of BeWo choriocarcinoma cells, which acquire characteristics of syncytiotrophoblasts. A DNA microarray analysis of BeWo cells undergoing forskolin-induced syncytialization revealed that among the induced genes, placental growth factor (PlGF) was 10-fold upregulated. We verified this result in two choriocarcinoma cell lines, BeWo and JEG-3, and also in first trimester placental villous explants by quantifying PlGF mRNA (real time PCR) and PlGF protein secreted into the supernatant (ELISA). Similar effects were noted for vascular endothelial growth factor (VEGF) mRNA and protein expression. Treatment with cholera toxin and the use of a specific inhibitor of protein kinase A (PKA) blocked these effects, indicating that the cAMP/PKA pathway is responsible for the cAMP-induced upregulation of PlGF and that one or more G protein coupled receptor(s) was involved. We identified two functional cAMP responsive elements (CRE) in the PlGF promoter and demonstrated that the CRE binding protein, CREB, contributes to the regulation of PlGF gene expression. We speculate that defects in this signaling pathway may lead to abnormal secretion of PlGF protein as observed in the pregnancy-related diseases preeclampsia and intrauterine growth restriction.

Early placental development is critical for successful pregnancy. Recently, we have reported that -70 genes were differentially expressed in human placental villi between 6- and 8- weeks of gestation in cDNA-based expression arrays for -400 PCR products, of which six specific gene products (COL4A4, CXCR4, ERBB2, HDAC1, HPRT1, and TNFRSF1A) appeared intriguing. In the present study we have examined expressions of these six candidate genes in placental villi obtained from 6-weeks, 7-weeks and 8-weeks (n = 6 for each group) human placental samples using quantitative real time RTPCR. We observed that there was considerable concordance (>95% confidence) in pair-wise analysis of transcript profiles between the two methods, however, absolute quantitative values as measured by quantitative RTPCR differed from those obtained from cDNA-based array analysis for 2 gene products (CXCR4 and ERBB2) out of 6 genes. No significant change was observed in the steady state expression of COL4A4 and HPRT1 during the time period examined. However, there was significant decrease in CXCR4 for 7-weeks (P < 0.01) and 8-weeks (P < 0.05) samples, and significant (P < 0.05) increase was seen for ERBB2 in 7-weeks and 8-weeks as compared to 6-weeks samples with no change between 7-weeks and 8-weeks samples. Moreover, significant (P < 0.05) increase for HDAC1 and decrease for TNFRSF1A was observed in 8-weeks samples as compared to 6-weeks samples with no change observed between 6-weeks and 7-weeks samples. We infer that it is essential that cDNA array-based data are verified in terms of quantitative estimates preferably by quantitative PCR before their use for any exploratory purpose. Taking together our previous array based data and the present study we conclude that a categorical balance exists between the expression of ERBB2 and HDAC1 genes affecting cell proliferation and differentiation in one hand, and CXCR4 and TNFRSF1A affecting chemotaxis, inflammatory response and apoptosis on the other hand. The expression of these genes appear important for the early development of human placental villi.

Background: Routes of trophoblast invasion seem to be clear, whereas specific invasive pathways need further elucidation. Extravillous trophoblasts (EVTs) transform spiral arteries to guarantee appropriate blood flow to the placenta in the second trimester. Embryo nutrition during the first trimester is thought to be histiotrophic, whereas proof that EVTs also invade uterine glands is lacking. We developed novel three-dimensional confrontation co-culture models to elucidate invasion of EVTs into uterine glands., Methods: First trimester decidua parietalis and placental villous explants were directly confronted and co-cultured for 72 h, or confronted indirectly after 72 h pre-culture for re-epithelialization of decidua pieces. Cryosections were stained by immunohistochemistry or immunofluorescent/immunohistochemical double labelling and compared with first trimester placentation sites in situ., Results: EVTs deeply invaded decidual tissues in direct confrontation assays and were found between the decidual epithelial cells and epithelial basement membrane. EVTs were also detected in the decidual stroma in direct proximity to glands, sometimes even replacing glandular epithelial cells. Similar observations were made in sections from the first trimester decidua/placental bed. In the invaded parts of sections of decidua basalis, 55% +/- 7% (mean +/- SEM; n = 10, range 6-11 weeks) of glandular cross sections were associated with or infiltrated by EVTs., Conclusions: Using novel confrontation co-culture assays, a potential new route of EVT invasion was detected. EVTs appear to break through the basement membrane of uterine glands to open their lumen towards the intervillous space. These data support the hypothesis of histiotrophic nutrition of the embryo prior to onset of maternal blood flow within the placenta.

The chorionic villi of human term placentae are a rich source of mesenchymal stem cells (PMSCs). The stem cell "niche" within the chorionic villi regulates how PMSCs participate in placental tissue generation, maintenance and repair, but the anatomic location of the niche has not been defined. A number of cell surface markers for phenotypic characterisation of mesenchymal stem cells (MSCs) were employed to identify the stem cell niche within the chorionic villi of first trimester and term human placenta. This included antibodies to pericyte cell surface markers STRO-1 and 3G5, which have been used to identify mesenchymal stem cells in other tissues, but have not been studied in placental tissues. PMSCs were isolated from term human placentae and shown to have stem cell properties by their ability to grow on untreated plastic culture ware, capacity for forming clones (i.e. clonogenicity) and their capability to differentiate into adipocytes, chondrocytes and osteocytes. Western analysis confirmed that STRO-1 and 3G5 are present in placental protein extracts and in PMSCs. Immunocytochemistry revealed PMSCs were positive for MSC cell surface markers (STRO-1, 3G5, CD105, CD106, CD146, CD49a, alpha-SMA) and negative for haematopoietic stem cell markers (CD117, CD34) and endothelial markers (CD34, vWF). Immunohistochemistry with antibodies to MSC cell surface markers on first trimester and term tissues revealed a vascular niche for PMSCs. Dual-label immunofluorescence analysis was used to compare STRO-1 antibody staining with that of endothelial cell marker vWF and found no significant overlap in staining. This indicated that some PMSCs have a pericyte-like phenotype. We propose that the vascular niche harbours a pool of PMSCs that can give rise to committed progenitors for tissue maintenance and repair, and that PMSCs contribute to vessel maturation and stabilization., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Disturbed trophoblast turnover, a key process in placental physiology, may lead to a number of pregnancy-associated pathologies. This study examines PCNA expression and describes and quantifies morphological changes during trophoblast turnover in preterm placentas and term placentas complicated by IUGR. The number ofCTF cells increased two-fold in preterm and IUGR placentas. A concurrent and slightly reduced proliferation rate of these cells was also found. The number of STF nuclei of terminal villi was lower by 21% in IUGR and by 18% in preterm placentas (P > 0.05). A statistically significant reduction of the number of syncytial knots by 50% as compared to the control placentas was observed. Correlations between PCNA-reactive CTF nuclei and syncytial knots, PCNA-reactive CTF nuclei and CCO activity, and CCO activity and syncytial knots were found. Moreover, a strong inverse relation was observed between syncytial knots and CTF cells, and CCO activity and CTF cells.

Human ADAM19 is a recently identified member of the ADAM family. It is highly expressed in human placentas, but its dynamic change and function at the human feto-maternal interface during placentation remain to be elucidated. In this present study, the spatial and temporal expression and cellular localization of ADAM19 in normal human placentas were first demonstrated, and the effects of ADAM19 on trophoblast cell adhesion and invasion were further investigated by using a human choriocarcinoma cell line (JEG-3) as an in vitro model. The data demonstrated that ADAM19 was widely distributed in villous cytotrophoblast cells, syncytiotrophoblast cells, column trophoblasts, and villous capillary endothelial cells during early pregnancy. The mRNA and protein level of ADAM19 in placentas was high at gestational weeks 8-9, but diminished significantly at mid- and term pregnancy. In JEG-3 cells, the overexpression of ADAM19 led to diminished cell invasion, as well as increases in cell adhesiveness and the expression of E-cadherin, with no changes in beta-catenin expression observed. These data indicate that ADAM19 may participate in the coordinated regulation of human trophoblast cell behaviors during the process of placentation.

Villous cytotrophoblast differentiation and subsequent fusion with the overlying syncytiotrophoblast depend on multiple factors such as growth factors, cytokines, hormones, protein kinases, transcription factors, structural membrane proteins and proteases. Caspase 8, an aspartate-specific cysteine protease, is mainly known for its role in programmed cell death, but was also demonstrated to be crucial for villous trophoblast differentiation. This study aimed to localize active caspase 8 in the villous trophoblast layer of human first trimester placenta. To this end, immunofluorescence double staining was performed, using a monoclonal rabbit antibody against cleaved caspase 8 in combination with antibodies against cytokeratin 7, chorionic gonadotropin beta subunit (beta hCG), beta-actin, placental protein 13 (PP13), alpha-fodrin and Ki-67. Immunofluorescence revealed cleaved caspase 8 in one out of 422 villous cytotrophoblasts resting on the basement membrane, in one out of 759 perinuclear regions within the syncytiotrophoblast and in few trophoblasts located between these two layers. Double staining of cleaved caspase 8 and Ki-67 antigen revealed that caspase 8 is activated only in cytotrophoblasts which have left the cell cycle. The staining suggests that caspase 8 is activated in villous cytotrophoblasts just prior to fusion of these cells and escorts the nuclei from the mononucleated to the syncytial state.

Background: Decoy receptor 3 (DcR3) is a soluble receptor belonging to the tumor necrosis factor receptor superfamily and can inhibit Fas ligand-induced apoptosis. DcR3 is related to carcinogenesis, but is found in human gestational tissues, where the function is uncertain. We aimed to determine DcR3 expression and regulation in endometrial cells in vitro, and throughout the menstrual cycle and pregnancy in vivo., Methods: Serum DcR3 levels were measured at various stages of the menstrual cycle (n = 40 women) and pregnancy (n = 20). DcR3 transcript and protein levels were analyzed in RL95-2 endometrial cells after treatment with estradiol (E(2)) (+/- anti-estrogen) and/or progesterone (+/- anti-progesterone). Finally, DcR3 protein and transcript were examined in decidua and chorionic villi from normal (n = 14) and anembryonic (n = 14) pregnancies., Results: We identified a trend towards cyclic changes of serum DcR3 levels in the normal menstrual cycle, peaking at the mid-luteal phase, and relatively lower, more stable serum levels throughout normal gestation. DcR3 protein levels were higher in decidua than chorionic villi in normal pregnancy (P = 0.001), and levels in decidua were significantly lower in anembryonic than in normal pregnancies (P = 0.034). Physiologic concentrations of E(2) and/or progesterone stimulated DcR3 transcripts in RL95-2 endometrial cells., Conclusions: This study suggested that DcR3 expression varies during the menstrual cycle and is regulated by sex steroid hormones in vitro in endometrial cells. Human gestational tissues showed a differential production of DcR3, and decidual DcR3 protein was lower in anembryonic than normal pregnancies, suggesting an active role of DcR3 in the regulation of successful pregnancies.

In the placental villus, cells attach to basement membrane via integrin alpha6beta4 and adhere both laterally and apically to their neighbours. The most prominent adhesive specialisation seen using the electron microscope is the desmosome, which connects cytotrophoblast cells (CTB) laterally and also contributes to the attachment of CTB to the overlying syncytium. However, numerous cadherins and other junctional proteins are also present in the corresponding plasma membrane domains, indicating a multiplicity of adhesive interactions. Integrins, tight junction components and cadherins are all found in the syncytial microvillous membrane, perhaps reflecting its ability to form intersyncytial bridges. There is a wide gulf to be filled between molecular anatomy and functional studies, with much to be learned about the role of adhesion molecules in regulating villous epithelial integrity, homeostasis and growth.

First trimester human villous vascularization is mainly studied by conventional two-dimensional (2D) microscopy. With this (2D) technique it is not possible to observe the spatial arrangement of the haemangioblastic cords and vessels, transition of cords into vessels and the transition of vasculogenesis to angiogenesis. The Confocal Laser Scanning Microscopy (CLSM) allows for a three-dimensional (3D) reconstruction of images of early pregnancy villous vascularization. These 3D reconstructions, however, are normally analyzed on a 2D medium, lacking depth perception. We performed a descriptive morphologic study, using an immersive Virtual Reality system to utilize the full third dimension completely. This innovative 3D technique visualizes 3D datasets as enlarged 3D holograms and provided detailed insight in the spatial arrangement of first trimester villous vascularization, the beginning of lumen formation within various junctions of haemangioblastic cords between 5 and 7 weeks gestational age and in the gradual transition of vasculogenesis to angiogenesis. This innovative immersive Virtual Reality system enables new perspectives for vascular research and will be implemented for future investigation.

We investigated whether the human placenta contributes to embryonic and fetal hematopoietic development. Two cell populations--CD34(++)CD45(low) and CD34( +)CD45(low)--were found in chorionic villi. CD34(++) CD45(low) cells display many markers that are characteristic of multipotent primitive hematopoietic progenitors and hematopoietic stem cells. Clonogenic in vitro assays showed that CD34(++)CD45( low) cells contained colony-forming units-culture with myeloid and erythroid potential and differentiated into CD56(+) natural killer cells and CD19(+) B cells in culture. CD34(+)CD45(low) cells were mostly enriched in erythroid- and myeloid-committed progenitors. While the number of CD34(++)CD45(low) cells increased throughout gestation in parallel with placental mass. However, their density (cells per gram of tissue) reached its peak at 5 to 8 weeks, decreasing more than 7-fold from the ninth week onward. In addition to multipotent progenitors, the placenta contained intermediate progenitors, indicative of active hematopoiesis. Together, these data suggest that the human placenta is potentially an important hematopoietic organ, opening the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.

In addition to its most well characterized biological role in the rescue and maintenance of corpus luteum function, human chorionic gonadotropin (hCG) also stimulates the onset of fetal gonadal steroidogenesis. However, excess hCG is teratogenic to fetal gonadal tissues, and therefore hCG must be tightly regulated. Although there is an anatomical barrier between the fetal vessels and maternal blood, other mechanisms may regulate hCG levels. In the present study, we investigated whether human chorionic villous macrophages degraded maternal hCG. Isolated human macrophages incorporated and degraded hCG in a time-dependent manner. Human placental villous macrophages and phorbol myristate acetate (PMA)-treated THP-1 cells expressed the gene encoding an exon 9-deleted form of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor; expression of the full-length receptor was not determined. While both PMA-treated or untreated THP-1 cells could uptake hCG into their cytoplasms, hCG degradation and excretion of its byproducts only progressed in PMA-treated THP-1 cells. In conclusion, hCG internalization and degradation are different processes in macrophages that protect fetal gonadogenesis from excess hCG. The exon 9-deleted LH/CG receptor, but not the full-length receptor, is involved in the degradation of cytoplasmic hCG by organ-specific, dominant-negative interactions.

Placenta vascular formation is important for fetal growth and development. Proper development of the placenta ensures the exchange of oxygen/nutrients and blood flow necessary for fetal growth. In this chapter, we will discuss the processes of vasculogenesis, angiogenesis, and pseudovasculogenesis during placental development and in pregnancies complicated by intrauterine growth restriction. Some of the factors controlling these processes include oxygen, the VEGF family of growth factors, and their receptors. Disruption in the balance of these controlling factors may explain the vascular malformations seen in pregnancies complicated by intrauterine growth restriction.