Functional variation among mesenchymal stem cells derived from different tissue sources.

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.

Stereological Analysis of Adolescent Placentas and Anthropometric Characteristics of Newborns.

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.

Role of ACE I/D polymorphism in pathological assessment of preeclampsia in Pakistan.

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.

Higher chromosomal aberration rate in miscarried conceptus from polycystic ovary syndrome women undergoing assisted reproductive treatment.

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.

The Density of Cell Nuclei at the Materno-Fetal Exchange Barrier is Sexually Dimorphic in Normal Placentas, but not in IUGR.

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.

High-throughput transcriptome-Seq and small RNA-Seq reveal novel functional genes and microRNAs for early embryonic arrest in humans.

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.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

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.

Computational modeling of the structure-function relationship in human placental terminal villi.

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.

Villus packing density and lacunarity: Markers of placental efficiency?

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, ß calculated as (ln (placental weight)/ln (birthweight)) from Kleiber's law [1]. Three ∼4.3 mm2 images each were obtained from 88 term placentas. Ranges of VPD and lacunarity were each correlated with ß (r = 0.31, p = 0.003, r = 0.23, p = 0.03 and respectively). The relationship between ß 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.

Expression of PAPPA2 in human fetomaternal interface and involvement in trophoblast invasion and migration.

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.

A microphysiological model of the human placental barrier.

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.

Adiponectin Inhibits Nutrient Transporters and Promotes Apoptosis in Human Villous Cytotrophoblasts: Involvement in the Control of Fetal Growth.

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.

Expression profile of micro-RNAs and functional annotation analysis of their targets in human chorionic villi from early recurrent miscarriage.

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.

Silencing of Paternally Expressed Gene 10 Inhibits Trophoblast Proliferation and Invasion.

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.

Statistical Mechanics of the Human Placenta: A Stationary State of a Near-Equilibrium System in a Linear Regime.

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.

High mobility group box 1 protein polymorphism affects susceptibility to recurrent pregnancy loss by up-regulating gene expression in chorionic villi.

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.

Propagation of pure fetal and maternal mesenchymal stromal cells from terminal chorionic villi of human term placenta.

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.

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2)...

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2)...

Dynamic changes in hyperglycosylated human chorionic gonadotrophin throughout the first trimester of pregnancy and its role in early placentation.

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.