Trophoblast Cell Populations Research Articles

Invasion and genome reproduction of the trophoblast cells of placenta junctional zone in the field vole, Microtus rossiaemeridionalis

In the field vole Microtus rossiaemeridionalis, like in other rodents, invasive secondary giant trophoblast cells (SGTC) form a continuous layer at the foeto-maternal interface in the beginning of placentation. However, in the field vole, at midgestation, clusters of junctional zone (JZ) trophoblast non-giant cells interrupt SGTC layer and progressively replace SGTC at the border of decidua basalis. As a result, 'border' cells form a continuous stratum of cytokeratin-positive glycogen-rich cells at the foeto-maternal interface. SGTC plunge into JZ and line the lacunae with maternal blood. SGTC are bound by their highly cytokeratin-positive sprouts forming a framework that holds other trophoblast cell populations. According to DNA cytophotometry, the 'border' cells show the highest ploidy among the JZ cells (up to 46% of 8c cells). Thus, in M. rossiaemeridionalis the role of barrier between semiallogenic foetal and maternal tissues is shifted from the highly endopolyploid (32c-1024c) SGTC to the specific subpopulation of glycogen-rich non-giant (2c-16c) 'border' trophoblast cells that, however, exceed the ploidy of the deeply located and/or proliferative JZ trophoblast cells.

Placental leptin in HIV-associated preeclampsia

ObjectiveHIV-associated preeclampsia reflects a combination of opposing influences on the immune status. The adipocyte hormone leptin has been implicated in the pathophysiology of preeclampsia and in enhancing immunity. This study is the first, to our knowledge, to determine whether leptin levels in the placenta differ between HIV-associated normotensive and preeclamptic pregnancies. The study also compares leptin levels between the exchange and conducting areas of the placenta. Study designPregnant women were recruited antenatally and grouped as follows: normotensive HIV uninfected (n=30), normotensive HIV infected (n=60), preeclamptic HIV uninfected (n=30) and preeclamptic HIV infected (n=60). Anthropometric data were collected and placental leptin was analysed by immunohistochemistry and ELISA. ResultsLeptin levels were similar in the central and peripheral regions of the placenta. Leptin immunoreactivity was observed amongst the different trophoblast cell populations. Both ELISA and immunohistochemistry of the placental exchange villi indicated that leptin levels were higher in preeclampsia compared to normotensive pregnancies (p<0.001). HIV status had no effect on leptin levels but levels were higher in participants on highly active antiretroviral treatment (HAART) compared to those on prophylaxis for prevention of mother to child transmission (PMTCT) with normotensive (p=0.006) and preeclamptic (p=0.002) pregnancies. The area of immunostaining was greater in the exchange compared to the conducting villi in HIV infected and uninfected preeclampsia. ConclusionsThis novel study establishes an elevation of leptin in preeclamptic placentae, irrespective of HIV status. Leptin elevation was not focal in that it occurred in both central and peripheral regions of the preeclamptic placenta. This suggests a role of leptin in the pathophysiology of preeclampsia.

Altered expression of interleukin-6, interleukin-8 and their receptors in decidua of women with sporadic miscarriage

Are alterations in decidual expression of interleukin (IL)-6 and IL-8 associated with sporadic miscarriage? IL-6 and IL-8 secretion from decidual uterine natural killer (uNK) cells and macrophages isolated from women with spontaneous miscarriage was reduced compared with normal controls. Miscarriage is a common gynaecological problem with huge financial and personal implications. Eleven to twenty per cent of all clinically recognized pregnancies are lost before the 20th week of gestation, with miscarriages often being divided into early (≤ 12 completed weeks from last menstrual period) and late (≥ 13 weeks). Spiral artery remodelling is a key feature of early pregnancy; failure of this process has been implicated in sporadic miscarriage. The molecular triggers that initiate spiral artery remodelling are not clear, although cytokines such as IL-6 and IL-8 may play a role. This was a laboratory-based study using decidual and placental bed biopsy samples from women with sporadic miscarriage (n = 30) and termination of pregnancy controls (n = 30). Total adherent decidual cells, CD10(+) stromal cells, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from apparently normal pregnancies that were terminated at either 8-10 or 12-14 weeks' gestation. In addition, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from sporadic miscarriage at 8-10 weeks' gestation. Secreted IL-8 was measured in all isolated cell populations, while IL-6 was measured in CD14(+) macrophages and CD56(+) uNK cells from both sporadic miscarriage and normal controls. Placental bed biopsies were taken from women after sporadic miscarriage or termination of pregnancy at ≤ 12 completed weeks' or >13 weeks' gestational age, formalin-fixed, paraffin-embedded and immunostained for IL-6, IL-6Rα, GP130, IL-8, CXCR1, CXCR2 and CD13 (aminopeptidase N). Staining intensity for each factor was assessed in extravillous trophoblast cell populations, myometrial and decidual stroma, myometrial and decidual spiral arteries and decidual glandular epithelium. A CPA model was used to assess the potential role of IL-6 and IL-8 in spiral artery remodelling. IL-8 was secreted by total adherent decidual cells, CD10(+) stromal cells and CD14(+) macrophages at both 8-10 and 12-14 weeks' gestation, with CD14(+) cells secreting the highest levels. Both CD14(+) and CD56(+) cells isolated from decidua of early sporadic miscarriage produced lower IL-6 (P = 0.04, P = 0.01, respectively) and IL-8 levels (P = 0.0007, P = 0.002, respectively) compared with normal cases. In addition, altered expression of IL-6, IL-8 and their receptors was observed in various cell types in placental bed (myometrial stroma, glandular epithelium, interstitial extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells) in sporadic miscarriage, particularly from later gestational ages. IL-6 and IL-8 disrupted vascular smooth muscle morphology and organization in an in vitro model of spiral artery remodelling. By the nature of sampling at the time of miscarriage, it was not possible to ascertain the cause or effect in the observed alterations of levels of IL-6 and IL-8 in sporadic miscarriage. Alterations in the expression of IL-6, IL-8 and their receptors may be associated with the aetiology of sporadic miscarriage, especially given the potential role of these cytokines in the regulation of trophoblast invasion and spiral artery remodelling. This project was supported by funding from Wellbeing of Women (RG1000). The authors have no competing interests to declare. Not applicable.

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al., in which a percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for human trophoblast cell isolation. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 7. Here, the isolated cells are then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro. The invaded cells are analyzed by immunocytochemistry and stained for counting.

Endopolyploid and proliferating trophoblast cells express different patterns of intracellular cytokeratin and glycogen localization in the rat placenta

The presence of keratin intermediate filaments is a characteristic of trophoblast differentiation. Meantime, their intracellular localization in the functionally different subtypes of placental trophoblast is poorly investigated in rodent, whereas their placentae are being broadly investigated in recent years as a model of the feto-maternal interaction. The purpose was to study the intracellular distribution of cytokeratin filaments in correlation with glycogen deposits, both being important constituents of the trophoblast cells in rat placenta. Different rat trophoblast cell populations exhibited different patterns of cytokeratin immunolocalization. The most intensive immunostaining was observed in the highly endopolyploid SGTCs (secondary giant trophoblast cells) at the border with decidua basalis. The most prominent cytokeratin-positive threads were found at the periphery of cytoplasm and in the extensive system of cytoplasmic sprouts by which the SGTC connect each other. Similar cytokeratin intensity and distribution was detected in the TSC (trabecular spongiotrophoblast cells) of the junctional zone of placenta that line the lacunae with the maternal blood. Clusters of highly proliferative pre-glycogen as well as glycogen cells showed some weaker cytokeratin signals mostly in the perinuclear and peripheral zones of cytoplasm. At the 11.5th to the 13.5th day of gestation, the interstitial and endovascular invasive endopolyploid TGTCs (tertiary giant trophoblast cells) prove the intensive cytokeratin staining throughout the cytoplasm and its sprouts. Meantime, the TGTCs were glycogen negative. By contrast, glycogen was heavily accumulated in the glycogen cells that belong both to the junctional zone of placenta and the cuff of the central arterial channel underlying the monolayer of endovascularly invading TGTCs. Thus, the TGTCs that are first to penetrate into the depth of the uterine wall do not contain glycogen but are accompanied by the glycogen-rich cells. The SGTC also contained the prominent deposits of glycogen at the periphery of cytoplasm and in the cytoplasmic sprouts. At the 16th day of gestation, an extensive interstitial invasion of the cytokeratin-positive glycogen trophoblast cells from the junctional zone was observed. The patterns of cytokeratin and glycogen intracellular localization are specific for each subtype of the rat trophoblast; that is, most probably, accounted for by the functional diversity of different trophoblast populations, i.e. patterns of invasion/phagocytosis and their involvement in a barrier at the feto-maternal interface.

Nodal Signals through Activin Receptor-Like Kinase 7 to Inhibit Trophoblast Migration and Invasion: Implication in the Pathogenesis of Preeclampsia

Trophoblast cell invasion into the uterus is an essential process for successful pregnancy, and shallow invasion of trophoblasts into the maternal decidua is linked to preeclampsia. We have reported that Nodal, a member of the transforming growth factor-β superfamily, acts through activin receptor-like kinase 7 (ALK7) to inhibit trophoblast proliferation and to induce apoptosis. In this study, we examined the spatial and temporal expression patterns of Nodal and ALK7 in human placenta from normal and preeclamptic pregnancies and investigated whether Nodal regulated trophoblast migration and invasion. Nodal and ALK7 were detected in villous and extravillous trophoblast cell populations in early gestation, and their levels were strongly up-regulated in preeclamptic placenta. Overexpression of Nodal or constitutively active ALK7 decreased cell migration and invasion, whereas knockdown of Nodal and ALK7 had the opposite effects. In placental explant culture, treatment with Nodal inhibited trophoblast outgrowth, whereas Nodal small-interfering RNA strongly induced the expansion of explants and the migration of extravillous trophoblast cells. Nodal stimulated the secretion of tissue inhibitor of metalloproteinase-1 and inhibited matrix metalloproteinase (MMP)-2 and MMP-9 activity. These findings suggest that the Nodal/ALK7 pathway plays important roles in human placentation and that its abnormal signaling may contribute to the development of preeclampsia.

Wide-ranging DNA methylation differences of primary trophoblast cell populations and derived cell lines: implications and opportunities for understanding trophoblast function.

Difficulties associated with long-term culture of primary trophoblasts have proven to be a major hurdle in their functional characterization. In order to circumvent this issue, several model cell lines have been established over many years using a variety of different approaches. Due to their differing origins, gene expression profiles and behaviour in vitro, different model lines have been utilized to investigate specific aspects of trophoblast biology. However, generally speaking, the molecular mechanisms underlying functional differences remain unclear. In this study, we profiled genome-scale DNA methylation in primary first trimester trophoblast cells and seven commonly used trophoblast-derived cell lines in an attempt to identify functional pathways differentially regulated by epigenetic modification in these cells. We identified a general increase in DNA promoter methylation levels in four choriocarcinoma (CCA)-derived lines and transformed HTR-8/SVneo cells, including hypermethylation of several genes regularly seen in human cancers, while other differences in methylation were noted in genes linked to immune responsiveness, cell morphology, development and migration across the different cell populations. Interestingly, CCA-derived lines show an overall methylation profile more similar to unrelated solid cancers than to untransformed trophoblasts, highlighting the role of aberrant DNA methylation in CCA development and/or long-term culturing. Comparison of DNA methylation and gene expression in CCA lines and cytotrophoblasts revealed a significant contribution of DNA methylation to overall expression profile. These data highlight the variability in epigenetic state between primary trophoblasts and cell models in pathways underpinning a wide range of cell functions, providing valuable candidate pathways for future functional investigation in different cell populations. This study also confirms the need for caution in the interpretation of data generated from manipulation of such pathways in vitro.

Maternal Immune Responses to Trophoblast: The Contribution of the Horse to Pregnancy Immunology

The horse has proven to be a distinctively informative species in the study of pregnancy immunology for several reasons. First, unique aspects of the anatomy and physiology of the equine conceptus facilitate approaches that are not possible in other model organisms, such as non-surgical recovery of early stage embryos and conceptuses and isolation of pure trophoblast cell populations. Second, pregnant mares make strong cytotoxic antibody responses to paternal major histocompatibility complex class I antigens expressed by the chorionic girdle cells, permitting detailed evaluation of the antigenicity of these invasive trophoblasts and how they affect the maternal immune system. Third, there is abundant evidence for local maternal cellular immune responses to the invading trophoblasts in the pregnant mare. The survival of the equine fetus in the face of strong maternal immune responses highlights the complex immunoregulatory mechanisms that result in materno-fetal tolerance. Finally, the parallels between human and horse trophoblast cell types, their gene expression, and function make the study of equine pregnancy highly relevant to human health. Here, we review the most pertinent aspects of equine reproductive immunology and how studies of the pregnant mare have contributed to our understanding of maternal acceptance of the allogeneic fetus.

Non-classical MHC-E (Mamu-E) Expression in the Rhesus Monkey Placenta

The aim of this study was to characterize the expression of the rhesus HLA-E ortholog Mamu-E, particularly at the maternal–fetal interface. Mamu-E expression was confirmed by locus-specific RT-PCR in the placenta as well as in peripheral blood mononuclear cells (PBMC) and other organs. We evaluated the utility of antibodies recognizing HLA-E (MEM-E/06 against native HLA-E, MEM-E/02 against denatured HLA-E) to detect Mamu-E by flow cytometry/immunofluorescence, Western blot, and immunohistochemistry (IHC). Western blot analysis of cells and selected transfectants confirmed the recognition of Mamu-E but not Mamu-AG by antibodies MEM-E/06 and HC10 but not MEM-E/02. Immunohistochemical staining of frozen sections of rhesus placenta with the MEM-E/06 antibody demonstrated expression in most populations of rhesus monkey trophoblast cells, including villous cytotrophoblasts (strong positive staining), apical membrane of syncytiotrophoblasts (light to moderate staining) and extravillous cytotrophoblasts (moderate to strong staining, especially endovascular trophoblasts in early pregnancy). Expression was not trophoblast cell-specific, especially at term, when endothelial cells in both the chorionic plate and placental villi showed strong staining for Mamu-E. Staining of rhesus extravillous trophoblast cells suggested the co-expression of Mamu-E and Mamu-AG (the rhesus HLA-G homolog) on these cells. MEM-E/06 was shown also to react with differentiating rhesus placental syncytiotrophoblasts in primary culture, detecting intracellular and weak surface expression of Mamu-E. We conclude that the gestation-dependent co-expression of Mamu-E with Mamu-AG in villous and extravillous trophoblast cells suggests important and perhaps complementary but distinct roles of these two non-classical MHC class I loci in pregnancy at the maternal–fetal interface. In addition, the MEM-E/06 antibody will be useful for the detection of Mamu-E at the maternal–fetal interface in the rhesus monkey.

Expression of TGF beta in the placental bed is not altered in sporadic miscarriage

Extravillous trophoblast invasion of uterine stroma and spiral arteries (SA) is essential for normal pregnancy and is reduced in preeclampsia and late miscarriage. The control mechanisms are not understood, but transforming growth factor-beta (TGF-β) may be a candidate. Placental and placental bed biopsies were obtained from early (8 +0–12 +6 weeks) euploid miscarriages ( n = 10), early aneuploid miscarriages ( n = 10), late (13 +0–19 +6 weeks) euploid miscarriages ( n = 10) and controls of the same gestation ( n = 20). Frozen sections were immunostained for TGF-β1, 2 and 3. Immunoreactivity of trophoblast and uterine cell populations was assessed semi-quantitatively. TGF-β1 immunolocalization was limited to extracellular matrix in cytotrophoblast islands and cytotrophoblast shell, perivascular fibrinoid and interstitial trophoblast and did not differ in miscarriage compared with controls. TGF-β2 was expressed additionally in endovascular trophoblast and multinucleate trophoblast giant cells. There was no aberrant TGF-β2 immunolocalization in late miscarriage, but TGF-β2 immunoreactivity was increased in extracellular matrix in cytotrophoblast islands in early miscarriage. TGF-β3 was absent from all cell populations. Stromal and extravillous trophoblast TGF-β2 immunolocalization suggests a more important role in trophoblast invasion than TGF-β1, but neither isoform was altered in miscarriage. Altered TGF-β localization is therefore unlikely to play a role in abnormal trophoblast invasion and SA transformation in miscarriage.

Epithelioid trophoblastic tumor of the uterus: a report of three cases

Epithelioid trophoblastic tumor of the uterus: a report of three cases

Clinicopathologic study of tumors of intermediate trophoblasts

To study the clinicopathologic features and immunophenotype of placental site trophoblastic tumor (PSTT) and epithelioid trophoblastic tumor (ETT). During the period from 1959 to 2005, a total of 1012 cases of gestational trophoblastic disease were diagnosed in Beijing Obstetrics and Gynecology Hospital. Six cases of PSTT and a case of ETT were retrieved from the archives of Beijing Obstetrics and Gynecology Hospital. Immunohistochemical study for cytokeratin 18, human chorionic gonadotropin (hCG), human placental lactogen (hPL), Mel-CAM (CD146), placental-like alkaline phosphatase (PLAP), epithelial membrane antigen (EMA), inhibin-alpha and proliferative cell nuclear antigen (PCNA) were performed. The morphologic features and immunohistochemical findings were compared with those of the controlled group which consisted of 20 cases of early gestational villi with decidua basalis and 20 cases of hydatidiform moles with implantation site. The mean age of patients with PSTT was 32.4, while the age of patients with ETT was 36. Major clinical findings included irregular vaginal bleeding and amenorrhea. Preoperative serum hCG level varied from normal to moderately elevated. Serum testosterone level was raised in 1 case. Uterine curettage could achieve an accurate pathologic diagnosis in 60% of cases. ETT involved mainly the lower uterine segment and endocervix. Histologically, PSTT cells permeated between the myometrial fibers and vessels either individually or connecting in cords or sheets in a manner reminiscent of the implantation site reaction. ETT composed of a relatively uniform population of mononuclear trophoblastic cells, clumping together in nests as the cell islets associating with eosinophilic, fibrillary and hyaline material and necrotic debris, forming a "geographic map" like pattern. Immunohistochemical study for hPL, hCG, Mel-CAM (CD146) and PLAP was most helpful for the differential diagnosis. The duration of follow-up varied from 14 months to 19 years. One case of PSTT developed metastasis in pancreas, 5 months after the operation. The remaining patients survived without tumor recurrence. PSTT is a tumor of implantation site intermediate trophoblasts while ETT differentiates towards chorionic-type intermediate trophoblasts. The different pathologic features and immunophenotype observed were closely related with the difference in tumor cell differentiation. An accurate pathologic diagnosis of the uterine curettage material is important for the clinical management. According to the limited follow-up data available, the clinical behavior of ETT is seemed similar to that of PSTT.

Cell reproduction and genome multiplication in the proliferative and invasive trophoblast cell populations of mammalian placenta

Spatiotemporal "time-table" of ways of cell reproduction (mitosis, restitutional mitosis, endomitosis, endoreduplication) of trophoblast cell populations is described. The populations of mitotically active trophoblast cells (diploid and low-polyploid) are located mostly out of contact with maternal tissues. In rodent placenta they mainly switch from mitotic cycle to polyploidizing (restitutional) mitoses and reach 4c-8c. Thereafter they switch to endoreduplication and reach 16c-64c. Following a series of endoreduplication cycles a part of this cell population sets apart and penetrates deeply into the decidualized endometrium and myometrium, their capabilities for replication being lost progressively (in rodent--256c-1024c). The invasive trophoblast cells that reach 256c-1024c via endoreduplication simultaneously form a barrier between semiallogenic fetal and maternal tissues. Arrest of mitoses and complete repression of DNA replication after a series of endoreduplication cycles makes hardly probable the renewal of mitotic activity in the deeply invading tertiary giant trophoblast cells, thereby preventing the possibility of their ectopic expanding in the maternal tissues during the normal pregnancy.

71 WHAT IS THE UTERINE RESPONSE IN A CLONED BOVINE PREGNANCY?

Bovine has a synepitheliochorial placenta and characteristically there is no invasion of the trophoblast, but there is migration of the binucleate trophoblast giant cells into the maternal endometrium. The feto-maternal interface occurs in placentome where a tridimensional organization permits interactions between maternal epithelium and trophoblast, and in the intercaruncular area it is possible to observe a few mini-placentomes and the uterine glands opening. The objective of the present investigation was to study the morphological aspects of the uterus in bovine that had a cloned cattle gestations to understand the differences with natural gestation. The uterus and fetal membranes from natural and cloned cattle gestations were collected, fixed in 10% formaldehyde, processed, and stained for light microscopy and immunohistochemistry. The morphological differences observed in the surrogate uterus were: extensive areas without placentome, hemorrhagic uterine areas, caruncular fusion giving a reduced number of caruncules, increase in size and weight (megacaruncules), and a significant number of mini-caruncules giving miniplacentomes (diameter &lt; 1 cm). In particular the mini-placentome showed functional trophoblastic cells with PAS+ granules in the binucleate trophoblast giant cells and an intense subepithelial capillary organization in maternal and fetal sides. The normal and clone placentomal cell populations were analyzed throughout pregnancy. The population of tetraploid and diploid trophoblastic cells was stained; detached cell cycle and DNA content was measured in FL2 using a FACscalibur flow cytometric system. We determined the percentage of cells in apoptosis (sub-G1), quiescent cells (G0/G1), synthesis (S), and proliferative cells (G2/M) with the aid of ModFit software. In addition, a cell cycle differential analysis was performed, and the tetraploid population presented statistical differences in cell cycle phases and populations relative to the apoptosis rate for the first (7.5 � 3.1%), second (15.2 � 5.0%) and third (17.3 � 4.3%) trimesters. The number of apoptotic cells increased significantly during pregnancy stages. The results showed that first trimester presented the majority of its cells in the G0-G1 phase, starting the cell cycle. On the other hand, the second and third trimesters presented the majority of their cells in the G2-M phase, ending the cell cycle. The relationship between cell cycle phases/rate of apoptosis in mononucleate cells, days of normal and cloned pregnancy, the number of binucleate cells, and their metabolic activity as well as their developmental kinetics could be important data in several studies that involve placental development in natural pregnancy or that derived from laboratory-manipulated embryos. This work was supported by FAPESP and CNPq.

Connexin31-deficient trophoblast stem cells: a model to analyze the role of gap junction communication in mouse placental development

The connexin (Cx) expression during placental development in rodents is subject to exacting spatiotemporal regulation. Following implantation, Cx31 characterizes the early trophoblast cell lineage and is expressed by the spongiotrophoblast during placental development until birth. Inactivation of the Cx31 gene results in a transient placental dysmorphogenesis with an imbalance in the trophoblast cell lineage differentiation in favor to giant cells [Dev. Biol. 231 (2001) 334]. In this study, we show that trophoblast stem (TS) cells exhibit the same connexin expression found in trophoblast cell lineage differentiation. Undifferentiated TS cells exclusively express Cx31 protein and Cx31.1 transcripts. Upon differentiation of TS cells, placental-specific Cx26 and Cx43 are induced. Cx31 knockout TS cells revealed an accelerated differentiation process to giant cells compared to controls, indicated by an overall shift in expression of connexins and marker genes such as Mash2, Pl-1, and Tpbpa. Moreover, proliferation was significantly reduced in Cx31 knockout TS cells upon differentiation. Both wild type and Cx31 knockout TS cells are able to invade and erode host vessels when injected into nude mice. We conclude that during trophoblast cell lineage differentiation, the Cx31 gap junction channel is involved in maintaining the proliferative diploid trophoblast cell population.

Galectin-1 and galectin-3 in the trophoblast of the gestational trophoblastic disease

While the presence and distribution of galectin-1 and galectin-3 in different normal trophoblast cell populations is known, no information is available regarding their occurrence in malignant trophoblast of gestational trophoblastic disease (GTD). Galectins-1 and -3 have, however, been implicated in malignancies of other tissues. Immunoreactivity for these galectins in the transformed trophoblast of invasive mole ( n=8), choriocarcinoma ( n=7) and one case of placental site trophoblastic tumor (PSTT) was compared to that of the invasive trophoblast of the normal first trimester of pregnancy implantation sites ( n=9). A large proportion of the transformed trophoblast cells of all GTD studied were positive for galectin-1 and galectin-3. Immunoreactivity was scored semiquantitatively to include both the prevalence among the trophoblast cells and the intensity of staining. Immunoreactivity for both galectin-1 and galectin-3 in gestational trophoblastic disease is increased (significant differences at p<0.05, Mann–Whitney Rank Sum Test). This finding may suggest a possible implication of galectins-1 and -3 in the invasiveness of the transformed trophoblastic cell, although the exact physiological significance of this finding remains to be determined.

Expression pattern of the CCAAT/enhancer-binding proteins C/EBP-alpha, C/EBP-beta and C/EBP-delta in the human placenta.

The human trophoblast has the capacity to invade maternal tissue in a controlled fashion and to produce a wide range of hormones. The transcription factors belonging to the CCAAT/enhancer-binding protein (C/EBP) family are regulators of intracellular processes and mediators of hormone action. C/EBP binding sites have been described in the promoters of several placenta-expressed target genes. In the present study, we used immunohistochemistry and Western-blot analysis to investigate the expression pattern of the three most important members of this family, C/EBP-alpha, -beta, and -delta, in the normal human placenta as well as in isolated trophoblast cell populations. We found C/EBP-alpha and C/EBP-beta expression in the villous syncytiotrophophoblast (ST) and the extravillous (intermediate) trophoblast (EVT), but it was absent from the villous cytotrophoblast (CT). Interestingly, expression of C/EBP-beta continued to be very strong up to the third trimester of pregnancy, especially in the ST. C/EBP-delta showed overall lower expression levels, stronger only in the EVT, while CT/ST showed very low/negative expression. These data show for the first time the expression pattern and tissue localization of C/EBP factors in the human placenta, indicating that these factors (especially C/EBP-beta) may play important roles in the regulation of placenta-specific genes and processes.

Elevated concentration of TNF-alpha induces trophoblast differentiation in mouse blastocyst outgrowths.

Elevated expression of tumour necrosis factor-alpha (TNF-alpha) is associated with adverse pregnancy outcome. This study has examined the expression of TNF-alpha and its receptors (TNF-Rs) by mouse blastocysts and blastocyst outgrowths from day 4 to 9.5 of pregnancy and investigated the effects of elevated TNF-alpha on the inner cell mass (ICM) and trophoblast cells of blastocyst outgrowths. RT-PCR demonstrated TNF-alpha mRNA expression from day 7.5 to 9.5, TNF-R1 from day 6.5 to 9.5 and TNF-R2 from day 5.5 to 7.5 of pregnancy, and in situ hybridisation revealed the trophoblast giant cells (TGCs) of the early placenta as the site of TNF-alpha expression. Day 4 blastocysts were cultured in a physiologically high concentration of TNF-alpha (100 ng/ml) for 72 h to the outgrowth stage and then compared to blastocysts cultured in media alone. TNF-alpha-treated blastocyst outgrowths exhibited a significant reduction in ICM cells (mean +/- SD 23.90+/-10.42 vs 9.37+/-7.45, t-test, P<0.0001) with no significant change in the numbers of trophoblast cells (19.97+/-8.14 vs 21.73+/-7.79, t-test, P=0.39). Within the trophoblast cell population, the TNF-alpha-treated outgrowths exhibited a significant increase in multinucleated cells (14.10+/-5.53 vs 6.37+/-5.80, t-test, P<0.0001) and a corresponding significant decrease in mononucleated cells (5.87+/-3.60 vs 15.37+/-5.87, t-test, P<0.0001). In summary, this study describes the expression of TNF-alpha and its receptors during the peri-implantation period in the mouse. It also reports that elevated TNF-alpha restricts ICM proliferation in the blastocyst and changes the ratio of mononucleated to multinucleated trophoblast cells. These findings suggest a mechanism by which increased expression of TNF-alpha during trophoblast differentiation may be detrimental to pregnancy.

ATP-evoked increase in intracellular calcium via the P2Y receptor in proliferating bovine trophoblast cells.

Bovine trophoblasts actively proliferate to elongate blastocysts before implantation. The trophoblast at this stage secretes cytokines and starts to differentiate into an endocrine cell (binucleate cell) for successful pregnancy. Intracellular calcium ([Ca2+]i) may act as a second messenger in the trophoblast response. In this study, we investigated [Ca2+]i signals in a bovine trophoblast cell line (BT-1) using fura-2 fluorescence. We found that an application of ATP (> or =1 microM) induced a transient increase in [Ca2+]i in BT-1 cells. The ATP-induced increase was not affected by the removal of extracellular Ca2+, but was suppressed by suramin (100 microM), an antagonist of P2 receptors. Pretreatment with pertussis toxin (0.1 or 1 microg/ml) partially inhibited the response to ATP. The order of potency to increase [Ca2+]i was ATP=UTP>ADP. ATP-induced [Ca2+]i responses preferentially occurred in cells at the periphery of the colony. The reduced responses at the center of the colony were associated with an increase in cell density and decrease in bromodeoxyuridine incorporation. These results indicated that ATP stimulated P2Y receptors coupled to pertussis toxin-sensitive and -insensitive G proteins, leading to an increase in [Ca2+]i as a result of release of Ca2+ from intracellular stores in BT-1 cells. The occurrence of ATP-induced [Ca2+]i signals depended on the cell confluence and reflected the high proliferative activity of the trophoblast cell population.

HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels.

During pregnancy, the human extra-villous trophoblast in the contact zone between maternal and fetal tissue in the placenta does not express the classical MHC class I and II molecules. Instead, HLA-G and -C, and possibly HLA-E, are expressed. HLA-G may modulate the immunological relationship between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed at a significantly lower level than the corresponding HLA-G mRNA isoforms with the 14-bp sequence deleted. Furthermore, characteristic HLA-G mRNA isoform expression patterns were associated with specific HLA-G genotypes and alleles. In the HLA-G*01012 and - G*01013 alleles that include the 14-bp sequence, an additional alternative splicing was observed, with the first 92-bp of exon 8 spliced out. This was most pronounced in HLA-G genotypes with G*01013. These findings may have functional implications for the recent reports of aberrant HLA-G expression and reproductive success.