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PubMed1978

Saccharide distribution in human trophoblast demonstrated using fluorescein-labelled lectins.

The reaction of six Flourescein-conjugated lectins with human trophoblast and foetal limb are described. The trophoblast had marked membrane fluorescence after treatment with wheatgerm and Lotus tetragonolobus lectins. Concanavalin A reacted strongly with the trophoblastic cytoplasm but did not react with the nuclei. Most of the lectins had only weak reactions with foetal tissue, apart from the cartilage matrix which fluoresced strongly after treatment with several of the lectins. In addition, concanavalin A reacted with amylase-labile material, probably glycogen, in the skin epidermis and chondrocyte cytoplasm. Various substances were tested in order to determine the nature of the wheatgerm lectin receptor of trophoblast. Several secretory and binding proteins were found not to be responsible, and the identity of the receptor remains unknown.

Carbohydrate Metabolism↗
PubMed1984

Further studies on immunological parameters in gestational trophoblastic neoplasia.

The total blood lymphocyte counts, T-cell counts, B-cell counts, percentage of T cells bearing Fc receptors for IgG (TG), lymphocyte responses to phytohaemagglutinin and concanavalin A and serum IgA levels were measured in 15 patients with hydatidiform mole, 32 patients with malignant trophoblastic disease, 30 pregnant controls, and 34 normal non-pregnant controls. The mean percentage of TG was similar in all four groups but the mean absolute number of TG in patients with malignant trophoblastic disease was significantly lower than that in normal non-pregnant controls, due to the lower mean lymphocyte count and T-cell count in the former group. An immune profile score based on the T-cell count, B-cell count, lymphocyte response to mitogens, and serum IgA level was useful in predicting the subsequent development of malignant trophoblastic disease in patients with hydatidiform mole but was not helpful in predicting resistance to chemotherapy in malignant trophoblastic disease.

Adolescent↗
PubMed2001

Transforming growth factor beta may act as an autocrine-survival-promoting factor for transformed trophoblasts.

Using five trophoblast cell lines of different differentiation status, we have shown that trophoblasts could constitutively release the transforming growth factor beta-1 (TGFbeta1), but not TGFbeta2. Treatment of the human tumorigenic, TL, and BeWo cells with a differentiating agent and a potent protein kinase C activator--the tumor-promoting agent--or the JEG-3 cells with cholera toxin--a potent cyclic adenosine 3':5'monophosphate (cAMP) inducer--or its analogue 8-bromo-cAMP, potentiates TGFbeta production, but the two signaling pathways appear to be mutually exclusive. Surprisingly, the JAR tell line failed to respond to either type of TGFbeta activator. Based on reverse transcriptase (RT)-polymerase chain reaction (PCR), it was found that only the JAR cell line expressed messenger ribonucleic acid for decorin, a natural inhibitor of TGFbeta, and none of the cell lines had detectable protein expression as determined by immunocytochemical studies. The tell number in cultures with decorin was invariably lesser than in those without decorin under serum-free conditions for all the cell lines tested. These results suggest that TGFbeta may act as an autocrine-survival factor for transformed trophoblasts by allowing the cells to survive longer under a microenvironment which is not favorable for growth. Lastly, our results indicate that decorin, acting in a paracrine manner, may also play an important negative regulatory role in the development of transformed trophoblasts by sequestering TGFbeta, thereby preventing its action.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1977

Effect of cyclic 3':5'-AMP derivatives prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture.

The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N6, O2'-dibutyryl cyclic 3':5'-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process, malignant trophoblast cultures were incubated with 3':5'-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmunoassayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretion of hCG, the N6--and O2'-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3':5'-GMP (cGMP), dbcGMP, 5'-AMP, adenosine, L-epinephrine and prostaglandins E1, E2, F1a and F2a were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast.

Adenine↗
PubMedDate not supplied

Interaction of mouse ectoplacental cone trophoblast and uterine decidua in vitro.

During the peri-implantation stages of mouse development, the secondary trophoblast invades into the uterine decidua. This uniquely controlled invasive process results in the formation of the placenta. We have analyzed this process in vitro using cultures of decidua and microdissected ectoplacental cones from Day 7 embryos. The results showed that the interaction between these two cell types is comparable to that seen in vivo. Morphologically, the decidua maintained close contact with the spreading trophoblast, limiting its invasion and producing a multilayered trophoblast outgrowth. Attachment to the decidua was not mediated through cell-matrix binding, but the subsequent invasion into the decidua was dependent on normal matrix interactions. Secretion of proteinases by the trophoblast also seemed to be a requirement for successful invasion, but not attachment.

Animals↗
PubMed2003

Effect of high concentrations of glucose on differentiation of rat trophoblast cells in vitro.

AIMS/HYPOTHESIS: Previous studies have shown that diabetic placentas are characterized by structural and biochemical anomalies, including defects in the differentiation of trophoblasts. In this study, the Rcho-1 cell line was used to investigate the impact of high glucose concentrations on different markers of differentiation of rat trophoblast cells in giant cells (endoreduplication, invasive phenotype and endocrine phenotype). MATERIALS: Rcho-1 cells were incubated for 12 days in medium supplemented with different concentrations of glucose and 10% horse serum to optimize differentiation. The cells were examined for the proportion of nuclei showing signs of apoptosis. The effect of high glucose was investigated on the endoreduplication process, on invasive phenotype (secretion of gelatinase B) and on endocrine phenotype (expression of placental lactogen I (PL-I) and II (PL-II) and progesterone secretion). RESULTS: Apoptosis was not induced by high glucose in Rcho-1. The number of cells was higher in the cultures exposed to high glucose (p<0.05) and their nuclei contained more DNA compared with control cells (p<0.001), while their nuclear size was smaller (p<0.001). Gelatinase B secretion increased during differentiation but no difference was found when gelatinase B secretion from trophoblasts exposed to high glucose was compared with the control cells. Rcho-1 cell cultures showed an increase in PL-I and PL-II mRNA expressions during differentiation and which was not affected by high glucose. Progesterone secretion increased during differentiation in control cultures. However, this increase was abolished when trophoblasts were cultured in high glucose. CONCLUSIONS/INTERPRETATION: Our data suggest that high glucose influences the endoreduplication process and the steroidogenesis during differentiation of rattrophoblasts.

Animals↗
PubMed2001

Decreased expression of fibroblast growth factor-4 and associated dysregulation of trophoblast differentiation in mouse blastocysts exposed to high D-glucose in vitro.

AIMS/HYPOTHESIS: Paracrine interactions are thought to operate between the inner cell mass and trophectoderm cell lineages to co-ordinate their expansion and differentiation during embryo implantation. We aimed to determine whether hyperglycaemia could interfere with this regulatory process. METHODS: Mouse blastocysts were pre-exposed to either control or high concentrations of d-glucose for 24 h in vitro and tested for their ability to attach and spread over a fibronectin-coated culture substrate. Quantitative immunocytochemistry was done on blastocysts to assess the protein expression of Fibroblast Growth Factor-4, a growth factor preferentially produced by the inner cell mass and thought to restrict trophectoderm differentiation into giant trophoblasts. Experiments were then done combining the pre-exposure to high D-glucose with the addition of recombinant fibroblast growth factor-4. RESULTS: Compared with control blastocysts, high D-glucose pre-treated embryos were found to form 18 % larger trophoblast outgrowths. These blastocysts also showed a 30 % reduction in the expression of fibroblast growth factor-4 protein by inner cell mass cells as they were outgrowing. The trophoblast surface area per outgrowth and the trophoblast nuclear area were corrected when the addition of recombinant fibroblast growth factor-4 was combined with the pre-exposure to high D-glucose. CONCLUSION/INTERPRETATION: The data suggests that trophectoderm differentiation is impaired by high D-glucose and that this effect is secondary to a deficiency in fibroblast growth factor-4 protein in the inner cell mass. These observations add a novel perspective to the study of the peri-implantation embryopathy associated with maternal diabetes

Animals↗
PubMed2005

The relation of increased uterine artery blood flow resistance and impaired trophoblast invasion in pre-eclamptic pregnancies.

OBJECTIVE: To assess the association between histopathologically confirmed vascular abnormalities developed during pre-eclampsia and abnormal arterial blood flows recorded during Doppler sonographies. MATERIALS AND METHODS: From pregnant women who attended our clinic between 01/03/2002 and 01/07/2002, a detailed medical history was obtained and routine biochemical blood tests, fetal ultrasonography and UA Doppler scans were performed. In addition, from pre-eclamptic and normal pregnant women who underwent cesarean sections, placental bed biopsies were taken. Thirty two pre-eclamptic [12 mild, 20 severe cases according to American College of Obstetricians and Gynecologists (ACOG) criteria] cases and as a control group 20 normal pregnancies were included in the study. In our study trophoblast invasion into decidual spiral arteries was observed in 75% of mild (9/12), and 55% of severe (11/20) pre-eclampsias. In the control group all the cases demonstrated trophoblast invasion in decidual spiral arteries. Trophoblast invasion in myometrial spiral arteries was noted in 50% (6/12) of mild and 25% (5/20) of severe pre-eclamptic pregnancies. It was seen in 16 cases out of 20 (80%) pregnancies. In the control group, decidual spiral artery invasion manifests significant differences (P<0.01) among groups studied. Invasion in decidual spiral arteries was seen in all normal pregnancies of the control group. There is not any significant difference between mild and severely pre-eclamptic groups (P>0.05). CONCLUSION: Doppler ultrasonography is not only a non-invasive method for evaluating fetal status in pre-eclamptic pregnancies, but it also correlates with partial trophoblastic invasion in spiral arteries, which contributes to the pathophysiologic mechanisms involved in pre-eclampsia.

Adult↗
PubMed1999

Aberrant positioning of trophoblast and lymphocytes in the feto-maternal interface with pre-eclampsia.

Pregnancy represents the growth of an allograft where fetal trophoblast cells evade immune rejection and invade maternal tissue. There should be a balance between fetal trophoblast and maternal immune-responsive cells and alterations in the proportion of these cells may relate to pregnancy disorders. To test this, the decidual tissue of placental bed biopsies was examined and trophoblast cells and lymphocytes were quantified morphometrically; spiral arteries were classified as unchanged, transformed or affected by acute atherosis. Normal pregnancy (n=19) was characterized by the transformation of about one half of all spiral arteries within the placental bed. We found that 40% of all lymphocytes were CD56+ uterine NK cells and 60%, CD3+ T-lymphocytes; about 30% of these were CD8+ T cells. Intrauterine growth retardation in the context of preeclampsia (n=15) was accompanied by reduced trophoblast numbers within smaller and more tortuous arteries and an increase in the proportion of CD56+ uterine NK cells and CD8+ T lymphocytes in the decidua (70% of all CD3+ cells). In the case of pre-eclampsia without fetal growth retardation (n=14) no increase in CD56+ uterine NK cells was seen, while CD8+ T lymphocytes were significantly increased compared with the normal level (50% of all CD3+ cells). Fetal growth retardation is associated with poor transformation of spiral arteries and characterized by an increase of uterine NK cells. Symptoms of pre-eclampsia are independently associated with an increase in the cytotoxic T subset of decidual lymphocytes. Pre-eclampsia and related fetal growth retardation are seemingly caused by an enhancement of the maternal cytotoxic defence against the fetal allograft.

Arteries↗
PubMed1999

Enrichment of fetal trophoblasts and nucleated erythrocytes from maternal blood by an immunomagnetic colloid system.

The aim of this study was to isolate fetal trophoblasts and nucleated erythrocytes from maternal blood using the immunomagnetic colloid system. About 25 ml of maternal blood was collected from pregnant women between of 14 and 20 weeks gestation. Nucleated erythrocytes (NRBCs) were isolated from 5 ml of maternal blood and a nested polymerase chain reaction for the Y chromosome was used to determine fetal origin. The sensitivity of the fetal gender diagnosis was 80% and the specificity was 86%. Both fetal trophoblasts and NRBCs were isolated from the remaining 20 ml of maternal blood. The fetal gender of the trophoblast-enriched fraction was determined using fluorescence in situ hybridisation (FISH) with dual-colour XY-specific DNA probes. XY-specific signals were observed in 0.38% of cells sorted from all pregnant women carrying male fetuses (n = 10). Simultaneous immunophenotyping for the fetal haemoglobin and FISH using XY probes were used to evaluate the fetal origin of cells enriched with anti-CD71. The mean percentage of male fetal erythroblasts was 0.24% and the number of fetal erythroblasts was estimated to be about 672 in 20 ml of maternal blood. The number of fetal erythroblasts detected in our study was greater than that detected by most other separation techniques. Our study shows that it would be feasible to use the immunomagnetic colloid system for the isolation of both trophoblasts and NRBCs from the same maternal blood sample with relatively good efficiency.

Amniocentesis↗
PubMed1996

Expression of HLA class I molecules in human first trimester and term placenta trophoblast.

Expression of HLA class I molecules in trophoblast cells from various locations in normal human first trimester and term placenta was investigated by immunohistochemistry with a panel of monoclonal antibodies against the heavy chains or complete HLA class I molecules complexed with beta2-microglobulin. These reagents were also employed to distinguish between the products of different HLA class I loci. In addition to previously characterized reagents, a novel monoclonal antibody against HLA-A molecules (TU155) was used. Various choriocarcinoma and transfected cell lines served as controls for the specificities of the monoclonal antibodies. Cells in close contact with maternal cells, such as invading trophoblast cells and cells of the basal plate, expressed beta2-m micro globulin in association with HLA-G and HLA-C heavy chains. These class I heavy chains may also have been present as isolated molecules, although not in each of the cells. In contrast, cells of the chorion laeve exclusively expressed HLA-G, and not HLA-A, -B, or -C antigens. Our data support the often discussed immune protective function and the regulatory function of the HLA-G molecule, during invasion. In addition, by using monoclonal antibodies HCA2 (anti-HLA-A and -G), HC10 (anti-HLA-B and -C), TU149 (anti-HLA-B, -C, and some -A alleles), SFR8-B6 (anti-HLA-Bw6 and some -C), LA45 (some HLA-A and -B), TU48 (anti-HLA-Bw4 and some -A), and TU155 (anti-HLA-A), we show the presence of HLA-C molecules in all extravillous trophoblast cells of the cell columns and in the basal plate; the trophoblast cells of the chorion laeve lack this antigen. The function of this molecule is not clear, although a protective function against natural killer cell activity in the endometrium is postulated.

Blotting, Western↗
PubMed1998

Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta.

The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and to adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of the latter enzymes belong to the family of matrix metalloproteinases. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases. We have studied the distribution patterns of matrix metalloproteinases-1, -2, -3, and -9 and their inhibitors TIMP-1 and TIMP-2 as compared to the distribution of their substrates along the invasive pathway of extravillous trophoblast of 1st, 2nd, and 3rd trimester placentas by means of light microscopy on paraffin and cryostat sections as well as at the ultrastructural level (only 3rd trimester placenta). The comparison of different methods proved to be necessary, since the immunohistochemical distribution patterns of these soluble enzymes are considerably influenced by the pretreatment of tissues. All three methods revealed immunoreactivities of both, proteinases and their inhibitors, not only intracellularly in the extravillous trophoblast but also extracellularly in its surrounding matrix, the distribution patterns depending on the stage of pregnancy and on the degree of differentiation of trophoblast cells along their invasive pathway. Within the extracellular matrix, immunolocalization of matrix metalloproteinases as well as their inhibitors showed a specific relation to certain extracellular matrix molecules.

Collagen↗
PubMed2000

X-chromosome inactivation in the human trophoblast of early pregnancy.

To investigate X-chromosome inactivation (XCI) in human trophoblasts during early pregnancy, trophoblast genomic DNA was extracted and analyzed for a Bst XI restriction endonuclease site polymorphism in the X-linked phosphoglycerate kinase gene, after digestion with methylation-sensitive Hpa II (control samples were digested instead with Afa I). Six villous trophoblast DNA samples were informative for the polymorphism (ie, heterozygous) and were derived from women homozygous for the polymorphism. These samples were then evaluated for XCI. In five of the six samples with Hpa II predigestion, the sizes of the two heterozygous band peaks differed; maternal X-chromosome (X(M))-derived alleles showed smaller peak sizes than paternal X-chromosome (X(P))-derived alleles, but the differences varied in degree. In samples obtained by microdissection from formalin-fixed, paraffin-embedded tissues (30 samples from different anchoring villi, and 38 samples from different branch villi), monoclonal band patterns of X(P)-derived alleles were observed more frequently than those of X(M)-derived alleles, but almost half of the samples showed polyclonal patterns. Our results suggest that a skewing of XCI exists in the human trophoblast; however, the degree of nonrandomness due to predominant X(P) inactivation appears to be restricted. It is probable that transcription of the X inactivation center (XIC) begins earlier in mice than in humans.

Clone Cells↗
PubMed1980

Utility of assay of alpha subunit of human chorionic gonadotropin in management of gestational, trophoblastic malignancies.

All patients who were treated with chemotherapy for malignant gestational trophoblastic neoplasms at the Southeastern Regional Trophoblastic Disease Center and who experienced remission during the period January 1, 1978, through December 31, 1979, were studied in an attempt to determine if the application of an assay for the alpha subunit of human chorionic gonadotropin (hCG-alpha) could predict those patients who would have recurrences. For each patient, the first three weekly serum samples with undetectable HCG by the hCG beta-subunit radioimmunoassay as well as the two preceding samples with detectable hCG were assayed in a homologous hCG-alpha assay. Results reaffirm previous data that the latter assay is useful only in patients receiving oral contraceptives for suppression of pituitary gonadotropin secretion. No patient who has remained in remission had a mean level of hCG-alpha in the three hCG-negative samples greater than 1 ng/ml. Of those patients who had recurrences of trophoblastic disease, two of three had hCG-alpha levels greater than 1.5 ng/ml. Although these data are preliminary, they suggest that the routine measurement of hCG-alpha in patients successfully treated for trophoblastic disease may aid in identification of a group of patients who require additional or more intensive follow-up and/or additional chemotherapy to prevent later recurrences.

Adolescent↗
PubMed1982

Treatment of nonmetastatic gestational trophoblastic disease: results of methotrexate alone versus methotrexate--folinic acid.

Two treatment regimens for nonmetastatic gestational trophoblastic disease are compared in this retrospective study. The course of 39 patients with nonmetastatic gestational trophoblastic disease treated with methotrexate alone is contrasted to that of 29 patients with nonmetastatic gestational trophoblastic disease who were treated with methotrexate alternated with folinic acid. Of those patients initially treated with methotrexate alone, 7.7% developed methotrexate-resistant disease and required a change in chemotherapy for induction of remission. In contrast, 27.5% of patients initially treated with methotrexate and folinic acid developed methotrexate-resistant disease and required a change in chemotherapy to achieve remission. Ultimately, remission was achieved in all patients. Methotrexate as single-agent chemotherapy was found to be consistently more toxic than methotrexate alternated with folinic acid. It is concluded that methotrexate with folinic acid at the dosage used in this study, while less toxic than methotrexate alone, is less effective than methotrexate alone in the induction of remission of nonmetastatic gestational trophoblastic disease.

Choriocarcinoma↗
PubMed1987

Immunohistochemical localization of epidermal growth factor receptor and myc oncogene product in human placenta: implication for trophoblast proliferation and differentiation.

Cytologic localization of epidermal growth factor receptor and myc oncogene protein product in developing human placenta was analyzed by avidin/biotin immunoperoxidase techniques with a monoclonal antibody to epidermal growth factor receptor and an affinity purified polyclonal antibody to myc protein product. Epidermal growth factor receptor was found to be almost exclusively localized to syncytiotrophoblast, paralleling the immunohistochemical localization of human chorionic gonadotropin. Since epidermal growth factor has been shown to stimulate human chorionic gonadotropin and human placental lactogen production by cultured early placental tissue, the present finding that epidermal growth factor receptor was localized to mitotically inactive syncytiotrophoblast suggests a role for epidermal growth factor receptor in the induction of differentiated function of trophoblast rather than trophoblast proliferation. By contrast, myc protein product was found to be predominantly localized to cytotrophoblastic cells, paralleling the autoradiographic distribution of replicating cytotrophoblast identified by tritiated thymidine labeling of placental explant. A close similarity between the cytologic localization of myc protein product and tritiated thymidine labeling of placental explant suggests that myc protein expression is linked to trophoblast proliferation. Furthermore, immunohistochemical cellular levels of both epidermal growth factor receptor and myc protein product were most pronounced in early placenta and declined in term placenta. Thus myc protein product and epidermal growth factor receptor seem to play a crucial role in the induction of trophoblast proliferation and differentiation, respectively, during the development of human placenta.

ErbB Receptors↗
PubMed1991

Forskolin and methotrexate induce an intermediate trophoblast phenotype in cultured human choriocarcinoma cells.

The human placenta and its associated membranes are vital to the maintenance, nutrition, and protection of the developing fetus. During placental development some cytotrophoblasts give rise to the chorionic membrane whereas others fuse to form a differentiated syncytium of cells that are responsible for placental protein and steroid hormone production. The mechanisms involved in the differentiation of the trophoblasts are unknown; however, an intermediate stage with a characteristic phenotype has been documented in vivo. We have observed that chemically dissimilar xenobiotic agents induced BeWo choriocarcinoma cells to change their usual cytotrophoblastic phenotype and acquire morphologic and functional characteristics typical of intermediate trophoblast. Incubation of BeWo cell cultures in the presence of 1 mumol/L methotrexate for 48 hours stimulated human chorionic gonadotropin secretion (2.3-fold) and aromatase activity (4.9-fold). Morphologic findings associated with these hormonal changes, including increased nuclear size and cytoplasmic expansion, were also observed. With the use of a computer-interfaced image analyzer, planimetric morphometry of the nuclear area of the cells revealed a 1.8-fold increase after incubation with methotrexate. The effect of forskolin, a direct activator of adenylyl cyclase, was also evaluated by means of this model of cytotrophoblast differentiation. The addition of 10 mumol/L forskolin to BeWo cultures also resulted in dramatic changes in trophoblast cell phenotype. Increases in human chorionic gonadotropin synthesis (7.3-fold), aromatase activity (13.5-fold), and nuclear area (3.0-fold) were induced over those of untreated cells. In addition, increases in [3H]thymidine incorporation (1.7-fold) were afforded by both treatments. These results suggest that biochemical and cytologic changes associated with human trophoblast differentiation can be induced in vitro via activation of the adenylyl cyclase pathway by forskolin and through unknown and apparently independent signals by methotrexate.

Cell Differentiation↗
PubMed1995

Insulin and glucose modulate glucose transporter messenger ribonucleic acid expression and glucose uptake in trophoblasts isolated from first-trimester chorionic villi.

OBJECTIVE: Our purpose was to determine the effects of insulin and glucose on glucose transport and expression of GLUT1 glucose transporter messenger ribonucleic acid in first-trimester human trophoblast-like cells. STUDY DESIGN: First-trimester human trophoblast-like cells were maintained as a continuous cell line. For 2[3H]deoxy-D-glucose uptake and messenger ribonucleic acid studies the cells were incubated in the presence or absence of insulin (10(-7) to 10(-11) mol/L) or D-glucose (0 to 50 mmol/L) for 0 to 24 hours. Glucose transport was measured by incubating cells with 0.1 mmol/L 2[3H]deoxy-D-glucose for 5 minutes. Specific uptake was determined by incubating companion cultures with 10 mumol/L cytochalasin B. The cells were then solubilized with sodium hydroxide and the radioactivity counted. Data were expressed as nanomoles of 2[3H]deoxy-D-glucose transported per milligram of protein per 5 minutes and analyzed by one-way analysis of variance with post hoc testing by the method of Tukey. GLUT1 messenger ribonucleic acid was measured by Northern blotting of total ribonucleic acid samples hybridized to a phosphorus 32-labeled complementary deoxyribonucleic encoding the rat GLUT1 glucose transporter. As a control for loading efficiency, blots were stripped and rehybridized to a 40-mer phosphorus 32-labeled beta-actin oligonucleotide probe. RESULTS: Insulin treatment resulted in a dose-dependent increase in the transport of 2[3H]deoxy-D-glucose at 24 hours (p < 0.001 at 10(-7) mol/L). This change was first detected at 12 hours of incubation. These data closely paralleled the insulin-induced increase in GLUT1 messenger ribonucleic acid seen in Northern blots. In contrast to insulin, increasing concentrations of D-glucose did not change the transport of 2[3H]deoxy-D-glucose. However, when cells were incubated in low concentrations of D-glucose (0 or 1 mmol/L), an enhancement in the uptake of 2[3H]deoxy-D-glucose (p < 0.001) was observed. Kinetic studies indicated that D-glucose augmentation of 2[3H]eoxy-D-glucose uptake was significant at 9 hours (p < 0.05). The effects of D-glucose on GLUT1 messenger ribonucleic acid expression paralleled the uptake of 2[3H]deoxy-D-glucose, although the modulation of GLUT1 messenger ribonucleic acid levels by glucose was much less pronounced than in insulin-treated cells. CONCLUSION: Although it has been assumed that the placenta has a limited role in influencing glucose transport to the fetus, our in vitro data demonstrate that both insulin and glucose can modulate glucose transport at the cellular level of the placental trophoblast. Thus maternal insulin and glycemic status may influence the expression of GLUT1, the major trophoblast glucose transporter protein, therefore directly affecting first-trimester placental glucose transport. These in vitro data may help explain the association between maternal glucose abnormalities and impaired fetal development during the first trimester when placental GLUT1 messenger ribonucleic acid expression is at its peak.

Analysis of Variance↗