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PubMed2004

The effect of leptin on mouse trophoblast cell invasion.

The hormone leptin is produced by adipose tissue and can function as a signal of nutritional status to the reproductive system. The expression of leptin receptor and, in some species, leptin, in the placenta suggests a role for leptin in placental development, but this role has not been elucidated. Leptin is required at the time of embryo implantation in the leptin-deficient ob/ ob mouse and has been shown to upregulate expression of matrix metalloproteinases (MMPs), enzymes involved in trophoblast invasion, in cultured human trophoblast cells. This led us to the hypothesis that leptin promotes the invasiveness of trophoblast cells crucial to placental development. We found that leptin stimulated mouse trophoblast cell invasion through a matrigel-coated insert on Day 10, but not Day 18 of pregnancy. Optimal stimulation occurred at a concentration of 50 ng/ml leptin, similar to the peak plasma leptin concentration during pregnancy in the mouse. Leptin treatment did not stimulate proliferation of mouse trophoblast cells in primary culture. Leptin stimulation of invasion was prevented by 25 muM GM6001, an inhibitor of MMP activity. Our results suggest that leptin may play a role in the establishment of the placenta during early pregnancy and that this function is dependent on MMP activity. This effect of leptin may represent one mechanism by which body condition affects placental development.

Animals↗
PubMed1990

Protein secretion by the mouse trophoblast during attachment and outgrowth in vitro.

Protein secretion from mouse blastocysts undergoing attachment and trophoblast outgrowth in vitro was assessed. When Day 5 blastocysts were cultured in serum-containing medium, secretion of several 'attachment-associated' proteins (PAS) was initiated within 24 h, coincident with attachment and outgrowth. Those proteins characteristic of the pre-attachment blastocyst disappeared or made-up only a small portion of the secretions once attachment began. The major secreted protein from attached embryos, PA1, is a 35,000-45,000 Mr acidic glycoprotein with multiple isoelectric forms. PA2, a group of basic 40,000 Mr proteins and PA3 a group of 72,000 Mr proteins were also produced during outgrowth. PAS were secreted during outgrowth on fibronectin-coated plastic in serum-free medium, but not by blastocysts held in a non-attachment state during culture in serum-free medium on uncoated plastic. In pre-attachment blastocysts, secreted proteins were produced by trophoblast vesicles, but not by isolated inner cell masses. Both trophoblast vesicles growing out in vitro and surgically isolated trophoblast from spreading blastocysts had secreted protein patterns qualitatively similar to those of intact blastocyst outgrowths. The results indicate that development of trophoblast protein secretion continues through the period of outgrowth and giant cell transformation. These changes are apparently dependent on attachment of the blastocyst to a suitable substrate, but not dependent on any other serum influence.

Animals↗
PubMed1991

Localization of placental lactogen-I in trophoblast giant cells of the mouse placenta.

The purpose of this investigation was to identify the cellular origin of placental lactogen-I (PL-I) expression in the mouse placenta and to cytologically define the transition from PL-I to PL-II expression during gestation. PL-I mRNA expression was assessed by in situ hybridization, and expression of PL-I and PL-II protein was determined by immunocytochemical analysis. PL-I mRNA and protein were localized to trophoblast giant cells. Trophoblast giant cells ceased producing PL-I at midgestation and began expressing PL-II. PL-I immunoreactivity was present in trophoblast giant cells on Days 9 and 10 of gestation but was not detectable in trophoblast giant cells on Day 11 of gestation. Immunoreactive PL-II-producing giant cells were detected first on Day 10 of gestation, continuing on Day 11 of gestation. Expression of PL-I and PL-II signals a significant functional transition in trophoblast giant cells of the developing mouse placenta.

Animals↗
PubMed1991

Assessment of expression of the receptor for colony-stimulating factor-1 (fms) in bovine trophoblast.

Bovine trophoblast was employed to assess the questions of whether the receptor for CSF-1 is expressed by noninvasive trophoblast and whether expression changes with differentiation within placentomes. Bovine placental poly(A) mRNA contained sequences cross-reactive with cDNA probes to c-fms and v-fms. Using a monoclonal antibody to v-fms, immunohistochemistry of postattachment bovine trophoblast showed expression of an fms-like protein between Day 29 and term. Expression occurred in both the intercotyledonary and cotyledonary trophoblast. Reactivity that was fms-like was also demonstrated on preattachment conceptuses flushed at Days 14 and 7 of gestation and on Day 7 embryos derived from in vitro oocyte maturation and fertilization. Unexpectedly, in the second half of pregnancy some cells, including binucleate cells, showed nuclear rather than cytoplasmic reactivity to the antibody. These data indicate expression of an fms-like protein in bovine placenta that does not correlate with properties of trophoblast cell invasiveness or major morphological differentiation. The data do support a universal role for this protein during mammalian placental development.

Animals↗
PubMed1993

Effects of growth factors and growth factor-extracellular matrix interactions on mouse trophoblast outgrowth in vitro.

A variety of cell types at the blastocyst implantation site produce growth factors that could play important role(s) in the implantation process. Recent evidence indicates that decidual cells and/or embryos produce transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and colony-stimulating factor (CSF-1). Furthermore, receptors for EGF, PDGF, and CSF-1 have been detected on embryonic and trophoblastic cells. The purpose of this study was to investigate the effects of these growth factors and possibly growth factor-extracellular matrix interactions on mouse blastocyst attachment and trophoblast outgrowth in vitro. Various dilutions of the growth factors TGF-alpha, EGF, PDGF, FGF, and CSF-1 were added to cultures of 5-day-old hatched blastocysts in fibronectin-coated plastic wells. Blastocysts were scored for attachment, trophoblast outgrowth, and surface area at 24, 48, and 72 h. Each of these growth factors significantly enhanced trophoblast outgrowth, and a cocktail containing all of the growth factors had a significantly stronger effect. PDGF and FGF, but no other growth factors, also enhanced trophoblast outgrowth following pulsatile incubation with the fibronectin matrix coating of the culture wells, indicating that interactions between these growth factors and extracellular matrix elements could influence implantation. This study suggests that various growth factors may play an important role in the implantation process, that synergistic effects may be obtained by combinations of growth factors, and that interactions between certain growth factors and extracellular matrix elements may be significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Ectopic transplantation of equine invasive trophoblast.

A system for transplanting invasive equine trophoblast (i.e., chorionic girdle) to ectopic sites has been developed as a means to study the differentiation of this tissue and to assess maternal immune responses to the conceptus tissue in a site outside the uterus. Chorionic girdle was isolated from Day 33 to 34 conceptuses and surgically placed into the vulvar mucosa or subdermal skin of recipient mares. Biopsy specimens of the graft sites for immunohistochemical staining were taken at weekly or biweekly intervals after grafting. Serum samples were collected from each recipient and tested for antibody to donor major histocompatibility complex (MHC) class I antigens using the lymphocyte microcytotoxicity assay. Transplanted trophoblast cells expressed differentiation markers associated with invading chorionic girdle and endometrial cup cells. The transplanted trophoblast cells were also labeled by an antibody to eCG. Strong cellular and humoral immune responses to the transplanted tissue were mounted by the recipients, similar to those occurring during normal equine pregnancy. Despite these responses, the invasive trophoblast transplants survived for at least 28 days after grafting and downregulated MHC class I antigens, as do the mature endometrial cup cells in equine pregnancy. These findings suggest that invasive equine trophoblast has the capacity to differentiate fully in equine nonuterine tissues, and that it can evade maternal immune responses independent of the physiological state of pregnancy and in sites other than the uterus.

Animals↗
PubMed2001

Influence of maternal smoking on trophoblast apoptosis throughout development: possible involvement of Xiap regulation.

Maternal smoking is associated with severe perinatal complications and significant placental pathologies with underlying ultrastructural changes. In this study, we examined the influence of maternal smoking on trophoblast apoptosis throughout development and correlated those findings with changes in expression of X-linked inhibitor of apoptosis protein (Xiap) as well as Fas and Fas ligand (FasL). Trophoblast apoptosis was determined by DNA fragmentation and TUNEL. Protein expression was assessed by Western blotting and immunohistochemistry. Maternal smoking was associated with increased trophoblast apoptosis in the first trimester but decreased trophoblast apoptosis near term. Placental Xiap levels decreased significantly throughout development in nonsmokers (P < 0.05) but remained elevated in smokers. Fas and FasL levels did not vary significantly throughout development nor between groups. However, procaspase-3 levels were significantly increased in smokers at term. Our results suggest that maternal smoking has different effects at different stages of trophoblast differentiation and that this is regulated in part through modulations in placental Xiap expression.

Apoptosis↗
PubMed2001

Monoclonal anti-annexin V antibody inhibits trophoblast gonadotropin secretion and induces syncytiotrophoblast apoptosis.

The pathogenic role of anti-annexin V antibodies remains unclear. Anti-annexin V antibodies are frequently associated with higher incidences of intrauterine fetal loss, preeclampsia, and arterial and venous thrombosis. The present study investigated the in vitro ability of anti-annexin V antibody to bind human trophoblast cells, to affect trophoblast gonadotropin secretion and invasiveness, and to induce placental apoptosis. Cytotrophoblast cells were dispersed in Ringer bicarbonate buffer containing trypsin and DNase I, filtered, and layered over a Percoll gradient in Hanks balanced salt solution. In the case of monoclonal anti-annexin V antibody, the highest binding was found when the cells displayed the greatest amount of syncytium formation. Anti-annexin V antibody, but not its negative control, induced trophoblast apoptosis and significantly reduced trophoblast gonadotropin secretion. These findings suggest that recognition by anti-annexin V antibody of adhered annexin V on trophoblast cell structures might represent a potential pathogenic mechanism by which these antibodies can cause defective placentation.

Annexin A5↗
PubMed2004

Epithelioid trophoblastic tumor of the broad ligament: a case report and review of the literature.

We report an epithelioid trophoblastic tumor, a recently delineated type of gestational trophoblastic tumor (GTT), discovered in the right broad ligament of a 41-year-old woman. The patient had gestational trophoblastic disease and was treated with methotrexate regimen 15 times 10 years earlier. The yellowish, spongy tumor with tiny hemorrhage spots was located in the right broad ligament, adherent to the right ovary. Microscopically, the tumor was circumscribed, with a pushing border, and the epithelial-differentiated tumor cells grew in cords, nests, and sheets within which were aggregates of hyaline material and necrotic debris. Most tumor cells were mononuclear with distinct cell borders, eosinophilic cytoplasm, and had nuclei with occasional indistinct nucleoli. Scattered multinucleated cells consistent with syncytiotrophoblastic cells were also present. Immunohistochemical staining revealed strong diffuse reactivity for cytokeratins (AE1/AE3, CAM 5.2, CK18), and focal reactivity, mainly in syncytiotrophoblastic cells, for beta-human chorionic gonadotropin, human placental lactogen, and inhibin-alpha. The histologic and immunohistochemical features were characteristic of epithelioid trophoblastic tumor and helped to distinguish the tumor from other trophoblastic tumors and squamous cell carcinoma. Our unusual findings in this case included a high level of serum beta-human chorionic gonadotropin, an unusual tumor location, and a higher Ki-67 proliferative index of 47.2%.

Adult↗
PubMed1995

In vitro chemosensitivity of paclitaxel and other chemotherapeutic agents in malignant gestational trophoblastic neoplasms.

This is the first report on the ATP cell viability assay as a chemosensitivity test system for gestational trophoblastic neoplasms (GTN). We obtained chemosensitivity profiles in two established trophoblastic cell lines and four fresh tumors. Ten drugs were tested in vitro in the two cell lines JAR and JEG-3. The IC50 values of the 10 chemotherapeutic agents tested were very similar for both cell lines. The three most active drugs in these cell lines were VP-16, paclitaxel and vincristine. This is the first report on the activity of paclitaxel in trophoblastic cell lines. We furthermore evaluated this assay for chemosensitivity testing in four fresh malignant GTN tumors: one placental site trophoblastic tumor, one chorocarcinoma and two invasive moles. The placental site trophoblastic tumor specimen revealed to be rather chemoresistant in vitro whereas the other three tumors were chemosensitive. From our cell line data we conclude that the ATP cell viability assay is a practicable assay for chemosensitivity testing of GTN cell lines and gives repeatable results. However, the value of this assay for fresh GTN chemosensitivity testing needs to be defined.

Adenosine Triphosphate↗
PubMed1986

Effects of prophylactic chemotherapy for persistent trophoblastic disease in patients with complete hydatidiform mole.

Seventy-one patients with complete hydatidiform mole were prospectively randomized into two groups: one group (39 patients) was treated with a single course of methotrexate and citrovorum factor rescue as chemoprophylaxis; the other group (32 patients) was not treated. After molar evacuation, four patients from the treated group (10.3%) and ten patients from the untreated group (31.3%) developed persistent trophoblastic disease. The time interval from evacuation of the mole to diagnosis of persistent trophoblastic disease was longer in the treated group than in the untreated group (9.5 +/- 2.4 weeks versus 5.1 +/- 1.6 weeks, P less than .05). Among high-risk patients, there was a lower incidence of persistent trophoblastic disease in the treated group than in the untreated group (14.3 versus 47.4%, P less than .05). Among low-risk patients there was no difference between the groups (5.6 versus 7.7%, P greater than .05). All 14 patients with persistent trophoblastic disease achieved complete remission with therapeutic chemotherapy. More courses of chemotherapy were required until complete remission in the treated group than in the untreated group (2.5 +/- 0.5 versus 1.4 +/- 0.5, P less than .005). These findings suggest that even though chemoprophylaxis reduces the incidence of persistent trophoblastic disease in patients at high risk, it increases tumor resistance and morbidity. Although prophylactic chemotherapy with methotrexate and citrovorum factor rescue may be helpful for high-risk patients who cannot be followed or whose compliance is in question, careful follow-up remains the most important way to identify patients who should receive chemotherapy.

Adult↗
PubMed1999

Dominant negative suppression of major histocompatibility complex genes occurs in trophoblasts.

BACKGROUND: Polymorphic class I and II major histo-: compatibility complex (MHC) genes are not transcribed in trophoblasts although many immune system cells express these genes constitutively. To study the molecular biology of MHC suppression for the purposes of potential transgenic animal development, we examined the effect on MHC expression in B cells by fusing them with trophoblasts. METHODS: Trophoblasts and B cells with separate selection markers were fused with polyethylene glycol. After growth in double selection media, the hybrids were analyzed for HLA-A, -B, -C, -DR, -DP, and -DQ expression by fluorescence-activated cell scanning and class I and II mRNA by Northern blotting. Class II promoter activity in trophoblasts was then analyzed by transfection of a lethal reporter construct and subsequently, the class II transactivator. RESULTS: Class I and II surface antigens and their corresponding mRNA were completely suppressed in the hybrids. The lethal reporter construct demonstrated that class II suppression resulted from lack of activation of the class II promoter. This in turn was caused by lack of functional class II transactivator. CONCLUSIONS: These data indicate that dominant negative trophoblast factors, either directly or indirectly, suppress expression of the MHC genes. If these factors can be cloned, the potential exists for developing transgenic animals that cannot express MHC or peptide antigen to T cell receptors through the MHC system.

Gene Expression↗
PubMed2005

The frequency and distribution of intratubular trophoblast in association with germ cell tumors of the testis.

Although the presence of intratubular trophoblast (ITT) has been reported, its frequency and distribution in association with germ cell tumors (GCT) have not been investigated. Beta human chorionic gonadotropin (hCG) is a sensitive immunohistochemical marker of syncytiotrophoblast. We therefore wished to investigate whether intratubular trophoblastic elements could be identified adjacent to invasive tumors using immunohistochemistry against hCG. Seventy-five GCTs were examined. Immunochemistry was performed for hCG. Both invasive tumor and seminiferous tubules were examined for positive staining. The seminomas showed ITT in five of 29 cases. All these cases had trophoblastic cells as part of the invasive tumor. Only one of 36 cases of nonseminoma and one of nine of the mixed GCTs (11%) showed ITT. Again, all of the positive cases had hCG-positive trophoblastic cells within the invasive tumor. ITT can be identified adjacent to GCTs in a significant number of cases. We suggest that this is a genuine in situ lesion, associated with seminomas with syncytiotrophoblastic cells. Differentiation toward trophoblastic elements in GCTs may occur at an earlier stage of their pathogenesis than has been previously recognized.

Chorionic Gonadotropin↗
PubMed2006

Cyclin E and p16 immunoreactivity in epithelioid trophoblastic tumor--an aid in differential diagnosis.

Epithelioid trophoblastic tumor (ETT) is a relatively uncommon trophoblastic tumor that can be confused with several trophoblastic and nontrophoblastic lesions, notably the placental site nodule and invasive squamous carcinoma of the cervix. In this report, we analyzed the immunoreactivity of two cell cycle-regulated proteins, cyclin E and p16, in ETTs, placental site nodules and cervical squamous carcinomas to determine whether they are useful in their differential diagnosis. Other trophoblastic lesions were also evaluated. Using an H-score based on both percentage of positively stained cells and immunointensity, we found that ETTs demonstrated a much higher cyclin E staining score than placental site nodules (P<0.0001) permitting distinction of ETTs and placental site nodules with a sensitivity of 94.7% at a specificity of 91.7% using a cutoff H-score value of >40. Only two placental site nodules had scores above the cutoff and both showed morphologic features that placed them in an intermediate position between a typical placental site nodule and an ETT, so-called "atypical PSN." p16 immunoreactivity, was not detected in any of the ETTs and placental site nodules, whereas it was strongly and diffusely positive in the vast majority of cervical squamous carcinomas examined (83/87 cases) (P<0.001). Therefore, cyclin E expression is useful in distinguishing an ETT from a placental site nodule and p16 expression is useful in distinguishing an ETT from a cervical squamous carcinoma. The majority of other types of trophoblastic lesions showed diffuse and intense nuclear immunoreactivity for cyclin E whereas none were positive for p16.

Carcinoma, Squamous Cell↗
PubMed1991

Susceptibility of cultured human trophoblasts to infection with human immunodeficiency virus type 1.

Primary cultures of essentially pure human term trophoblasts were studied to determine their ability to support the expression of complete proviral clones of human immunodeficiency virus (HIV) and their permissiveness to this virus. Transient expression of molecular clones derived from two biologically distinct strains, BRU and NDK, resulted in the release of comparable amounts of infectious virions, which were rescued by cocultivation with permissive CEM-SS cells. Trophoblasts were inoculated with three HIV-1 isolates, RF, 3B and NDK, which differ in their cytopathogenicity on T lymphoblastoid cells. Infection of cells by all three strains was demonstrated by the presence of virus-specific proteins in the trophoblasts and the detection of virus gag gene-related DNA sequences by the polymerase chain reaction (PCR), but cells were more susceptible to infection with the RF and NDK strains than with the 3B strain. The virus was readily transmitted to the CEM-SS cells with simultaneous formation of syncytia between the two cell types. Flow cytometry and direct radioimmunoassay revealed no trace of the CD4 receptor on the surface of the cultured trophoblasts and CD4 mRNA could not be detected by Northern blot hybridization, although a minimal amount of CD4-associated mRNA was detected by PCR. Our data suggest that infection of trophoblasts occurs independently of the pathway mediated by CD4.

Base Sequence↗
PubMed1994

Basal and Tat-transactivated expression from the human immunodeficiency virus type 1 long terminal repeat in human placental trophoblast rules out promoter-enhancer activation as the partial block to viral replication.

We have analysed the capacity of the trophoblast-derived malignant cell lines BeWo, JAR and JEG-3, and primary cultures of highly purified trophoblast cells to support the basal and Tat-mediated trans-activation-enhanced transcriptional activity of two distinct human immunodeficiency virus type 1 (HIV-1) isolates. Kinetic studies based on expression of long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) constructs revealed that LTRs of both the prototype strain 3B and the highly cytopathic Zairean variant NDK were activated significantly in all target cells. Overall, the strongest activation was observed in primary trophoblasts. A novel modification of quantitative PCR was used to normalize LTR expression for transfection efficiency, enabling the calculation of specific expression rates in terms of muU CAT enzyme per fmol of transfected DNA. Using the latter criterion we determined that LTRs of both viruses were activated in decreasing order from trophoblasts to JAR, JEG-3 and BeWo cells; furthermore, the expression of HIV-1 3B LTR always significantly surpassed that of HIV-1 NDK. The effects of trans-activation on either of the LTRs, when assayed in cotransfection assays with various amounts of HIV-1 NDK-Tat expression vector, increased in a dose-dependent fashion and were comparable in a particular neoplastic cell line. Furthermore, the cell-specific LTR activity patterns did not correspond to the abundance of transcription factors binding specifically to the viral NF kappa B and SP1 motifs. Unlike SP1-binding proteins which were relatively abundant, substantially smaller amounts of proteins with NF kappa B specificity were found in all cells. Despite this apparent deficit in NF kappa B activity, trophoblasts supported a high basal activity of both LTRs. These data indicate that an insufficiency of basal or Tat-trans-activated LTR activity cannot account for the low level of HIV-1 replication in this important cell type.

Base Sequence↗
PubMed2005

Id helix-loop-helix proteins are differentially expressed in gestational trophoblastic disease.

AIMS: To assess the expression of Id proteins in trophoblastic tissues and to correlate this with clinical parameters, proliferative and apoptotic indices as well as to related oncogene expression. METHODS AND RESULTS: Immunohistochemistry for Id1, Id2, Id3 and Id4 was performed on 83 trophoblastic tissues including 17 normal first-trimester placentas, seven term placentas, 47 hydatidiform moles (HM), and 12 spontaneous miscarriages. The four Id proteins were predominantly expressed in the villous and implantation site intermediate trophoblast. Expression of Id1 in HM was significantly higher than that in normal placenta (P = 0.0006) and spontaneous miscarriage (P = 0.0001) but did not correlate with subsequent development of gestational trophoblastic neoplasia (GTN). Id1 expression correlated with the proliferation index as assessed by MCM7 (P = 0.003) and Ki67 (P = 0.017) and with the apoptotic activity assessed by TUNEL (P = 0.001) and M30 CytoDeath antibody (P = 0.013). Moreover, the expression of Id1 correlated with the expression of p53 (P = 0.004), p21(WAF1) (/CIP1) (P = 0.003) but not with p16 (P = 0.107). CONCLUSIONS: Id proteins may play a role in the regulation of proliferative and apoptotic activity in trophoblastic tissue and are potentially useful in differentiating molar and non-molar gestation, but are not helpful in predicting GTN.

Cell Cycle Proteins↗
PubMed1994

A study of placental bed spiral arteries and trophoblast invasion in normal and severe pre-eclamptic pregnancies.

OBJECTIVE: To investigate trophoblast invasion and vascular changes in placental bed spiral arteries in normal and severe pre-eclamptic pregnancies. DESIGN: A histological and immunohistochemical study of placental bed biopsies containing spiral arteries. SETTING: The University Hospital, Leuven, Belgium. SUBJECTS: Twenty-one placental bed biopsies from 21 normal pregnancies and 24 placental bed biopsies from 24 severe pre-eclamptic pregnancies, taken at caesarean section. OBSERVATIONS: Histological and immunohistochemical appearance of spiral arteries (stained with haematoxylin and eosin), periodic acid schiff, and a monoclonal antibody to low molecular weight cytokeratin. RESULTS: One hundred and twenty-seven spiral arteries were studied. In the 21 biopsies from clinically normal pregnancies at term, 100% of the decidual spiral arteries and 76% of the myometrial arteries showed trophoblast invasion. In the 24 biopsies from women with severe pre-eclampsia, trophoblast invasion was seen in 44% and 18% of the decidual and myometrial segments, respectively. Endovascular trophoblast invasion was complete, partial or isolated. A variety of morphological features was present not only in different spiral arteries from the same biopsy but also in different segments of the same artery. The vascular change most commonly associated with normal pregnancies was physiological change and subintimal thickening of both segments of the spiral arteries. In pre-eclampsia medial disorganisation and hyperplasia in the myometrial arteries and acute atherosis in decidual arteries were common. CONCLUSION: Endovascular trophoblast did not show an all or none invasive phenomenon in normal and pre-eclamptic pregnancies. More decidual than myometrial arteries were invaded in both groups of patients, and there was a gradient in the percentage of decidual and myometrial arteries invaded from normal pregnancy to pre-eclampsia. Morphological features in one spiral artery may not necessarily be representative of all of those in a placental bed.

Arteries↗