Decidual NK cell-derived conditioned medium enhances capillary tube and network organization in an extravillous cytotrophoblast cell line.

Extravillous cytotrophoblast (EVT) migration, invasion and endovascular differentiation are regulated by a variety of growth factors, cytokines and adhesion molecules. Decidual natural killer cells (dNK) and their secreted cytokines probably modulate these processes. In this study, we used dNK-derived conditioned medium (dNK-CM) to investigate whether or not (i) dNK-CM was able to enhance capillary tube and network formation of an EVT cell line, HTR8/SVneo, on Matrigel, (ii) PI3K/AKT pathway and p38 MAPK pathway activation were involved, and (iii) HTR8/SVneo surface ICAM-1 played a role in the process of HTR8/SVneo endovascular differentiation. The results demonstrated that HTR8/SVneo constitutively form 'vascular' tubes and networks after culture on Matrigel. dNK-CM enhanced and maintained tube and network formation, acquiring an endothelium-like angiogenic morphology followed by increased VEGF-C production. HTR8/SVneo cell expression level of VE-cadherin, PECAM-1, VCAM-1 and alphavbeta3 was unaltered by dNK-CM, whereas ICAM-1 expression level was increased. Anti-human ICAM-1 blocking antibody inhibited HTR8/SVneo migration and partially reversed dNK-CM-mediated enhancement of HTR8/SVneo tube and network formation. PI3K/AKT and p38 MAPK pathways were activated in dNK-CM-mediated enhancement of HTR8/SVneo tube and network formation. The PI3K/AKT and p38 MAPK pathway inhibitors (LY294002 and SB202190, respectively) decreased dNK-CM-stimulated ICAM-1 induction, HTR8/SVneo migration, and reversed tube and network formation. The results suggest that dNK cell-secreted growth factors and cytokines participate in the regulation of HTR8/SVneo endothelium-like tube formation. Adhesion molecules, particularly ICAM-1, expressed on EVT may participate in the process. To our knowledge, this is the first report of a role for ICAM-1 in EVT angiogenesis, as previously reported for endothelial cells.

Expression of ADAMTS-5/implantin in human decidual stromal cells: regulatory effects of cytokines.

BACKGROUND: The restricted expression of ADAMTS-5 (A Disintegrin And Metalloproteinase with ThromboSpondin repeats-5) to the maternal-fetal interface in mice has led to this novel metalloproteinase being assigned the trivial name 'implantin'. METHODS: As a first step in determining whether ADAMTS-5 also contributes to the implantation process in humans, we have examined the spatiotemporal expression of this ADAMTS subtype in the endometrium during the menstrual cycle and pregnancy by immunohistochemical analysis. A quantitative competitive PCR (QC-PCR) strategy and western blotting were subsequently used to determine whether interleukin (IL)-1beta and transforming growth factor (TGF)-beta1, two cytokines involved in the formation of the maternal-fetal interface, were capable of regulating ADAMTS-5 messenger RNA (mRNA) and protein levels in primary cultures of stromal cells isolated from first trimester decidual tissues. RESULTS: ADAMTS-5 expression in the stroma of the human endometrium correlates with decidualization of this cellular compartment in vivo. IL-1beta was found to increase (P < 0.05) whereas TGF-beta1 decreased (P < 0.05) ADAMTS-5 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies directed against either cytokine. CONCLUSIONS: ADAMTS-5 expression is restricted to decidualized stromal cells of the human endometrium in vivo and is subject to regulation by cytokines in vitro.

Methylation status and expression of E-cadherin and cadherin-11 in gestational trophoblastic diseases.

The clinical significance of cadherins in gestational trophoblastic diseases (GTD) is not fully understood. In this study, the expression of E-cadherin and cadherin-11 in 12 normal placentas, 32 cases of hydatidiform mole (HM) including 15 complete HMs and 17 partial HMs, and five choriocarcinomas was investigated by immunohistochemistry and correlated with follow-up of HMs. Cases with available frozen blocks were further analyzed by western blot and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Methylation of E-cadherin was investigated by methylation-specific PCR in six normal first trimester placentas, 19 HMs and their associated deciduas. E-cadherin expression was localized to cytotrophoblast and intermediate trophoblast whereas cadherin-11 was expressed in syncytiotrophoblast, intermediate trophoblast, and decidua. Immunoreactivity of E-cadherin was reduced in choriocarcinoma and complete HM when compared with that in normal first trimester placenta (P < 0.01, P = 0.04). Hypermethylation of E-cadherin was demonstrated in three complete HMs with the lowest level of E-cadherin. Compared with normal first trimester placenta, immunoreactivity of cadherin-11 was higher in complete HM (P = 0.02), but lower in choriocarcinoma (P = 0.02). Such differential expression was confirmed by western blot and semiquantitative RT-PCR. No obvious association was observed between the development of persistent trophoblastic disease with the expression of E-cadherin and cadherin-11.

alpha-, beta-, gamma-catenin, and p120(CTN) expression during the terminal differentiation and fusion of human mononucleate cytotrophoblasts in vitro and in vivo.

The cadherins play key roles in the formation and organization of the mammalian placenta by mediating cellular interactions and the terminal differentiation of trophoblastic cells. Although cadherin function is regulated by the cytoplasmic proteins, known as the catenins, the identity and expression pattern(s) of the catenins present in the trophoblastic cells of the human placenta have not been characterized. In these studies, we have determined that alpha-, beta-, gamma-catenin, and p120(ctn) expression levels are high in villous cytotrophoblasts isolated from the human term placenta but decline as these cells undergo aggregation and fusion to form syncytium with time in culture. In contrast, the expression levels of these four catenin subtypes remained constant in non-fusing JEG-3 choriocarcinoma cells at all of the time points examined in these studies. alpha-, beta-, gamma-catenin, and p120(ctn) expression was further immunolocalized to the mononucleate cells present in these two trophoblastic cell cultures. Similarly, intense immunostaining for all four catenins was detected in the mononucleate villous cytotrophoblasts of the human first trimester placenta. Collectively, these observations demonstrate that the expression levels of alpha-, beta-, gamma-catenin, and p120(ctn) are tightly regulated during the formation of multinucleated syncytium in vitro and in vivo.

N-cadherin is regulated by gonadal steroids in adult sexually dimorphic spinal motoneurons.

Gonadal steroids influence the morphology and function of neurons in the adult spinal cord through cellular and molecular mechanisms that are largely unknown. The cadherins are cell adhesion molecules that participate in the formation and organization of the CNS during embryonic development, and recent evidence suggests that the cadherins continue to regulate neural structure and function in adulthood. Using degenerate oligonucleotides coding conserved regions of the catenin-binding domain of classical cadherins in a RT-PCR cloning strategy, we identified several cadherin subtypes, the most frequently cloned being N-, E-, and R-cadherin, suggesting that these are the major classical cadherin subtypes present in the adult male rat lumbosacral spinal cord. We then examined cadherin expression levels of these cadherin subtypes under steroid conditions known to induce plastic changes in spinal motoneurons. Semiquantitative PCR revealed that mRNA levels of N-cadherin, but not E-cadherin or R-cadherin, are elevated in castrated rats treated with testosterone, 17 beta-estradiol, or dihydrotestosterone relative to castrate rats not treated with steroids. Immunolocalization of N-cadherin revealed that steroid treatment increased N-cadherin expression levels in functionally related neural populations whose morphology and function are regulated by steroids. These results suggest a role for N-cadherin in steroid-induced neuroplastic change in the adult lumbar spinal cord.

Differential expression of the coxsackievirus and adenovirus receptor regulates adenovirus infection of the placenta.

The molecular mechanisms and pathologic significance of placental viral infections are poorly understood. We investigated factors that regulate placental infection by adenovirus, which is the most common viral pathogen identified in fetal samples from abnormal pregnancies (i.e., fetal growth restriction, oligohydramnios, and nonimmune fetal hydrops). We also determined the pathologic significance of placental adenovirus infection. Northern hybridization, flow cytometry, and immunostaining revealed that placental expression of the coxsackievirus and adenovirus receptor (CAR) varied with gestational age and trophoblast phenotype. The CAR was continuously expressed in invasive or extravillous trophoblast cells but not in villous trophoblast cells. We postulate that the villous syncytiotrophoblast, which does not express CAR and is resistant to adenovirus infection, limits the transplacental transmission of viral pathogens, including adenovirus. Conversely, extravillous trophoblast cells underwent apoptosis when infected by adenovirus in the presence of decidual lymphocytes (which simulated the maternal immune response to viral infection). Thus, adenovirus infection and/or the maternal immune response to adenovirus infection induced the death of placental cell types that expressed CAR. Consequently, we speculate that adenovirus infection of extra-villous trophoblast cells may negatively impact the process of placental invasion and predispose the mother and fetus to adverse reproductive outcomes that result from placental dysfunction.

N-cadherin is regulated by gonadal steroids in the adult hippocampus.

In the adult hippocampus, gonadal steroids induce neural remodeling through cellular and molecular mechanisms that are largely unknown. The calcium-dependent cell adhesion molecule N-cadherin, which participates in the developmental organization of the nervous system, has recently been localized to hippocampal synapses and is suspected to participate in adult synaptic physiology. Little is currently known about the regulation of cadherins in the adult central nervous system, although posttranslational modifications are thought to account for variability in N-cadherin expression levels. To evaluate the possibility that gonadal steroids regulate N-cadherin in the adult hippocampus, we examined hippocampal N-cadherin mRNA levels and protein expression in castrated adult male rats treated with testosterone, or its metabolites 17beta-estradiol or dihydrotestosterone. Northern blot analysis indicated increased hippocampal N-cadherin mRNA levels in the adult rat hippocampus after treatment with 17beta-estradiol but not testosterone or dihydrotestosterone. Increased N-cadherin immunoreactivity was observed in CA1 and CA3 pyramidal cells after 17beta-estradiol treatment. Additionally, both 17beta-estradiol and testosterone treatment increased N-cadherin immunoreactivity in the neuropil of the stratum lacunosum-moleculare, which includes apical dendrites from pyramidal cells. In contrast, dihydrotestosterone treatment had no effect on levels of N-cadherin protein expression in CA1 or CA3 pyramidal cells or in the stratum lacunosum-moleculare. These results demonstrate that, in the hippocampus, expression levels of N-cadherin are dynamic in adulthood. To our knowledge, there have been no other demonstrations of steroid regulation of cadherin expression in neural populations. These results suggest a possible adhesive mechanism for steroid-induced plasticity of the adult nervous system.

Regulation of beta-catenin mRNA and protein levels in human villous cytotrophoblasts undergoing aggregation and fusion in vitro: correlation with E-cadherin expression.

The cellular mechanisms underlying the formation and organization of the human placenta remain poorly understood. Recent studies have demonstrated that E-cadherin, in association with the cytoplasmic protein known as beta-catenin, plays an integral role in the differentiation of the trophectoderm in the murine and bovine embryo. Although E-cadherin expression is regulated during the aggregation and fusion of human villous cytotrophoblasts, the expression of beta-catenin during the terminal differentiation of these primary cell cultures has not been determined. In this study, beta-catenin mRNA concentrations and protein expression were examined in primary cultures of human villous cytotrophoblasts using northern and western blot analysis. beta-catenin mRNA concentrations and protein expression were high in freshly isolated mononucleate cytotrophoblasts but decreased as these cells underwent aggregation and fusion to form syncytium. A similar pattern of expression was observed for the E-cadherin mRNA transcript and protein species present in these cell cultures. Immunoprecipitation studies demonstrated that the beta-catenin and E-cadherin protein species present in the mononucleate cytotrophoblasts were capable of forming intracellular complexes. In contrast, beta-catenin and E-cadherin mRNA and protein expression in JEG-3 choriocarcinoma cells remained constant over time in culture. beta-catenin and E-cadherin expression was subsequently immunolocalized to the aggregates of mononucleate cells present in both of these trophoblastic cell cultures and the villous cytotrophoblasts of the human first trimester and term placenta. Taken together, these observations indicate that the E-cadherin-beta-catenin complex plays a central role in the terminal differentiation of human trophoblasts in vitro and in vivo.

Antisteroidal compounds and steroid withdrawal down-regulate cadherin-11 mRNA and protein expression levels in human endometrial stromal cells undergoing decidualisation in vitro.

The cellular mechanisms by which steroids and antisteroidal compounds modulate the function and/or integrity of the human endometrium remain poorly understood. We recently determined that the expression of the novel cadherin subtype, known as cadherin-11, is tightly regulated in endometrial stromal cells undergoing decidualisation in vivo and in vitro. To determine whether the actions of antisteroids on the endometrium are mediated, at least in part, by their ability to regulate the expression of this cell adhesion molecule, we examined the effects of the antiprogestin RU486 and the antiestrogen ICI 182,780 on cadherin-11 mRNA and protein expression levels in human endometrial stromal cells undergoing decidualisation in vitro. RU486 decreased the levels of the cadherin-11 mRNA transcript and protein species present in these cell cultures in a dose- and time-dependent manner. Similarly, ICI 182,780 was capable of reducing stromal cadherin-11 mRNA and protein expression levels in a dose-dependent manner, suggesting that the progesterone-mediated increase in cadherin-11 expression levels in human endometrial cells undergoing decidualisation in vitro is dependent on the presence of estrogens. Cadherin-11 expression levels also were reduced in endometrial stromal cell cultures subjected to progesterone withdrawal, an in vitro model for menstrual breakdown. These studies not only give us useful insight into the mechanism(s) by which progesterone regulates stromal cadherin-11 expression, but they strengthen our hypothesis that this cell adhesion molecule plays a central role in the remodeling processes that occur in the human endometrium in response to fluctuations in the levels of gonadal steroids.

Cadherin-11 is a hormonally regulated cellular marker of decidualization in human endometrial stromal cells.

Cultured human endometrial stromal cells respond to the gonadal steroids, progesterone and 17beta-estradiol, with morphological and biochemical changes that are characteristic of decidualization in vivo. To date, the cellular mechanisms involved in the terminal differentiation of human endometrial stromal cells into decidual cells remain poorly understood. We have recently determined that the novel cadherin subtype, known as cadherin-11, is expressed by endometrial stromal cells undergoing decidualization during the luteal phase of the menstrual cycle and the decidua of pregnancy. In these studies, we have examined cadherin-11 mRNA and protein expression levels in human endometrial stromal cells undergoing steroid-mediated decidualization in vitro. Progesterone or a combination of progesterone and 17beta-estradiol increased stromal cadherin-11 mRNA and protein expression levels with time in culture. Maximum levels of cadherin-11 expression in these cell cultures correlated with a marked increase in IGFBP-1 mRNA levels, a biochemical marker of decidualization. In contrast, 17beta-estradiol had no effect on stromal cad-11 mRNA and protein expression or the levels of the IGFBP-1 mRNA transcript. Taken together, these observations demonstrate that cadherin-11 mRNA and protein expression levels are up-regulated during the terminal differentiation of endometrial stromal cells-suggesting that this cell adhesion molecule may serve as a useful cellular marker for decidualization.

17Beta-estradiol potentiates the stimulatory effects of progesterone on cadherin-11 expression in cultured human endometrial stromal cells.

Cadherin-11 (cad-11) is a novel member of the cadherin gene superfamily of calcium-dependent cell adhesion molecules. To date, the factors capable of regulating this cell adhesion molecule remain poorly characterized. We have recently determined that cad-11 expression in the human endometrium is tightly regulated during the menstrual cycle. The spatiotemporal expression of cad-11 in the stromal cells of the human endometrium during the menstrual cycle suggests that gonadal steroids regulate the expression of this endometrial cell adhesion molecule. In view of these observations, we have examined the ability of progestins, estrogens, and androgens, alone or in combination, to regulate cad-11 expression in isolated human endometrial stromal cells using Northern and Western blot analyses. In these studies, we have determined that progesterone, but not 17beta-estradiol or dihydrotestosterone, is capable of regulating cad-11 messenger RNA and protein expression levels in isolated endometrial stromal cells. In addition, 17beta-estradiol, but not dihydrotestosterone, was capable of potentiating the stimulatory effects of progesterone in a dose-dependent manner. Taken together, these observations suggest that both 17beta-estradiol and progesterone are required for maximal cad-11 expression in human endometrial stromal cells in vitro.

Regulated expression of cadherin-6 and cadherin-11 in the glandular epithelial and stromal cells of the human endometrium.

The cadherins are key morphoregulators. A switch in the cadherin subtype(s) expressed by a population of cells has been associated with the differentiation and formation of tissues during embryonic development. To date, the role(s) of the cadherins in the highly regulated remodeling processes which occur in the human endometrium in preparation for the implanting embryo remain poorly characterized. Here we report that two atypical cadherins, known as cadherin-6 and cadherin-11, are spatiotemporally expressed in the human endometrium during the menstrual cycle. Cadherin-6 levels are high in both the glandular epithelium and stroma of the endometrium during the follicular phase and decline as the cycle enters the luteal phase. The down-regulation of cadherin-6 in the glandular epithelium during the luteal phase does not effect the levels of cadherin-11 in this cell type. In contrast, the loss of cadherin-6 expression in endometrial stroma cells is concomitant with an increase in the levels of cadherin-11. Collectively, these observations suggest that multiple factors regulate the expression of these two endometrial cadherins. As a first step in identifying these factors, we examined the effects of progesterone on cadherin-6 and cadherin-11 expression in isolated endometrial stromal cells. Progesterone was capable of differentially regulating the expression of these two stromal cell adhesion molecules. These findings lend further support to our hypothesis that steroids are key regulators of cadherin expression in mammalian tissues.

Type 2 cadherins in the human endometrium and placenta: their putative roles in human implantation and placentation.

PROBLEM: The cadherins are a gene superfamily of calcium-dependent cell adhesion molecules. To date, the role(s) of the cadherins in human implantation remains poorly defined. METHOD OF STUDY: The spatiotemporal expression of the type 2 cadherins, known as cadherin-11 and cadherin-6, in the endometrium and placenta was examined using the reverse transcriptase-polymerase chain reaction. RESULTS: Cadherin-6 and cadherin-11 are differentially expressed in the endometrial stroma during the menstrual cycle. The switch between cadherin-6 and cadherin-11 expression in the endometrial stroma occurs during the late secretory phase. Maximum cadherin-11 mRNA levels were observed in the decidua of early pregnancy but were markedly reduced at term. In the placenta, cadherin-11 is expressed in the syncytial trophoblast and extravillous cytotrophoblast columns. However, cadherin-6 seems to be the predominant cadherin subtype present in highly invasive extravillous cytotrophoblasts. CONCLUSION: Cadherin-11 and cadherin-6 may play a central role in the formation and organization of the human endometrium and placenta.

Regulated expression of cadherin-11 in human extravillous cytotrophoblasts undergoing aggregation and fusion in response to transforming growth factor beta 1.

Transforming growth factor beta 1 is believed to be a key regulator of extravillous cytotrophoblast invasion during the first trimester of pregnancy. In addition, this growth factor has been shown to regulate cellular differentiation and fusion in cultured extravillous cytotrophoblasts. To date, the cellular mechanisms by which transforming growth factor beta 1 promotes these developmental processes remain poorly understood. Recent studies indicate that the expression of the novel cadherin subtype, known as cadherin-11, is associated with the terminal differentiation and fusion of villous cytotrophoblasts isolated from the human term placenta and human myoblasts in vitro. In this study, cadherin-11 mRNA and protein expression were examined in primary cultures of human extravillous cytotrophoblasts cultured in the presence of increasing concentrations of transforming growth factor beta 1 using northern and western blot analysis, respectively. Transforming growth factor beta 1 was shown to increase cadherin-11 mRNA and protein expression in these cultured extravillous cytotrophoblasts in a dose-dependent manner. Cadherin-11 was further localized to the large cellular aggregates and multinucleated cells that formed in response to increasing concentrations of transforming growth factor beta 1 using immunocytochemistry. Collectively, these observations suggest that the morphogenetic effects of transforming growth factor beta 1 on cultured extravillous cytotrophoblasts are mediated, at least in part, by an increase in cadherin-11 expression. This study not only adds to the understanding of the cellular mechanisms by which transforming growth factor beta 1 promotes trophoblast differentiation and fusion but provides useful insight into the cell biology of the cadherins.

Progesterone regulates beta-catenin mRNA levels in human endometrial stromal cells in vitro.

Cadherin-catenin complexes mediate cell-cell interactions and may play a central role in intracellular signaling. To date, the factors capable of coordinately regulating cadherin and catenin expression levels within a mammalian cell remain poorly characterized. We have recently determined that progesterone is a key regulator of cadherin-11 mRNA and protein expression levels in cultured human endometrial stromal cells. As a first step in determining whether gonadal steroids are also capable of regulating stromal catenin expression, we have examined the ability of progestins, estrogens, and androgens to regulate beta-catenin mRNA levels in these endometrial cell cultures. Here we report that progesterone, but not 17beta-estradiol or dihydrotestosterone, increased beta-catenin mRNA levels in cultured human endometrial stromal cells. The stimulatory effect of progesterone on the levels of the stromal beta-catenin mRNA transcript could not be potentiated by 17beta-estradiol. These studies not only demonstrate that gonadal steroids are capable of regulating beta-catenin mRNA levels in human endometrial stromal cells, but may also give us useful insight into the cellular mechanisms by which gonadal steroids regulate the cyclic remodeling processes that occur in the human endometrium during each menstrual cycle.

Hormonal regulation of N-cadherin mRNA levels in rat granulosa cells.

We have examined the ability of hormones to modulate the steady-state levels of N-cadherin mRNA transcripts in aggregated and dispersed rat granulosa cell populations. Estradiol and follicle-stimulating hormone (FSH) had no effect on the levels of N-cadherin mRNA transcripts in aggregated granulosa cells. In contrast, these two hormones stimulated N-cadherin mRNA levels in dispersed granulosa cells. This is the first report that estradiol and FSH are capable of regulating N-cadherin mRNA levels. The results also suggest that the N-cadherin mRNA levels in dispersed and aggregated granulosa cells are regulated by different mechanisms.

Estrogens potentiate the stimulatory effects of follicle-stimulating hormone on N-cadherin messenger ribonucleic acid levels in cultured mouse Sertoli cells.

Gonadal steroids and FSH are key regulators of Sertoli cell function. N-Cadherin (N-cad) is a calcium-dependent cell adhesion molecule that mediates Sertoli cell-germ cell interactions. We recently demonstrated that steroids, in particular estradiol, are potent regulators of testicular N-cad messenger RNA (mRNA) levels in vivo. In view of the cooperative effects of steroids and FSH on Sertoli cell-germ cell interactions, we examined the combined effects of these hormones on N-cad mRNA levels in cultured mouse Sertoli cells. FSH was capable of increasing N-cad mRNA levels 2-fold in these cells. The effects of FSH on N-cad mRNA levels in cultured Sertoli cells were mimicked by cAMP-inducing agents. Treatment of the Sertoli cell cultures with FSH and estradiol stimulated N-cad mRNA levels 3-fold, whereas steroids alone had no effect on N-cad mRNA levels. These studies demonstrate that FSH and estradiol in combination are required to achieve maximal N-cad mRNA levels in cultured Sertoli cells. The results obtained from these studies substantiate the hypothesis that estrogens play a pivotal role in regulating spermatogenesis.

Regulated expression of cadherin-11 in human epithelial cells: a role for cadherin-11 in trophoblast-endometrium interactions?

Cadherin-11 is a novel member of the cadherin supergene family. Cadherin-11 expression is localized to mesenchymal tissue and specific regions of the neural tube during mouse embryogenesis. Here we report that cadherin-11 is spatiotemporally expressed in the epithelial cells of the human placenta. Cadherin-11 mRNA levels were low in freshly isolated cytotrophoblast cells but increased as the cytotrophoblast cells aggregated and fused to form syncytiotrophoblast cells in vitro. The increase in cadherin-11 mRNA levels was concomitant with a decrease in E-cadherin expression. Cadherin-11 was localized to the syncytial trophoblast and extravillous cytotrophoblasts, but not the villous cytotrophoblasts of the human placenta by immunohistochemistry. As both of the former cell types have intimate interactions with the endometrium, we examined cadherin-11 expression in the human endometrium. Cadherin-11 was detected in the glandular and surface epithelium of the endometrium at all stages of the menstrual cycle. However, cadherin-11 was abundant only in the stroma in the late secretory stage of the menstrual cycle. The accumulation of cadherin-11 in the stroma correlated with decidualization. Taken together, our observations demonstrate that cadherin-11 is expressed in certain epithelial cell lineages and suggest the possibility that cadherin-11 plays an important role in mediating trophoblast-endometrium interactions.

Transduction of human trophoblast cells by recombinant adenoviruses is differentiation dependent.

To explore the feasibility of adenoviral (Ad)-mediated gene transfer to the human placenta, we examined the ability of two recombinant Ad vectors to transduce isolated human cytotrophoblast cells and two choriocarcinoma cell lines (BeWo and JEG-3, which have different potentials to undergo morphological differentiation in response to cAMP). Recombinant Ad efficiently transduced cytotrophoblast cells. However, there was a marked reduction in the transduction efficiency of these vectors after the terminal differentiation of the mononucleate cytotrophoblasts into multinucleate syncytial trophoblast. BeWo and JEG-3 cells were readily transduced with the recombinant Ad, but a striking reduction in transduction efficiency of the Ad vector was observed in BeWo cells following cAMP-stimulated cellular differentiation, which includes cell fusion to form syncytia. In contrast, JEG-3 cells, which are not induced to fuse in the presence of cAMP, did not show a reduced transduction efficiency when exposed to the cyclic nucleotide. Reporter gene copy number increased with Ad-mediated gene transfer into undifferentiated Bewo cells but was low in cells that had been previously exposed to cAMP. In contrast, both undifferentiated and cAMP-treated BeWo cells were capable of expressing a reporter gene when transfected with an Ad-based plasmid. Taken together, these results demonstrate that the reduction in transduction efficiency of the Ad vectors in cAMP-treated BeWo is the result of reduced infectivity rather than of a reduction in the transcription/translation efficiency of the exogenous genes. Our findings demonstrate that recombinant Ad vectors will not be useful for the transfer of genes into differentiated trophoblast cells because these cells are resistant to Ad infection. This may limit the utility of Ad-based vectors for placental gene therapy. However, we have documented that less-differentiated trophoblast cells are susceptible to Ad-mediated gene transfer. Our observations also suggest a mechanism by which differentiated human trophoblast cells resist Ad infection and prevent fetal infection by maternally derived Ad.