Adhesive and degradative properties of human placental cytotrophoblast cells in vitro.

Human fetal development depends on the embryo rapidly gaining access to the maternal circulation. The trophoblast cells that form the fetal portion of the human placenta have solved this problem by transiently exhibiting certain tumor-like properties. Thus, during early pregnancy fetal cytotrophoblast cells invade the uterus and its arterial network. This process peaks during the twelfth week of pregnancy and declines rapidly thereafter, suggesting that the highly specialized, invasive behavior of the cytotrophoblast cells is closely regulated. Since little is known about the actual mechanisms involved, we developed an isolation procedure for cytotrophoblasts from placentas of different gestational ages to study their adhesive and invasive properties in vitro. Cytotrophoblasts isolated from first, second, and third trimester human placentas were plated on the basement membrane-like extracellular matrix produced by the PF HR9 teratocarcinoma cell line. Cells from all trimesters expressed the calcium-dependent cell adhesion molecule cell-CAM 120/80 (E-cadherin) which, in the placenta, is specific for cytotrophoblasts. However, only the first trimester cytotrophoblast cells degraded the matrices on which they were cultured, leaving large gaps in the basement membrane substrates and releasing low molecular mass 3H-labeled matrix components into the medium. No similar degradative activity was observed when second or third trimester cytotrophoblast cells, first trimester human placental fibroblasts, or the human choriocarcinoma cell lines BeWo and JAR were cultured on radiolabeled matrices. To begin to understand the biochemical basis of this degradative behavior, the substrate gel technique was used to analyze the cell-associated and secreted proteinase activities expressed by early, mid, and late gestation cytotrophoblasts. Several gelatin-degrading proteinases were uniquely expressed by early gestation, invasive cytotrophoblasts, and all these activities could be abolished by inhibitors of metalloproteinases. By early second trimester, the time when cytotrophoblast invasion rapidly diminishes in vivo, the proteinase pattern of the cytotrophoblasts was identical to that of term, noninvasive cells. These results are the first evidence suggesting that specialized, temporally regulated metalloproteinases are involved in trophoblast invasion of the uterus. Since the cytotrophoblasts from first trimester and later gestation placentas maintain for several days the temporally regulated degradative behavior displayed in vivo, the short-term cytotrophoblast outgrowth culture system described here should be useful in studying some of the early events in human placen

Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity.

RNA amplification with transcript sequencing (RAWTS) is a rapid and sensitive method of direct sequencing that involves complementary DNA synthesis, polymerase chain reaction (PCR) with a primer or primers containing a phage promoter, transcription from the phage promoter, and reverse transcriptase-mediated sequencing. By means of RAWTS, it was possible to sequence each of four tissue-specific human messenger RNAs (blue pigment, factor IX, phenylalanine hydroxylase, and tyrosine hydroxylase) in four cell types examined (white blood cells, liver, K562 erythroleukemia cells, and chorionic villus cells). These results indicate that there is a basal rate of transcription, splicing, and polyadenylation of tissue-specific mRNAs in adult and embryonic tissues. In addition to revealing sequence information, it is possible to generate a desired in vitro translation product by incorporating a translation initiation signal into the appropriate PCR primer. RAWTS can be used to obtain novel mRNA sequence information from other species as illustrated with a segment of the catalytic domain of factor IX. In general, the ability to obtain mRNA sequences rapidly across species boundaries should aid both the study of protein evolution and the identification of sequences crucial for protein structure and function.

Localization, secretion, and action of inhibin in human placenta.

Inhibin is a gonadal glycoprotein hormone that regulates the production of follicle-stimulating hormone (FSH) by the anterior pituitary gland and exhibits intragonadal actions as well. The present study shows that inhibin-like immunoreactivity (inhibin-LI) is present in cells of the cytotrophoblast layer of human placenta at term and in primary cultures of human trophoblasts. Human chorionic gonadotropin (hCG) stimulated secretion of inhibin-LI from these cultured placental cells. This effect was mimicked by 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP), forskolin, and cholera toxin, suggesting that the mechanism of hCG induction of placental inhibin-LI secretion is cAMP-dependent. Incubation with an antiserum that binds the alpha-subunit of human inhibin increased the secretion of hCG and gonadotropin-releasing hormone-like immunoreactivity (GnRH-LI) from trophoblast cells in culture, suggesting a local tonic inhibitory action of endogenous inhibin on hCG and GnRH-LI release. The action of inhibin on hCG secretion may partially require the presence of placental GnRH, as suggested by evidence that a synthetic GnRH antagonist partially reverses the hCG increase induced by inhibin immunoneutralization. Results suggest paracrine roles for both inhibin and GnRH in the regulation of placental hCG production.

Characterization and histologic localization of human growth hormone-variant gene expression in the placenta.

The human growth hormone-variant (hGH-V) gene is one of five highly similar growth hormone-related genes clustered on the short arm of chromosome 17. Although the pattern of expression of the adjacent normal growth hormone (hGH-N) and chorionic somatomammotropin (hCS) genes in this cluster are well characterized, the expression of the hGH-V gene remains to be defined. In previous studies, we have demonstrated that the hGH-V gene is transcribed in the term placenta and expressed as two alternatively spliced mRNAs: one is predicted to encode a 22-kD hormone (hGH-V), the other retains intron 4 in its sequence resulting in the predicted synthesis of a novel 26-kD hGH-V-related protein (hGH-V2). In the present report, we document the expression of both of these hGH-V mRNA species in the villi of the term placenta, demonstrate an increase in their concentrations during gestation, and directly sublocalize hGH-V gene expression to the syncytiotrophoblastic epithelium of the term placenta by in situ cDNA-mRNA histohybridization. The demonstrated similarity in the developmental and tissue-specific expression of the hGH-V gene with that of the related hCS gene suggests that these two genes may share common regulatory elements.

Immunocytochemical localization of relaxin in human decidua and placenta.

Specimens from 20 human term placentas were stained with 4 different antisera produced against porcine relaxin (Rlx) using the avidin-biotin immunoperoxidase procedure. Cells of the parietal decidua adherent to the fetal membranes, cells of the chorionic cytotrophoblast, as well as cells of the placental basal plate consistently stained with all 4 anti-Rlx sera. Occasionally, Rlx was detected in epithelial cells lining the amniotic membrane. The syncytiotrophoblast stained for Rlx in 2 specimens only. This response was seen only in syncytiotrophoblast that lined villi in close proximity to the basal plate. Syncytiotrophoblast of the chorionic villi either did not stain at all or gave very weak positive immunostaining with the anti-Rlx sera in all specimens. No difference was noted in staining patterns among placentas delivered by elective cesarean section or vaginal delivery.

Localization of alpha-interferon in the human feto-placental unit.

The distribution of alpha-interferon in human placental tissue was investigated by immunocytochemical study of paraffin wax-embedded tissue sections using a sheep alpha-interferon antiserum. Fifty-eight placentas of gestational ages from 8 to 40 weeks were examined. alpha-Interferon was present in the syncytiotrophoblast of the chorionic villi of all placentas and was also in macrophages in 28 cases. The appearances suggest production of interferon in human placental trophoblast and, in view of its diverse biological effects, support the concept of a role for alpha-interferon in the complex series of events required for successful gestation.

Characterization of the tumour necrosis factor receptor in human placenta.

A specific receptor for tumour necrosis factor present in a purified plasma membrane preparation from the villous tissue of human placenta has been characterized. The data fit a one-class-of-sites model exhibiting a KD = 28.4 (+/- 0.002) nM, with 8.1 (+/- 0.05) X 10(11) receptors mg-1 membrane protein. These data provide evidence for the existence of a specific receptor for a major endotoxin-induced cytokine in the placenta.

Immunohistochemical demonstration of epidermal growth factor (EGF) receptors in normal human placental villi.

With an avidin-biotin-peroxidase (or glucose oxidase) complex method using anti-epidermal growth factor receptor monoclonal antibody (528 IgG), the tissue and cellular distribution of the receptors for epidermal growth factors (EGF) in normal human placental villi, from 6 to 42 weeks of gestation, were studied. EGF receptors were mainly localized on the free surface of the syncytiotrophoblast that directly faced to intervillous space of the maternal circulation. The cell surface of cytotrophoblasts, except for the region that was adjacent to the basal lamina, was also positive for EGF receptors. The receptors were in close contact to the fetal vessels in the villous stroma. The EGF receptors on the syncytiotrophoblast were thought to be involved in the production and secretion of human chorionic gonadotropin and placental lactogen, probably under the control of maternal EGF. The receptors on cytotrophoblasts may play a role in trophoblastic proliferation, possibly mediated by EGF in the fetal circulatory system.

Fetal and maternal blood volumes in shed human placentae: discrepant results comparing morphometry to haemoglobin content.

The maternal blood volume (MBV) and fetal blood volume (FBV) of shed human placentae delivered by caesarean section at term were measured using a morphometric technique and from the placental content of adult and fetal haemoglobin. MBV was 35.3 +/- 1.5 (s.e.m.) per cent by the former and 11.0 +/- 1.5 per cent by the latter technique. FBV was 11.0 +/- 0.7 and 9.0 +/- 0.6 per cent respectively (n = 6). Measurement of the dimensions of individual villi initially photomicrographed in 0.9 per cent NaCl and subsequently re-photographed in fixative suggested that individual villi shrank to 0.7 of their initial volume during fixation. It is suggested that morphometric measurement of MBV may lead to approximately a threefold overestimate because of relative MBV expansion and villous tissue shrinkage during the process of fixation without alteration in overall placental volume.

Proteins of the apical and basal plasma membranes of the human placental syncytiotrophoblast: immunochemical and electrophoretic studies.

Proteins of the basal and microvillous plasma membranes of the human placental syncytiotrophoblast were compared to elucidate the basis for structural and functional differences in the two membranes. Among the proteins common to both membranes were actin, alkaline phosphatase, albumin, transferrin, immunoglobulin G, proteins of Mr 35,000, 55,000 and 180,000, and five immunochemically detected proteins. Each membrane also contained unique proteins. Major microvillous cytoskeleton proteins of Mr 68,000 80,000 and 105,000 (alpha-actinin) were lacking or absent from basal membrane cytoskeletons which instead contained unique proteins of Mr 14,000, 16,000, 220,000 and 240,000. In addition, immunochemical analyses revealed four glycoprotein antigens unique to microvillous membrane and five unique to basal membrane. Fibronectin was also found to be exclusive to basal membrane. The difference in membrane-associated cytoskeletal proteins correlated with the different organization of actin microfilaments at the two membranes. The protein antigens unique to each of the two membranes provided further evidence for the polarization of membrane proteins and functions in the syncytium.

Ultrastructural changes and immunocytochemical analysis of human placental trophoblast during short-term culture.

Trophoblastic cells, of at least 95 per cent purity by immunofluorescence and morphological criteria, were obtained from human term placenta by a simple trypsinisation method without the additional purification steps or complex culture conditions used by others. The differentiation of these cells was followed over four days in culture by fluorescence immunocytochemistry, by scanning and transmission electron microscopy and by light microscopy. The results support the idea that the isolated cells are cytotrophoblast and that these differentiate during this time into cells with characteristics of villous syncytiotrophoblast. This process involved first the formation of a multicellular layer of mononucleated cells, then the development of a syncytium of multinucleated cells and, not necessarily concurrently, functional differentiation. This may be a useful model for the study of syncytiotrophoblast function.

Isolation of a transferrin receptor structure from sodium deoxycholate-solubilized human placental syncytiotrophoblast plasma membrane.

A human placental transferrin receptor structure has been isolated as a complex with doubly-radiolabelled (125I, 59Fe) human transferrin following gel filtration and affinity chromatography of sodium deoxycholate-solubilized protein from isolated syncytiotrophoblast microvillous plasma membrane vesicle preparations. The molecular weight of this complex has been determined to be 150 000 by Sepharose 6B gel filtration. The molecular weights of the constituent components of the complex have been determined by sodium dodecyl sulphate/polyacrylamide disc gel electrophoresis to be 80 000 (representing transferrin) and 65 000 (representing the deoxycholate-solubilized receptor structure). The binding activity of this placental transferrin receptor structure is maintained in sodium deoxycholate and is not requisite on being an integral component of the cell membrane.

Immunological studies of transferrin and transferrin receptors of human placental trophoblast.

In primate pregnancy, fetal iron is derived from maternal transferrin; however, the mechanisms by which iron is taken up by the human placenta have not yet been established. In the present study, transferrin was demonstrated on the microvillous surface of human trophoblast in immunohistological studies of 130 mature and immature placentae from both normal and abnormal pregnancies. Similar results were found for baboon placentae. Upon short-term culture of placental tissue, the amount of trophoblast transferrin decreased and no incorporation of 14C lysine into transferrin could be detected by radioimmunoelectrophoresis. Thus this transferrin apparently was not synthesized by the placenta. When transferrin was removed from cryostat sections of placenta by treatment with chaotropic agents, subsequently added transferrin bound in an identical distribution. The specificity of this reaction was confirmed by the lack of binding of other serum proteins and by displacement procedures in which trophoblast transferrin was shown to be dislodged by transferrin added in vitro. These findings suggest that placental iron transport is predicated by binding of transferrin to specific receptors on trophoblast.

Proteins of human placental microvilli: II. Identification and topology of the plasma membrane proteins.

We have investigated the location of proteins in the transverse plane of the plasma membrane of microvilli isolated from the syncytiotrophoblast layer of the human term placenta. Microvillous proteins were labelled with 125I under reaction conditions where those proteins exposed on the maternal-facing surface of the microvilli were most heavily labelled. The proteins were then solubilized and subjected to one- and two-dimensional electrophoresis followed by protein staining and autoradiography. More than 65 proteins, differing in molecular weight or isoelectric point or both, were identified, and these were classified into three groups: weakly, moderately heavily, and heavily labelled. The microvilli were in the form of intact vesicles that were correctly orientated ('right-side out'). Thus the extent of labelling of each protein could be used as an indication of the extent of its exposure on the maternal-facing surface of the microvilli. Human serum albumin was present on the surface of the isolated, washed microvilli, but was probably a contaminant originating from maternal blood.

The distribution of intermediate filament proteins, actin and desmoplakins in human placental tissue as revealed by polyclonal and monoclonal antibodies.

The distribution of intermediate filament proteins (cytokeratin, vimentin, desmin), actin, and desmoplakins in various placental compartments was studied by immunofluorescence microscopy using polyclonal and monoclonal antibodies. Trophoblast cells (cytotrophoblast, syncytiotrophoblast, isolated trophoblast cells, trophoblastic giant cells) were strongly stained by all types of cytokeratin antibodies. Antibodies to desmoplakins revealed the presence of desmosomes at all membranes, except the basal membrane of cytotrophoblast cells, and at the basal as well as the lumen-oriented membrane of the syncytiotrophoblast. After disappearance of the cytotrophoblast cell layer the distribution of desmosomes in the syncytiotrophoblast was unaltered. Isolated trophoblast cells contained desmosomes around their entire circumference. Amnion epithelial cells were heterogeneous with respect to cytokeratin composition as revealed, for example, by polyclonal antibodies with a broad range of cytokeratin reactivity and by monoclonal antibodies to cytokeratin No. 18. With the latter, a heterogeneous staining of amnion epithelial cells was achieved. Desmosomes (spots reactive with desmoplakin antibodies) were present at the lateral membranes of the amnion epithelial cells. In addition, vimentin filaments were coexpressed in these cells. Large vessels of the chorionic plate and stem villi showed thick walls consisting of vimentin-, desmin- and actin-positive cells. They were surrounded by mantles rich in vimentin-, desmin- and actin-positive cells, resembling myofibroblasts. This indicates that these cells may play a role in villous contractility and modulation of the intervillous space with effect on both maternal and fetal placental circulation.

Proteins of human placental microvilli: I. Cytoskeletal proteins.

Microvilli isolated from the syncytiotrophoblast surface of the human term placenta were separated into two fractions, one of which contained microvilli lacking a visible cytoskeleton on electron microscopy. One- and two-dimensional electrophoresis showed that a number of proteins were present in reduced amounts in the fraction lacking a visible core structure. The possibility that these proteins were cytoskeletal components was investigated by further electrophoretic studies in conjunction with 125I labelling of proteins of intact and disrupted microvillous vesicles, digestion of external proteins with immobilized, insoluble trypsin, and selective solubilization of plasma membrane proteins by Triton X-100. From the results of these studies, eight proteins of molecular weights 103 000, 80 000, 70 000, 69 000, 43 000, 36 000, 25 000 and 18 000 were tentatively assigned to the cytoskeleton. The differences between our findings for the cytoskeletal proteins of human placental microvilli and the results reported by others for the well-studied cytoskeletal proteins of the intestinal microvilli of the rat are likely to reflect differences in structure and function of the microvilli from the two sources.

Impact of first-trimester chromosome, DNA, and metabolic studies on pregnancies at high genetic risk: experience with 1,000 cases.

We describe the results and follow-up of chorionic villus studies in 1,034 pregnancies at risk for chromosome or metabolic disorders. Direct chromosome studies were successful in 99.7% and yielded results within a few days. Fifty pregnancies at risk for an unbalanced translocation, inherited from parents with many small reciprocal translocations, were a good test for the quality of the direct method. The 101 metabolic studies involving 28 disorders were correct in 99 pregnancies in the first trimester. In two cases a correct diagnosis was obtained by the confirmatory amniocentesis. DNA studies were carried out in pregnancies of male fetuses at risk for Duchenne muscular dystrophy and a few metabolic disorders. The abortion rate after chorionic villus sampling was 5.1% but more than half of the pregnant women were greater than or equal to 36 years old and have a spontaneous abortion rate of 10% between the 10th and 14th week according to Gustavii [Lancet 1:562, 1984]. Follow-up studies confirmed results of all chromosome studies after termination when there was fetal cell growth; the outcome of 504 consecutive continuing pregnancies showed no discrepancies of the phenotype after birth. It was concluded that first-trimester chorionic villi studies gave reliable results and were increasingly preferred by the patients, while the sampling can be considered a safe procedure based on the currently available data.

Antenatal diagnosis of thalassemia major in Sardinia.

In this report, we summarized our experience, carried out in Sardinia, with antenatal diagnosis in one thousand pregnancies in which the fetus was at risk for homozygous beta-thalassemia. In the majority of these cases, the thalassemia lesion segregating in the family was the nonsense mutation at the codon corresponding to amino-acid 39. At the outset (976 cases) we used globin chain synthesis analysis by column chromatography on fetal blood obtained by placental aspiration, and recently (24 cases) we employed the synthetic oligonucleotide method on amniocyte DNA. Apart from 126 pregnancies still in progress, in all the other cases the diagnosis has been confirmed. In the majority of the cases (99%), we obtained sufficient fetal blood for the analysis. The fetal mortality associated with placental aspiration was 6.1%. The biochemical analysis gave reliable results. We had two misdiagnoses (0.2%): one due to a nonglobin protein comigrating with the beta chains and the other for a misclassification of the type of thalassemia segregating in the family. The oligonucleotide method gave clear-cut results in all the cases tested. The method was sensitive enough to detect the mutation directly in the DNA isolated from 20-25 ml of amniotic fluid in 75% of the pregnancies tested. In one case, we successfully employed this method for the analysis of the DNA isolated from chorionic villi. The oligonucleotide method seems to be the best procedure for monitoring the pregnancies at risk for beta-thalassemia in places where one or a few beta-thalassemia lesions are prevalent.

Maternal serum alpha-fetoprotein screening after chorionic villus sampling.

To evaluate whether maternal serum alpha-fetoprotein (MSAFP) concentrations at 15-18 weeks' gestation are influenced by chorionic villus sampling performed three to ten weeks earlier, MSAFP levels were determined for 417 postchorionic villus sampling patients and 967 control subjects without previous chorionic villus sampling. Statistical comparison of the medians, distributions, and proportions above defined multiples of the median demonstrated no significant difference between the two populations. These results indicate that MSAFP screening in postchorionic villus sampling patients should be reliable and that values may be interpreted using criteria similar to those used for the general population.

Tetraploidy and aneuploidy after observation of diploid fertilization in vitro.

A case is described in which a tetraploid and aneuploid conceptus resulted from dipronuclear embryos from in vitro fertilization. This finding strongly suggests that, after a meiotic error in either the egg or sperm, polyploidy has occurred as the result of a postfertilization mitotic error.