Placental Amnion Research Articles

Pre-B cell colony enhancing factor (PBEF/NAMPT/Visfatin) and vascular endothelial growth factor (VEGF) cooperate to increase the permeability of the human placental amnion

Fluid efflux across the region of the amnion overlying the placenta is an essential component of the intramembranous absorption pathway that maintains amniotic fluid volume homeostasis. Dysregulation of this pathway may result in adverse pregnancy outcomes, however the factors controlling amnion permeability are unknown. Here, we report a novel mechanism that increases placental amnion permeability. Pre-B Cell Colony Enhancing Factor (PBEF) is a stress-responsive cytokine expressed by the human amnion, and is known to induce Vascular Endothelial Growth Factor (VEGF) production by other cell types. Interestingly, VEGF is up-regulated in the ovine amnion when intramembranous absorption is augmented. In this study, we show that PBEF induced VEGF secretion by primary human amniotic epithelial cells (AEC) derived from the placental amnion, as well as from the reflected amnion that lines the remainder of the gestational sac. Further, PBEF treatment led to the increased expression of VEGFR2 in placental AEC, but not reflected AEC. To test the hypothesis that PBEF and VEGF increase placental amnion permeability, we monitored the transfer of 2′,7′-dichlorofluorescein (DCF) from the fetal to the maternal side of human amnion explants. A treatment regimen including both PBEF and VEGF increased the rate of DCF transfer across the placental amnion, but not the reflected amnion. In summary, our results suggest that by augmenting VEGFR2 expression in the placental amnion, PBEF primes the tissue for a VEGF-mediated increase in permeability. This mechanism may have important implications in amniotic fluid volume control throughout gestation.

MiR-143 Regulation of Prostaglandin-Endoperoxidase Synthase 2 in the Amnion: Implications for Human Parturition at Term

BackgroundThe human amnion plays a pivotal role in parturition. Two of its compartments, the placental amnion and the reflected amnion, have distinct transcriptome and are functionally coordinated for parturition. This study was conducted to determine the microRNA (miRNA) expression pattern and its significance in the placental amnion and the reflected amnion in association with labor at term.Methodology/Principal FindingsMicroRNA microarray, real-time quantitative RT-PCR (qRT-PCR), and miRNA in situ hybridization analyses of the placental amnion and the reflected amnion (n = 20) obtained at term were conducted. Luciferase assay, transfection, and qRT-PCR analyses of primary amnion epithelial cells (AECs) and amnion mesenchymal cells (AMCs) were performed. MicroRNA microarray analysis demonstrated differential expression of 32 miRNAs between the placental amnion and the reflected amnion after labor. Thirty-one (97%) miRNAs, which included miR-143 and miR-145, a cardiovascular-specific miRNA cluster, were down-regulated in the reflected amnion. Analyses of miR-143 and miR-145 by qRT-PCR confirmed microarray results, and further demonstrated their decreased expression in the reflected amnion with labor. Interestingly, expression of miR-143 and miR-145 was higher in AMCs than in AECs (p<0.05). Luciferase assay and transfection confirmed miR-143 binding to 3′ UTR of prostaglandin-endoperoxidase synthase 2 (PTGS2) mRNA and miR-143 regulation of PTGS2 in AMCs.ConclusionsWe report region-specific amniotic microRNAome and miR-143 regulation of PTGS2 in the context of human labor at term for the first time. The findings indicate that miRNA-mediated post-transcriptional regulation of gene expression machinery in the amnion plays an important role in the compartments (placental amnion vs reflected amnion) and in a cell type-specific manner (AECs vs AMCs) for parturition.

Identification of Periplakin as a New Target for Autoreactivity in Idiopathic Pulmonary Fibrosis

Injury to alveolar epithelial cells is central to the pathophysiology of idiopathic pulmonary fibrosis (IPF). An abnormal autoimmune response directed against antigens of the alveolar epithelium may contribute to the disease. To detect circulating autoantibodies (autoAbs) directed against epithelial structures. We performed immunoblot by separating human placental amnion extract or alveolar epithelial cell (A549 cell line) proteins on polyacrylamide gels, blotting on nitrocellulose membranes, and incubating with serum from patients with IPF (n = 40) or healthy subjects (n = 40). Proteomic analysis and mass spectrometry characterized the target protein. Inhibition experiments performed with the correspondent recombinant protein confirmed our results. We identified IgG autoAbs recognizing a 200-kD protein in the serum of patients with IPF. Proteomic analysis identified this protein as human periplakin (PPL), a component of desmosomes. Anti-PPL Abs were found by immunoblot in both serum and bronchoalveolar lavage in patients with IPF: 16/40 (40%) of them were positive versus none of the control subjects. Immunohistochemistry revealed that PPL was strongly expressed in bronchial and alveolar epithelium, but that PPL exhibited changes in intracellular localization among normal and fibrotic alveolar epithelium. In an alveolar epithelial wound repair assay, an anti-PPL IgG decreased cell migration. Recombinant PPL induced bronchoalveolar lavage T lymphocyte proliferation. Patients with IPF with anti-PPL Abs had a more severe respiratory disease, despite no difference in survival. We found a new circulating autoAb directed against PPL in patients with IPF, associated with a more severe disease.

Evidence for a Spatial and Temporal Regulation of Prostaglandin-Endoperoxide Synthase 2 Expression in Human Amnion in Term and Preterm Parturition

Prostaglandin-endoperoxide synthase 2 (PTGS2) is a key enzyme involved in parturition. PTGS2 mRNA was found to be differentially expressed between placental amnion (amnion overlying the placental disc) and reflected amnion (amnion of the extraplacental chorioamniotic membranes) in term placentas. The aim was to evaluate the spatial and temporal regulation of PTGS2 expression in the amnion and the chorion-decidua. PTGS2 expression was analyzed in the amnion and chorion-decidua obtained from 32 women: term not in labor (n = 12), term in labor (n = 12), and preterm labor (n = 8), by immunoblotting and densitometry. Prostaglandin E(2) (PGE(2)) in the amnion and chorion-decidua was measured by a specific immunoassay. Compared to preterm labor cases, PTGS2 expression increased at term before the onset of labor far more prominently in placental amnion (4.5-fold; P = 0.002) than in reflected amnion (1.4-fold; P = 0.007). There was a significant increase in PTGS2 expression in reflected amnion (2.9-fold; P < 0.01) but not in placental amnion with labor at term. PTGS2 expression was higher in reflected amnion than in chorion-decidua in labor at term (2.9-fold; P < 0.01). PTGS2 was barely detected in amnion and chorion-decidua with preterm labor. Expression of PGE(2) showed a good correlation with PTGS2 expression (r = 0.722; P < 0.001). PTGS2 expression in the amnion shows a distinct spatial and temporal regulation. Spontaneous labor at term and pathological preterm labor clearly differ in amniotic PTGS2 and PGE(2) abundance. Our observations underscore the biological significance of the amnion and amniotic fluid in human parturition.

Surfactant Protein-A as an Anti-Inflammatory Component in the Amnion: Implications for Human Pregnancy

The mechanism of mouse parturition is thought to involve myometrial infiltration by amniotic fluid (AF) macrophages, activated by surfactant protein-A (SP-A). In humans, the concentration of AF SP-A decreases during labor, and no fetal macrophages are found in the myometrium after labor. Therefore, it appears that the mechanisms of labor in mice and humans are different. We investigated a potential role for SP-A in human pregnancy and parturition by examining SP-A expression patterns in AF and amnion. High molecular mass (>250 kDa) oligomeric SP-A was increased in AF with advancing gestation. Interestingly, these oligomers were more abundant in placental amnion before labor at term, while they increased primarily in reflected amnion during labor (p < 0.05). Immunoblotting showed a binding of high molecular mass SP-A in AF to amnion. In C57BL/6 mice, oligomeric SP-A was also readily detected in AF from E15 onwards, but not in amnion. Macrophage density in mice myometrium did not change with advancing gestational age. Microarray analysis of human amnion explants incubated with SP-A revealed a molecular signature of inhibited cytokine-cytokine receptor interaction with downregulation of IL-1beta, CXCL2, and CXCL5 mRNA expression. The findings in this study strongly suggest that SP-A signals amniotic anti-inflammatory response via AF during pregnancy. We propose that an SP-A interaction among AF, placental amnion, and reflected amnion is a unique mechanism for immunoregulation in human pregnancy akin to that established in lung biology. However, AF SP-A and fetal macrophages by themselves do not seem to be exclusive effectors of parturition in humans.

Pregnant women have increased incidence of IgE autoantibodies reactive with the skin and placental antigen BP180 (type XVII collagen)

BP180 (type XVII collagen) is a transmembrane protein expressed in a variety of cell types. It is also the target of autoantibodies in cutaneous autoimmune disease including bullous pemphigoid and pemphigoid gestationis, a disease unique to pregnancy. The purpose of this study was to determine the prevalence and specificity of cutaneous autoantibodies in a cohort of pregnant women. De-identified sera were collected from pregnant women (n=299) and from non-pregnant controls (n=134). Sera were analyzed by ELISA for the presence of IgG and IgE autoantibodies directed against several cutaneous autoantigens. IgE antibodies against the NC16A domain of BP180 were detected in 7.7% of pregnant women, compared to 2.2% of healthy controls (p=0.01). No increase in total or cutaneous autoantigen specific IgG was seen. Total serum IgE was within the normal range. Full-length BP180 was detected by western immunoblot in epidermal, keratinocyte, placental and cytotrophoblast (CTB) cell lysates. Furthermore, flow cytometry and indirect immunofluorescence confirmed the expression of BP180 on the surface of cultured CTBs. Finally, it was demonstrated that IgE antibodies in the pregnancy sera labeled not only cultured CTBs, but also the placental amnion and cutaneous basement membrane zone using indirect immunofluorescence. We conclude that some pregnant women develop antibodies specific for BP180, and that these autoantibodies are capable of binding both CTB and the placental amnion, potentially affecting placental function.

Circulating Levels of Placental Protein 12 (PP 12) in Diabetic Pregnancy Complicated by Retinopathy*)

Placental protein 12 (PP 12) is a soluble tissue antigen. Immunohistochemical studies have localized PP 12 in the placental syncytiotrophoblast, chorion and amnion, and also in the decidua. During normal pregnancy serum-PP 12 is already raised in the first trimester, there is then a peak at 18 weeks, a gradual fall until 32 weeks, and a moderate increase thereafter. The mean of the healthy control group at 18 weeks of pregnancy was 122.9 +/- 47.5 micrograms/l. The mean of the diabetic group with retinopathy at the same time was 192.2 +/- 78.8 micrograms/l. There was no significant difference between background retinopathy and the proliferative form of diabetic retinopathy. At all times during pregnancy the median values of PP 12 in diabetic pregnancies were significantly (p less than 0.01) above the control values. Increased PP 12 levels in diabetic pregnancy complicated by retinopathy are probably caused by decidual, placental and amniotic leakages.

Region-Specific Gene Expression Profiling: Novel Evidence for Biological Heterogeneity of the Human Amnion1

The amnion plays an important role during pregnancy and parturition. Though referred to as a single structure, this fetal tissue is regionally divided into placental amnion, reflected amnion, and umbilical amnion. Histological differences between placental amnion and reflected amnion led us to hypothesize that the amnion is biologically heterogeneous. The gene expression profiles of placental amnion and reflected amnion were compared in patients at term with no labor (TNL; n = 10) and in labor (TIL; n = 10). Real-time quantitative RT-PCR revealed a higher expression of IL1B mRNA in reflected amnion than in placental amnion in TNL cases but not in TIL cases. Extended screening using microarrays showed differential expression of 17 genes in labor, regardless of the region. Interestingly, 839 genes were differentially expressed between placental amnion and reflected amnion. Pathway analysis identified 19 signaling pathways, such as mitogen-activated protein kinase and transforming growth factor beta pathways, associated with region. Lipopolysaccharide (LPS) treatment of the amnion explants showed more robust activation of mitogen-activated protein kinase 3/1 (extracellular signal-regulated kinase 1/2) in placental amnion of TNL but not in TIL cases. Placental amnion from TNL and TIL cases showed a significant difference in the amplitude of IL1B mRNA induction by LPS. We report that the anatomical region has a substantial impact on the transcriptional program and the biological properties of the amnion. Labor-associated switching to a proinflammatory signature is a feature particular to placental amnion. The novel observations herein strongly suggest that the seemingly homogeneous amnion is biologically heterogeneous and compartmentalized, with implications for the physiology of pregnancy and parturition.

Amnion Degeneration over Fetal Placental Surface Vessels Possibly Resulting from Focal Hypoxia: A Case Report

The monoamnionic placenta of this twin gestation had focal amnion necrosis, but this was present only over the fetal surface vessels of one twin; this twin also developed cerebral atrophy. We hypothesize that this degeneration is due to a more severely reduced oxygen tension in its vessels. The placental amnion epithelium may undergo several degenerative processes, including amnion nodosum and changes due to meconium staining. Sonography had disclosed what appeared to be a dividing membrane, but this was not found at birth when monoamnionic twins with entangled cords presented. The amnion degeneration was present only over the large surface fetal vessels of the placenta of that twin who also developed central nervous system degeneration, and macrophage infiltration was confined to the same lesions. Focal hypoxia from entangling cords may have caused this defect.

Bullous pemphigoid antigen II (BP180) and its soluble extracellular domains are major autoantigens in mucous membrane pemphigoid: the pathogenic relevance to HLA class II alleles and disease severity

Mucous membrane pemphigoid (MMP), a chronic autoimmune subepithelial blistering disease, is associated with circulating IgG and/or IgA autoantibodies against several basement membrane zone antigens. The heterogeneity of clinical presentation and diversity of target autoantigens have contributed to difficulties in characterizing this condition immunologically. To analyse serum autoantibody profile and HLA class II alleles in MMP patients and to correlate this with the clinical presentation of disease. Well-defined subgroups consisting of 124 patients with MMP were examined for IgG and IgA reactivity with immunoblotting using human epidermal, dermal and placental amnion proteins. The results were further analysed on the basis of detailed clinical (sites of involvement and disease severity) and immunopathological criteria (immunofluorescence study and HLA class II alleles). Immunoblot assay revealed that the majority of MMP patients had IgG (93 of 124, 75%) and/or IgA autoantibodies (63 of 124, 51%) to BP180 (including its soluble ectodomains, 120-kDa LAD-1 and 97-kDa LABD97 antigens). Other antigens targeted predominantly by IgG autoantibodies included: BP230 in 34 (27%), beta4 integrin in 26 (21%), and laminin 5 in three (2%). All the BP230+ sera and 23 (88%) beta4 integrin+ sera also reacted with at least one of the BP180 antigens. Over 85% of patients with reactivity to beta4 integrin had ocular involvement. In most cases of MMP, more severe clinical features were associated with antibody reactivity to multiple basement membrane zone antigens, as well as reactivity to multiple BP180 component antigens. Dual BP180/LAD-1 reactivity with IgG and IgA was associated with a more severe phenotype. In addition, the subset-dependent autoantibody reactivity correlated well with specific HLA class II alleles, DQB1*0301, DRB1*04 and DRB1*11. Our results confirmed that BP180 is a major autoantigen targeted by the sera of patients with MMP. The disease-prevalent HLA class II alleles and humoral autoimmune response against the particular subsets of antigenic epitope(s) within BP180 ectodomain may contribute to the clinicopathological significance and disease severity of MMP.

Human placental amnion is a novel substrate for detecting autoantibodies in autoimmune bullous diseases by immunoblotting.

Identification of antigens by immunoblotting techniques, using epidermal and dermal extracts, is regarded as essential for making a definitive diagnosis in autoimmune bullous diseases (AIBDs). These procedures involve epidermal-dermal separation for subsequent protein extraction, which may result in partial loss of some antigenic polypeptides and changes in the conformational epitopes targeted by autoantibodies in AIBDs. It may therefore be necessary to use different substrates for consistent results. Objectives To evaluate the usefulness of human placental amnion extract as a substrate for immunoblotting in the diagnosis of AIBDs. We checked the structural components of the desmosomes and basement membrane zone (BMZ) of amnion by electron microscopy. Using immunofluorescence and immunoblotting techniques, we tested the amnion immunoreactivity with antibodies to desmosomal and BMZ proteins, and with sera from 76 patients with AIBDs including pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid (BP), pemphigoid gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, paraneoplastic pemphigus and mucous membrane pemphigoid. The desmosomes and BMZ of the amnion tissue were ultrastructurally similar to those in skin. Antigen mapping confirmed that amnion contains all the proteins that were recognized by a panel of monoclonal and polyclonal antibodies. Immunoblotting showed that the antibodies clearly detected bands corresponding to desmogleins 1 and 3, desmocollins 1 and 2, desmoplakins 1 and 2, three subunits (alpha3, beta3 and gamma2) of laminin 5, BP antigens 1 and 2, the 97-kDa LAD antigen and type VII collagen. In addition, most of the patient sera (82%) reacted exclusively with their respective antigens. Harvesting proteins from amnion does not require epidermal-dermal separation, and a sufficient yield of desmosomal and hemidesmosomal proteins can be obtained. Therefore, amnion may be a more reliable source of substrate than skin samples for immunoblot analysis of AIBDs.

Fetal membrane distention: Determination of the intrauterine surface area and distention of the fetal membranes preterm and at term

Objective: This study was undertaken to obtain an accurate measurement of the intrauterine surface area and the degree of distention of the apposed fetal membranes preterm and at term. Study Design: Serial longitudinal images of the uterus in 23 women between 25 and 41 weeks’ gestation were obtained by ultrasonography. A Mathematica (Wolfram Research, Inc, Champaign, Ill) program assembled a 3-dimensional image and calculated the intrauterine surface area for each patient. The surface areas of the placental amnion and membranes were measured in vitro after delivery. From these measurements the degree of distention of each fetal membrane in vivo was calculated. Results: The mean calculated intrauterine surface areas were as follows: 1037 ± 70 cm2 (25-29 weeks’ gestation, n = 4), 1376 ± 121 cm2 (30-34 weeks’ gestation, n = 4), and 1876 ± 307 cm2 (37-41 weeks’ gestation, n = 15, P = .0021 by Wilcoxon rank sum test). The surface areas of the expelled membranes at 25 to 29, 30 to 34, and 37 to 41 weeks’ gestation were 737 ± 61 cm2, 855 ± 77 cm2, and 1115 ± 149 cm2, respectively. The ratios of intrauterine surface area to the area of the expelled membrane and hence a measure of the degree of distention in vivo were 1.4 ± 0.05 at 25 to 29 weeks’ gestation (n = 4), 1.6 ± 0.2 at 30 to 34 weeks’ gestation (n = 4), and 1.7 ± 0.3 at term (n = 15). Conclusion: The intrauterine surface area in vivo increases during gestation. The surface area of the fetal membranes as measured in vitro increases to a lesser extent. The fetal membranes are therefore distended in vivo. (Am J Obstet Gynecol 2000;182:128-34.)

The anchoring zone in the human placental amnion: bunches of oxytalan and collagen connect mesoderm and epithelium.

This study deals with the examination of the elastic fibre system as well as collagen fibrils and collagen type IV in the amnion of the human chorionic plate of uncomplicated pregnancies at term. In organs other than placenta, the elastic fibre system comprises elastic fibres, elaunin and oxytalan microfibrils. The investigation was performed by light and electron microscopy and immunocytochemistry. Abundant oxytalan fibres were present in all amnionic layers, while no elastic fibres were found. Oxytalan microfibrils formed a broad sub-epithelial layer and were intermingled with collagen fibrils in the subjacent compact layer and in the amnionic mesoderm. Light microscopically, bunches containing orcein-stained oxytalan and collagen-type-IV-immuno-stained microfibrils were seen rising from the amnionic mesoderm perpendicularly towards the epithelial layer, where they obviously inserted. It can be assumed that the subepithelial microfibrillar layer and the following compact layer form an anchoring zone between the amnionic mesoderm and the epithelium that may contribute to the maintenance of strength. The ultrastructure of the bunches clearly showed collagen fibrils mixed with oxytalan microfibrils. No collagen type I-immunostaining was found in the bunches. After pretreatment of cryostat sections with elastase, oxytalan-orcein-staining was absent, but collagen type IV-immunoreactivity was not altered. Furthermore, after oxytalan-orcein-staining resp. anti-collagen type IV incubation, all positive fibres revealed an identical morphological pattern. We propose that oxytalan and collagen type IV may represent further members of the microfibril complex.

Multiple-pulse-mediated electrofusion of intact erythrocyte onto human term placental amnion

The creation of surface modified human term placental amnion by electrofusing human cells onto its surface has been thought of. A multiple-pulse electrofusion protocol with 10 square pulses of 10-μs pulse length, and electric field of 0.2 kV cm −1, can make erythrocyte–amnion tissue electrofusion possible. The protocol devised merge the cell-tissue-adherence steps with fusogenic pulse. The finding opens up a new avenue of cell electrofusion onto human tissue with minimal procedural complexities.

Thromboxane Modulates Endothelial Permeability

The study tests the role of thromboxane in modulating microvascular permeability in vitro. Cultured monolayers of bovine aortic endothelial cells were challenged with the thromboxane (Tx) mimic U46619. This led to disassembly of actin microfilaments, cell rounding, border retraction and interendotheHal gap formation. Pretreatment with the Tx receptor antagonist SQ 29,548 prevented the Tx mimic-induced cytoskeletal changes. The Tx mimic also altered endothelial cell barrier function. Increased permeability was indicated by the increased passage of labelled albumin across monolayers cultured on microcarriers, relative to untreated endothelial cells (p < 0.05). Furthermore, electron microscopy of endothelial cells cultured on the basement membrane of human placental amnion indicated increased permeability based on wide, interendotheHal gap formation and transit of the tracer horseradish peroxidase. Quantification of interendothelial gaps revealed an eleven-fold increase with the Tx mimic relative to untreated endothial cells (p < 0.05) and prevention by pretreatment with the Tx receptor antagonist (p < 0.05). These data indicate that Tx directly modulates the permeability of endothelial cell in vitro.

Parathyroid hormone-related protein mRNA in avascular human amnion.

Previously, we found that preproendothelin-1 mRNA is present in human avascular amnion tissue. In this investigation, we evaluated the possibility that another vasoactive protein, namely, parathyroid hormone-related protein (PTH-rP), also is produced in amnion. Using a specific cDNA probe, we identified PTH-rP mRNA in amnion tissue and found that the level of PTH-rP mRNA in placental amnion was greater than that in reflected amnion tissue obtained from the same pregnancy. PTH-rP mRNA also was demonstrable in chorion laeve and in decidua, but the levels of this mRNA in these tissues were much lower than that in amnion. By radioimmunoassay, we found that human amnion cells in primary monolayer culture secrete immunoreactive PTH-rP into the medium. Importantly, the placental amnion covers the chorionic vessels that traverse over the chorionic plate prior to branching into the cotelydons; specifically, there is no intervening tissue between placental amnion and the adventitial tissue of the chorionic vessel wall. Thus, the potential exists for the production of endothelin-1, a potent vasocontractant, and PTH-rP, a vasorelaxant, in placental amnion for export to the adventitial surface of the chorionic vessels. Moreover, amnion cells respond to a number of agents (e.g., transforming growth factor-beta) that effect changes in the expression of these two peptides. Therefore, the possibility should be considered that amnion-derived vasoactive proteins may modulate fetal chorionic, umbilical, and villous vessel tone and thereby fetal-placental blood flow.

Distribution of CA 125 in placental tissues.

The presence of the tumor marker CA 125 was studied in different compartments of the human placenta. Levels of CA 125 in the cytosol of chorionic villi ranged from 27-17100 U/g (median 560 U/g). In the placental amnion and chorion concentrations ranged from 175-29000 U/g, median 1060 U/g and were not statistically different. In the umbilical cord values were significantly lower (range 44-7600 U/g; median 180 U/g). Maternal serum probes were above the upper limit of normal in all cases (range 48-500 U/ml; median 131 U/ml). Immunohistochemistry detected CA 125 exclusively within the amniotic cells of the placenta and the umbilical cord. This might be because CA 125 fixes more to insoluble structures in the amnion or because of contamination of chorionic villi with the underlying decidua.

37] Cultures as epithelial models: Porous-bottom culture dishes for studying transport and differentiation

In this chapter, methods are described for fabricating porous-bottom culture dishes with porous bottoms composed of cellulose ester filters (Millipore), polycarbonate filters (Nucleopore), collagen membranes, and a similar arrangement in which the porous growth surface is a placental amnion that has been denuded of its epithelial cells. In all of these culture dishes, the medium above the cells is separate from the medium below the porous membrane, facilitating transport measurements. They can be manipulated easily under sterile conditions in a laminar flow hood so that short-circuit current, potential difference, and transepithelial resistance as well as other noninvasive measurements can be performed and the culture returned to the incubator so that the same culture can be studied many times. It is suggested that filter-bottom cups are easiest to fabricate on a large scale and have been used for many studies of epithelial development, s transport, and for cell fractionation to prepare vesicles for flux studies.

Labor in humans: 1. Progesterone, 20 alpha-dihydro-progesterone, estrone and 17 beta-estradiol in near placental and most distant human amnion and chorion laeve in various stages of labor at term.

In some species the onset of labor is regulated by changes in the estrogen/progesterone ratio. The same change in fetal membranes has been suggested to be one of the triggering mechanisms of labor in humans. In order to examine if a gradient in steroid concentration existed in fetal membranes and if changes in concentrations could be observed with the onset and advancing labor, the concentration of progesterone (P), 20 alpha-dihydro-progesterone (20 alpha-OHP), estrone (E1) and 17 beta-estradiol (E2) were determined by specific radioimmunoassay in the near placental and most distant regions of human amnion and chorion laeve obtained at term, before, at the onset and in advanced labor. Both estrogen and progestin concentrations in the chorion were higher than in the amnion. No gradient in the concentration of steroids was found. No statistically significant differences in estrogen and progestin levels were associated with the onset of labor.

Intercellular junctions in the human fetal membranes. A freeze-fracture study.

Freeze-fracture replicas of the human reflected and placental amnion and chorion laeve at term were studied in order to give a systematic survey of the nature and extension of the intercellular junctions in the fetal membranes. No differences could be detected between the reflected and placental amniotic epithelium. In both the replicas never displayed plasma membrane differentiations typical of occluding junctions, while communicating junctions were occasionally and desmosomes frequently seen. In the chorionic trophoblast maculae occludentes, communicating junctions and desmosomes were regularly encountered. It is assumed that the maculae occludentes are remnants of occluding junctions which early in gestation possibly seal off the chorionic cavity; it appears improbable that they contribute significantly to the permeability properties of the chorionic trophoblast, since it is known from previous ultrastructural studies that large open intercellular channels cross the chorionic trophoblast. Thus the absence of occluding junctions, which could act as effective permeability barriers, in both epithelial components of the fetal membranes suggests that the factors able to influence the amniotic fluid turnover or the paraplacental protein exchange are the geometrical relationships and physico-chemical properties of the intercellular channels in the amniotic epithelium and chorionic trophoblast. In addition, communicating junctions were present between fibroblasts in the chorion laeve but not in the amnion, possibly indicating differences in the functional state of these cells and/or their extracellular microenvironments.