Type VII collagen associated with the basement membrane of amniotic epithelium forms giant anchoring rivets which penetrate a massive lamina reticularis.

In human amnion a simple cuboidal epithelium and underlying fibroblast layer are separated by an almost acellular compact layer rich in collagen types I and III. This (>10 µm) layer, which may be a thick lamina reticularis, apparently presents an unusual set of conditions. Integration of the multilaminous tissue across it is apparently achieved by waisted structures which we have observed with the light microscope in frozen, paraffin-wax and semi-thin resin sections. We have also captured transmission and scanning electron micrographs of the structures. These structures which cross the compact layer we call "rivets". The composition of these "rivets" has been examined immunocytochemically and in three dimensions using the confocal laser scanning epi-fluorescence microscope. The rivets contain type VII collagen and an α6 integrin. They associate with type IV collagen containing structures (basement membrane lamina densa and spongy coils) and a special population of fibroblasts which may generate, maintain or anchor rivets to the underlying mesenchymal layer. Although type VII collagen is well known to anchor basal lamina to underlying mesodermal collagen fibres these "rivets" are an order of magnitude larger than any previously described type VII collagen containing anchoring structures. Intriguing possible functions of these features include nodes for growth of fibrous collagen sheets and sites of possible enzymatic degradation during regulated amnion weakening approaching term. If these sites are confirmed to be involved in amnion degradation and growth they may represent important targets for therapeutic agents that are designed to delay preterm premature rupture of the membranes a major cause of fetal morbidity and mortality.

Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors Type 1 and 2 in human and rhesus monkey fetal membranes.

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-1 and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA was also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour.

Confocal and conventional immunofluorescence and ultrastructural localisation of intracellular strength-giving components of human amniochorion.

Key cytoskeletal polypeptides of human fetal membranes have been localised at subcellular level using confocal and conventional indirect immunofluorescence microscopy. Correlation with electron microscope data has allowed us to examine how cellular compartments of this multilaminar tissue maintain their mechanical integrity until the time of membrane rupture at parturition. Evidence is presented for myofibroblastic characteristics of cells in both the fibroblast and reticular layers which may therefore have tension-generating, position-adjustment and wound-healing roles in the amniochorion. Desmin and vimentin are coexpressed in these cells, but a small localised population of cells in the fibroblast layer contains vimentin alone. An interaction of cytokeratin filaments with nuclei and desmosomes of amniotic epithelium in vivo is demonstrated, indicating that nuclei of cells of ectodermal origin are integrated into a mechanical structure extending throughout the tissue as a whole. Cells of the basal 1 or 2 layers of trophoblast have been shown to have a more extensive and better integrated cytoskeletal organisation than those overlying and forming the boundary with decidua. Structures within the trophoblast, identified previously as degenerate villi, contain cells with intermediate filaments with similar immunofluorescence properties to those of the neighbouring reticular layer and thus may represent papillae that prevent shearing at this interface.

Micro-trabeculae, macro-plaques or mini-basement membranes in human term fetal membranes?

Immunocytochemical confocal laser scanning microscopy and ultrastructural analysis, including immunoelectron microscopy, reveals the distribution of structures in human term amniochorion similar in some respects to basement membranes but with unusually restricted dimensions. On the basis of their immunoreactivity, these trabecular structures, found on the fibroblast layer side of the spongy layer of human term amniochorion and adjacent reticular layer, have been shown to contain type IV collagen, laminin, and nidogen. The origin of these components may be from primitive epithelial structures which pumped fluid into the lakes that eventually coalesced to form the extraembryonic coelom separating the extraembryonic somatic mesoderm from the extraembryonic splanchnic mesoderm. Such a theory of their origin might link them with the mysterious 'cellular layer', a single-cell-thick layer of cells which is usually no longer present in fetal membranes at term. The similarity in composition but not in size of these structures to anchoring plaques for type VII collagen is possible support for the view that these structures are integrators of extracellular matrix polymeric proteins. The 'pseudobasement' membrane associated with the trophoblast layer, on investigation, appears to be typical by six criteria. 'Coiled' fibrous structures in the extracellular matrix of the spongy layer may aid adjustments under tension at this shear surface by a detachable 'Velcro' or 'two spring' fastening system. The coils are rich in fibronectin. The suggestion is made that the compact layer is a giant lamina reticularis associated with the amniotic epithelial basement membrane.

Confocal and conventional immunofluorescent and immunogold electron microscopic localization of collagen types III and IV in human placenta.

Confocal and conventional indirect immunofluorescence and immunogold electron microscopic methods were applied to examine the distribution of extracellular matrix constituents (collagens types III and IV) in the villi of immature and term human placentae. The immunofluorescence study revealed that collagen type III is more distinct in the villous stroma of term placenta as compared with that of the first trimester. Collagen type IV was detected mainly in endothelial and epithelial basement membranes and interestingly also to a certain extent in the stroma. Results obtained using immunoelectron microscopy support the proposal that collagen types III and IV are characteristic of stromal and basement membranes, respectively. Stromal collagen type IV is apparently localized in association with the interstitial types of collagen (I and III), in the villous stroma of term placenta.