Capillary volume fraction is the principal determinant of villous membrane thickness in the normal human placenta at term.

The thickness of the villous membrane is known to be an important factor in determining the morphometric diffusing capacity of the placenta at term. As yet it is not certain how areas of the villous membrane specialised for gaseous exchange, the vasculo-syncytial membranes, differentiate. One mechanism suggested is that they arise through obtrusion of the fetal capillaries contained within the stromal core. As a result the bulk of the overlying trophoblast is displaced laterally, reducing the thickness of the villous membrane to as little as 1-2 microns at these points. To test this hypothesis the relationship between the vascularity of the villi, as determined by the villous capillary volume fraction, and the mean thickness of the villous membrane was investigated. Data were taken from a recent study in which placental villi were biopsied from normal term placentae within 1 min of caesarean delivery and at 5, 10, 15 and 20 min thereafter. Since intuitively the membrane has both a maximum and a minimum thickness a sigmoid relationship was fitted to the data using least squares regression analysis. Estimates of arithmetic and harmonic mean thicknesses were then predicted from the capillary volume fraction for a large number of placentae using data from previous studies. These all employed similar stereological techniques but were performed over a number of years by several workers in this laboratory. The predicted values were tested against the measured values using paired 't' tests, but no significant differences (P greater than 0.05) were detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of laminin and fibronectin isoforms in human placental villi.

We studied the localization of laminin alpha1, alpha2, alpha3, alpha5, beta1, beta2, and gamma1 chains and extradomain A- (EDA), EDB-, and oncofetal fibronectin by immunohistochemistry in human placental villi during placental development. The laminin alpha2, alpha5, beta1, beta2, and gamma1 chains were detected in the trophoblastic basement membrane (BM) at all stages of gestation, suggesting the presence of laminin-2, -4, -10, and -11 trimers. The laminin alpha1 chain was selectively found at sites where the villous BM was in contact with proliferating cells in trophoblastic islands or columns. EDA-Fn, but not other Fn isoforms, was found in the trophoblastic BM during the first trimester. The laminin alpha2, beta1, beta2, and gamma1 chains were detected in the villous stroma and capillaries throughout placental development, while the laminin alpha5 chain emerged distinctly during development. Extensive EDA-Fn immunoreactivity was found in first-trimester villous stroma, but distinctly fewer Fn isoforms were seen in the villous stroma during the later stages of gestation. Our results also suggest that, during the formation of new villi, laminins are not found in trophoblastic sprouts before the ingrowth of the villous mesenchyme. Rather, laminins may be deposited at the villous epithelial-mesenchymal interface. Furthermore, the results show that distinct changes occur in the localization of various laminin and Fn isoforms during the maturation of villous trophoblastic and capillary BMs.     

Ultrastructural observations on the maturation of the placental labyrinth of the golden hamster (days 10 to 16 of gestation).

Morphogenesis of the labyrinthine part of the chorioallantoic placenta of the golden hamster between day 10 of gestation and term (day 16) was studied by light and electron microscopy. During this period the labyrinth increases greatly in both size and complexity. Trabeculae of the labyrinth, thin partitions composed of trophoblastic tissue and fetal capillaries which delimit the maternal blood spaces, apparently proliferate both by appositional and interstitial growth. From the time of its formation (day 9 of gestation) until term the labyrinth is hemotrichorial in organization (i.e. three layers of trophoblast separate maternal blood from fetal capillaries). Both the inner and intermediate layers of trophoblast (layers III and II, respectively) are syncytial. The outer trophoblastic layer (III), which is in direct contact with maternal blood, remains cellular, although many of its component cells grow to giant cell dimensions ("labyrinthine giant cells"). Between the tenth and fourteenth days of gestation the anatomical barrier to diffusion between maternal and fetal blood is progressively reduced. This is accomplished both by gradual attenuation of the trophoblastic layers and fetal capillary endothelium and by the formation of discontinuities (gaps) in layer I, and diaphragmed fenestrae in fetal capillary endothelium. The labyrinthine placental barrier is fully developed and probably attains maximal functional efficiency by the fourteenth day of gestation. Late in the fifteenth day of gestation, a few hours before parturition, distinct degenerative changes are apparent in the trophoblastic layers and fetal capillaries of the trabeculae. The factors responsible for initiation these degenerative changes and the onset of parturition are still controversial.     

Immunofluorescence study of the extracellular matrix of the human placenta

Distribution of collagen types I, III, IV, V and fibronectin in human placental villi has been studied by indirect immunofluorescence. During 9-12 weeks of pregnancy the extracellular matrix of villi represents a network of filaments organized in bundles and aggregates that contain collagen types I and III and finer filaments of collagen types IV and V. Collagen type IV is regularly detected in basal membrane of capillaries and particularly in villous epithelium, collagen type V and fibronectin are occasionally detected there. Marked immunofluorescent reaction on collagen types IV and V and fibronectin, and weak reaction on collagen type III is observed in cellular islets around cytotrophoblasts. In the fetus born in term placental villi have uniform immunofluorescence in thick basal membranes of fetal capillaries and of chorionic epithelium. The immunofluorescent reaction specific for all collagen types is uniform in villous stroma. Distribution of different collagen types and fibronectin, including the unusual localization of membrane collagen type IV, in villous stroma and cellular islets of early and mature placenta is discussed.     

Chorion laeve trophoblasts of preeclamptic fetal membranes: histochemically detectable enzyme activities do not change at a subcellular level.

We examined the subcellular localization of ADP-degrading activity and cytochrome c oxidase (CCO) activity in chorion laeve trophoblasts from term and near term human fetal membranes, and compared them with those from severe preeclamptic fetal membranes. The methods used for the detection of enzyme activities were the lead nitrate method for ADP-degrading activity and the diaminobenzidine method for CCO. Precipitates indicative of ADP-degrading activity were visible on surface microvillous plasma membranes of chorion laeve trophoblasts both from normal and preeclamptic fetal membranes. The intensity and distribution patterns were the same in the normal and preeclamptic subjects. CCO labeling was visible in almost all laeve trophoblastic mitochondria both in normal and preeclamptic cases. Previously, we demonstrated that in preeclamptic villous trophoblasts there were decreases in ADP-degrading activity and the presence of CCO-negative mitochondria, which were proposed to lead to dysfunction of each villous trophoblast, and finally to placental insufficiency in preeclampsia. Reductions or changes in enzyme intensities/distribution patterns, which are characteristic features of preeclamptic villous trophoblasts, were absent in chorion laeve trophoblasts in preeclampsia. These results suggest that in preeclampsia there are no, or at least less severe, abnormalities in the enzyme activities of chorion laeve trophoblasts, compared with villous trophoblasts, as far as enzyme-histochemically detectable enzymes are concerned.     

Morphology of the mitochondria and endoplasmic reticula of chorion laeve cytotrophoblasts: their resemblance to villous syncytiotrophoblasts rather than villous cytotrophoblasts

We examined the morphological features of the mitochondria and endoplasmic reticula of chorion laeve cytotrophoblasts from term human fetal membranes, and compared them with those of syncytiotrophoblasts and cytotrophoblasts from human placental villi. Ultrastructural enzyme histochemistry of cytochrome c oxidase and glucose-6-phosphatase were used as cytochemical markers for these intracellular organelles. Chorion laeve cytotrophoblasts possessed abundant endoplasmic reticula, and small mitochondria with a few cristae, which were characteristic of villous syncytiotrophoblasts rather than villous cytotrophoblasts. As for these organellar structures, statistical analysis confirmed similarities between chorion laeve cytotrophoblasts and villous syncytiotrophoblasts, but significant differences between laeve cytotrophoblasts and villous cytotrophoblasts. Though these two cytotrophoblasts originated from one common cell in early placental development, they exhibited quite different organellar morphology during placental/chorioamniotic differentiation. Considering previous data, we concluded that chorion laeve cytotrophoblasts were metabolically active cells, similar to villous syncytiotrophoblasts, performing many functions in fetal membrane physiology.     

Quantitative characteristics of components of the placental barrier of women with uncomplicated full-term pregnancies]

Placentas from four healthy women have been studied electron microscopically after urgent spontaneous deliveries. In every placenta 130-200 resorptive villi of the chorion were photographed. The results on morphometric analysis of 1,205 electronograms of the placental barrier are presented. At full-term uncomplicated pregnancies all components of the placental barrier (syncytium, cytotrophoblast, basal membrane of the trophoblast, stromal connective-tissue layer, basal membrane and endothelium of fetal capillaries) have been stated to demonstrate rather stable quantitative characteristics (see Table in the text). The quantitative data obtained can serve for electron microscopic investigation of placenta at different pathologic states of the mother and the fetus, make it possible to differentiate more precisely physiological and pathological changes in placental ultrastructure.     

Comparative distribution of four lysosomal enzymes within various structures of the human placenta

The histochemical activity of 4 lysosomal enzymes, acid phosphatase (AP), nonspecific esterase (NE), aryl sulphatase (AS), and beta-glucuronidase (BG), was compared in following structures of the human placenta: syncytiotrophoblast, villous stromal cells, fetal capillaries and larger blood vessels, cells of basal plate, macrophages, and Hofbauer cells. In spite of a general similarity in distribution of the investigated enzymes, differences concerning particular structures were found. Thus positively stained granules in endothelia of capillary vessels were revealed only in the reaction for BG, although contours of capillaries were also outlined by the diffuse reaction product for AS. The muscular layer of larger vessels reacted strongly for AS and weakly for NE with the remaining reactions being negative. In syncytiotrophoblast, BG appeared much less active than the other 3 enzymes. The possible significance of the BG positive granules in endothelial of capillaries and of the occasional divergence in distribution of the classical lysosomal markers (AP and BG) is discussed.     

Syncytiotrophoblast is a barrier to maternal-fetal transmission of herpes simplex virus

Herpes simplex virus (HSV)-1 has been discovered in placental tissue from spontaneous miscarriages, but reports of transplacental transmission and fetal infection are extremely rare. Previously, we demonstrated that the villous syncytiotrophoblast, which forms a continuous layer between the maternal and fetal circulation, is resistant to HSV entry. Here, we tested our hypothesis that the villous syncytiotrophoblast prevents transplacental transmission of HSV secondary to decreased expression of HSV entry mediators (HveA, HveB, and HveC). In addition, we investigated the ability of HSV to infect extravillous trophoblast cells, which mediate placental attachment to the uterine wall, and the expression of HSV receptors in these cells. We performed fluorescence-activated cell sorting (FACS) analyses and immunostaining to demonstrate that HveA, HveB, and HveC were not expressed in third-trimester villous trophoblast cells. Consequently, villous explants obtained from third-trimester placentas were resistant to infection by a recombinant HSV-1 vector, HSV-1 KOS, but approximately 20% of mesenchymal cells within the villous core were infected when villous explants were pretreated with trypsin to disrupt the villous trophoblast layer. Conversely, FACS analysis and immunostaining demonstrated that extravillous trophoblast cells expressed HveA, HveB, and HveC, and these cells were efficiently infected by HSV vectors. Infection of extravillous trophoblast cells by HSV-1 was not reduced when the cells were pretreated with an antibody against HveA but was partially reduced when the cells were pretreated with antibodies directed against HveB and HveC. Thus, the decreased expression of herpesvirus entry mediators in villous syncytiotrophoblast prevents placental villous infection, thereby limiting maternal-fetal transmission of HSV.     

Morphogenesis of the fetal membranes of an American mole, Scalopus aquaticus.

Scalopus membranes are characterized by: Superficial nidation; antimesometrial orientation of the embryonic disc; amniogenesis by folding; an extensive but transitory choriovitelline placenta; a large yolk sac with late and incomplete inversion; large persistent allantoic vesicle; a very broad, thin, villous, epitheliochorial chorioallantoic placenta of annular shape interrupted mesometrially, dotted with numerous areolae, and bordered by a nonvillous sparsely vascular chorioallantoic membrane connected with the persistent bilaminar omphalopleure by a very narrow rim of chorion. There is no decidua. Electron microscopy shows that at 8 mm, CR, (limb bud embryo) the uterine epithelium of the interhemal membrane may be 0.5 micron or less in thickness, but that it shows no signs of degeneration. Trophoblastic microvilli often penetrate the epithelium to within 0.2 micron of its base. At this time there is active secretion by the uterine glands, and cellular hypertrophy and cytolysis of the epithelium at the gland mouths, with active phagocytosis by the areolar cytotrophoblast. The occurrence of absorptive areolae in an insectivore emphasizes the probable primitiveness of this widely distributed placental mechanism. In spite of similarities of the yolk sac to that of rabbits and rodents, the bilaminar omphalopleure produces no invasive trophoblastic giant cells. The definitive membranes of Parascalops breweri and Scapanus latimanus are like those of Scalopus. The placentae of Talpa europaea, Condylura cristata, and Neurotrichus gibbsii are discoid and relatively much smaller, thicker and more complex in internal structure. There is some reason to believe that the fetal membrane systems of moles and shrews (Soricoidea) are more like those of the ancestral mammalian stock than are those of any other recent eutherians.     

Ultrastructural differentiation of stromal and vascular components in early macaque placental villi

In this study, closely staged placental villi from rhesus monkeys between 19 and 60 days of gestation were used to examine 1) the origin of endothelial cells and the mechanism of angiogenesis in villi; 2) the origin of placental macrophages (Hofbauer cells), and 3) the origin of the reticular cells that compartmentalize the stroma. The results did not support the concept that early stromal cells in the villi were derived by in situ delamination from cytotrophoblast. The extraembryonic mesodermal (mesenchymal) cells at the earliest of ages examined contained considerable granular ER. These cells organized into groups and formed primitive intercellular junctions, thus giving rise to the early angioblastic masses. The angioblastic masses were cellular, not syncytial; and lumen formation was not the result of intracellular vacuolization, but rather was the result of the acquisition of junctionally defined spaces. The earliest capillaries lacked intravascular blood cells and a basal lamina. Later, blood cells were evident in the lumina. At about 45-50 days of gestation, fetal capillaries began to indent the basal surface of the trophoblast. The basal lamina of the fetal capillaries still had not developed by 60 days of gestation. Between 35 and 40 days of gestation, significant morphological changes took place in the villous stroma. Evidence was obtained that the mesenchymal cells differentiated into the reticular cells that subdivided the stroma into fibril-rich and fibril-free compartments. At the same time, Hofbauer cells were observed for the first time; they occupied the fibril-free regions of the stroma. We did not observe any clear indications of intermediate stages of differentiation between other stromal cell types and Hofbauer cells. It is suggested that placental macrophages may have an origin independent of other stromal types; one possibility is that they are derived from blood monocytes as in other tissues. It is further suggested that the activities of the macrophages and reticular cells may be important in remodeling the extracellular matrix and may be related to the process of branching morphogenesis in the villi.     

Placentation in cloned cattle: structure and microvascular architecture

To elucidate the morphological differences between placentas from normal and cloned cattle pregnancies reaching term, the umbilical cord, placentomes and interplacentomal region of the fetal membranes were examined macroscopically as well as by light and scanning electron microscopy. In pregnancies established by somatic nucleus transfer (NT), the umbilical cord and fetal membranes were edematous. Placentomal fusion was common, resulting in increased size and a decreased number of placentomes. Extensive areas of the chorioallantoic membrane were devoid of placentomes. An increased number of functional or accessory microcotyledons (<1 cm) were present at the maternally oriented surface of fetal membranes. Extensive areas of extravasated maternal blood were present within the placentomes and in the interplacentomal region. The crypts on the caruncular surface were dilated and accommodated complexes of more than one primary villus, as opposed to a single villus in non-cloned placentae. Scanning electron microscopy of blood vessel casts revealed that there was also more than one stem artery per villous tree and that the ramification of the vessels failed to form dense complexes of capillary loops and sinusoidal dilations as in normal pregnancies. At the materno-fetal interface, however, the trophoblast and uterine epithelium had normal histology. In conclusion, the NT placentas had a range of pathomorphological changes; this was likely associated with the poor clinical outcome of NT pregnancies.     

Evolutionary transformations of chorioallantoic placental characters in rodentia with special reference to hystricognath species

The aim of this paper is to reconstruct the evolution of chorioallantoic placental characters in Rodentia. The analysis is based on pre-existing hypotheses of rodent relationships and the tracing of character evolution. Data on 64 rodent species of 49 genera are derived from the literature. New results refer to the hystricognath species Petromus typicus A. Smith, 1831 and Octodon degus (Molina, 1782). This comprehensive analysis confirms that the stem species pattern of Rodentia is characterised by a haemochorial placenta which is divided horizontally. Inside the placental labyrinth, fetal vessels and their trophoblastic external border build up a network through which the maternal blood flows. The trophoblastic tissue is one-layered, syncytial and possess a considerable surface extension. Within Rodentia, evolutionary transformations occurred on the macroscopic as well as the fine structural level. The results suggest that the stem species of Hystricognathi underwent transformations only on the macroscopic level, i.e., forming a ring-shaped arrangement of placental regions with centrally situated maternal arteries and the acquisition of a subplacenta. By contrast, in Muridae the chorioallantoic placenta shows derived features only in regard to the fine structure of the labyrinth, i.e. the interhaemal membrane is modified in composition, and the fetal capillary endothelium is fenestrated. Geomyoidea underwent transformations on both levels. Macroscopically, their placenta is modified into a hillock shape. Microscopically, the interhaemal membrane is formed by the cytotrophoblast. In addition to the mentioned transformations, some aspects of other fetal membrane differentiation in Rodentia are briefly discussed.     

Ultrastructural development of chorioallantoic placenta in the Indian Miniopterus bat, Miniopterus schreibersii fuliginosus (Hodgson).

The chorioallantoic placental interhemal membrane of Miniopterus schreibersii fuliginosus has been described electron-microscopically. Morphologically there are three main types of placentae which develop in chronological sequence. They are (1) primary placenta, (2) secondary placenta and (3) tertiary placenta. In neural groove and limb-bud embryos the primary placenta consists of the following elements which separate the maternal and fetal circulations: (1) a continuous ectoplasmic layer, (2) intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The primary placenta degenerates until term when it consists of a thin syncytiotrophoblastic layer resting on basal lamina. Mesenchyme does not show the presence of fetal capillaries. The secondary placenta is formed in early limb-bud embryos. The electron microscope has revealed that the placenta is of the endotheliomonochorial type and (1) consists of a well-developed maternal endothelium, (2) the trophoblast surrounding the maternal blood tubule is cellular, not syncytial as previously thought and the apical plasma membrane of these trophoblastic cells is in direct contact with the discontinuous interstitial membrane, (3) basal lamina, (4) mesenchyme and (5) fetal endothelium. Tertiary placenta at full term stage is of the hemodichorial type having the following elements: (1) thin ectoplasmic layer, (2) a thick intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The definitive chorioallantoic placental barrier in this bat thus differs from the organization earlier proposed by Chari and Gopalakrishna [Proc. Indian Acad. Sci. 93: 463-483, 1984] on the basis of light-microscopic observations: (1) the absence of maternal endothelium in the primary placenta from the neural groove and early limb-bud embryos, (2) the existence of only cellular trophoblast in the secondary placenta throughout the gestation and (3) the presence of well-developed hemodichorial tertiary placenta is the unique feature of the interhemal membrane in higher Chiroptera.     

Differential expression of the extra domain-containing form of cellular fibronectin in human placentas at different stages of maturation

The distribution of the extra domain-containing form of cellular fibronectin was studied in human placentas at different stages of maturation by using the monoclonal antibody 52DH1 in indirect immunofluorescence. In early chorionic tissue (7 to 10 weeks post menstruationem) cellular fibronectin was codistributed with laminin and type IV collagen in the trophoblastic basement membranes. At weeks 11 to 12 the trophoblastic basement membranes were negative but positivity was typically revealed in distinct aggregates in the stromal tissue. In second-trimester and term placentas the immunoreactivity was confined to the vessel endothelia of villous stroma. Extravillous trophoblast cells seen in placentas at different stages did not show positivity. Double staining with the 52DH1 monoclonal antibody and polyclonal fibronectin antibodies showed that both in the early and term placentas there was much fibrillar positivity only revealed with the polyclonal antibodies. The present results show that cellular fibronectin is a prominent component of early trophoblastic basement membranes and may thus play a special role in the maturation of chorionic villi.     

Differential expression of the extra domain-containing form of cellular fibronectin in human placentas at different stages of maturation

Summary The distribution of the extra domain-containing form of cellular fibronectin was studied in human placentas at different stages of maturation by using the monoclonal antibody 52DHl in indirect immunofluorescence. In early chorionic tissue (7 to 10 weeks post menstruationem) cellular fibronectin was codistributed with laminin and type IV collagen in the trophoblastic basement membranes. At weeks 11 to 12 the trophoblastic basement membranes were negative but positivity was typically revealed in distinct aggregates in the stromal tissue. In second-trimester and term placentas the immunoreactivity was confined to the vessel endothelia of villous stroma. Extravillous trophoblast cells seen in placentas at different stages did not show positivity. Double staining with the 52DHl monoclonal antibody and polyclonal fibronectin antibodies showed that both in the early and term placentas there was much fibrillar positivity only revealed with the polyclonal antibodies. The present results show that cellular fibronectin is a prominent component of early trophoblastic basement membranes and may thus play a special role in the maturation of chorionic villi.     

The permeability coefficient of the wall of a villous membrane

Summary The equations hitherto used to correct the permeability coefficient for the unstirred layer influence are valid only for flat membranes. Therefore, appropriate equations for membranes with a villous surface (e.g., small intestine) have been derived. They take into account the non-linear concentration gradient in the intervillous part of the unstirred layer. Quantitative information about the geometry of the villous surface and the unstirred layer thickness are needed to calculate the permeability coefficient of the membrane wall (e.g., intestinal epithelium). The concentration of highly permeable substances drops sharply already in the upper part of the intervillous space, so that the tips of the villi function as effective absorbing area. The intervillous concentration gradient of a substance with a low permeability coefficient is so small, that such a substance is absorbed by the total surface area of the villous membrane. The effective absorbing area of substances with intermediate permeability coefficient lies between the described limits.     

Fine structure of the paracellular junctions of terminal villous capillaries in the perfused human placeta

Summary Selected lobules of human term placentae were extracorporeally perfused for a recovery period of 20 min, fixed by perfusion and mordanted with ferrocyanide prior to processing for transmission electron microscopy. The lateral membranes of the endothelial cells of the terminal villous capillaries were found to be separated by paracellular clefts of mean width 15.6 nm. At tight junctional regions (1–4 sites per cleft) the two membranes approached each other more closely and frequently appeared to fuse. However, tilting of the sections in the electron microscope stage showed that the membranes were separated by a gap of mean width 4.1 nm in at least 94% of tight junctional profiles. When individual tight junctions were studied by a combination of serial sectioning and goniometric tilting, they were seen to widen abruptly within a distance of three to seven consecutive thin sections, indicating they were not continuous throughout the axial length of the capillaries. The wide regions of the clefts usually showed linkers, strands of glycocalyx-like material spanning the gap. Linkers may contribute to cell adhesion and possibly form part of a filter within the tortuous paracellular pathway provided by the discontinuous network of tight junctional strands. Human term placental capillaries appear to resemble closely other continuous non-brain capillaries.     

FINE STRUCTURE OF THE OCTOPUS RETINA

The fine structure of the visual and the supporting cells and of the blood capillaries in the octopus retina is described. Lamellated structures contained in the proximal segment of the visual cell consist of compact arrays of dense membranes each of which is quintuple-layered and divides at its margins into two thinner sheets or membranes which are connected directly with the agranular or granular endoplasmic reticulum. Proximal to the deeper extremities of the rhabdomeres, the lateral plasma membranes of two adjoining visual cells contact each other forming a quintuple-layered compound membrane, which results in occlusion of the intercellular space. The central layer of the compound membrane is of high density, so that the membrane, as a whole, appears to be a single thick layer at low magnifications. The supporting cells are connected with the neighboring visual cells by two types of junctions. Long slender processes extend from the supporting cells to the surface of the retina through narrow spaces among the distal segments of the visual cells. The capillary endothelial cells are characterized by luminal surfaces irregularly contoured and by lateral surfaces elaborately interdigitated. The functional significance of the close contact between adjoining visual cells is discussed.