Branching patterns of human placental villous trees: perspectives of topological analysis.

Topological analysis was applied to investigate the branching pattern of three specimens obtained from early human placenta (6, 9, and 16 weeks p.m.) reconstructed on the basis of semi-thin sections. Centripetal Horton-Strahler and centrifugal branching order nomenclature was used for topological description of the analysed tree-like structures. Bifurcation ratio and vertex ratio were determined for all three cases and were found to be relatively constant. It was shown that branching pattern is closely related to the model of random segment branching that implicates a high level of asymmetry and a small level of space limitation for branching. The significance of this approach for the analysis of development of the villous tree, for the analysis of mesenchymal villous heterogeneity, and for the estimation of physiological parameters for fetoplacental exchange is discussed. We suggest that topological analysis can lead to a new quantitative classification of branching patterns of the human placental villous trees in normal and pathologic pregnancies.

The human placenta is encircled by a ring of smooth muscle cells.

The marginal zone of the human term placenta was studied by transmission electron microscopy and immunohistochemistry using antibodies against cytoskeletal filaments, extracellular matrix molecules and endothelial markers. The marginal sinus of the intervillous space is separated from the chorionic and basal plates by a layer of cells expressing vimentin, desmin, alpha- and gamma-smooth muscle actins, and smooth muscle myosin. Also ultrastructurally, these cells share all features with smooth muscle cells. This muscular ring is continuous with the media of uteroplacental veins entering the marginal sinus. In the basal plate the muscle cells may extend far into the central parts of the placenta. The muscular ring is separated from the intervillous space by a layer of endothelial cells. They are continuous with the maternal endothelium of the marginal uteroplacental veins. Moreover this endothelium covers neighbouring parts of the chorionic and basal plates, locally extending to the surfaces of large stem villi. The data suggest (1) that the marginal zone of the intervillous space ('marginal sinus') represents the dilated and merged parts of uteroplacental veins and (2) that lateral growth of the human placenta partly takes place by expansion into the uteroplacental veins. The functional importance of this muscular ring remains unknown.

Pregnancy-induced de-differentiation of media smooth muscle cells in uteroplacental arteries of the guinea pig is reversible after delivery.

The majority media of smooth muscle cells of uteroplacental arteries of the guinea pig is not destroyed during trophoblast invasion. Rather, most of these cells de-differentiate during pregnancy-induced arterial dilatation, forming a population of mesenchyme-like myoblasts ready to reconstitute the media after birth. We have studied the re-differentiation of these cells after delivery by means of transmission electron microscopy and immunohistochemistry using antibodies against a panel of cytoskeletal proteins. The data reveal that post partum re-differentiation of the media myoblasts starts immediately after birth where some of the invasive trophoblast cells are still present. The process of re-differentiation is completed at day 8 after parturition. Post partum re-differentiation can be subdivided into two steps: until day 5 after parturition, the central parts of the media are reconstituted out of the reservoir of vimentin-positive myoblasts by stepwise acquisition of desmin, alpha-smooth muscle actin, gamma-smooth muscle actin and smooth muscle myosin. Only thereafter the same re-differentiation takes place in the peripheral parts of the media. On the ultrastructural level immunohistochemical re-expression of cytoskeletal proteins is accompanied by reconstitution of the intracellular contractile apparatus. The data support our earlier notion that the majority of media smooth muscle cells in the guinea pig uterus does not degenerate during trophoblast-invasion but rather de-differentiate temporarily.

The distribution of macrophages in spiral arteries of the placental bed in pre-eclampsia differs from that in healthy patients.

Placental bed biopsies taken during caesarean section from 10 patients with pre-eclampsia and six healthy pregnancies were studied. We applied antibodies against cytokeratin and different macrophage markers to analyse the distribution of invasive extravillous trophoblast cells as compared to that of macrophages in myometrial segments of uteroplacental arteries. The data were evaluated quantitatively. We found a clear inverse relationship between local infiltration with macrophages and trophoblast invasion. In pre-eclampsia, vessel cross-sections prevailed which were characterized by large numbers of macrophages but a low degree of trophoblast invasion. In contrast, in normal third trimester pregnancies the respective arterial segments had a high degree of trophoblast invasion but were largely void of macrophages. These data suggest causal links between macrophages and inhibition of intra-arterial trophoblast invasion in pre-eclampsia.

Pressure dependence of so-called transtrophoblastic channels during fetal perfusion of human placental villi.

To test the influence of perfusion pressures on structural preservation of human placental villi and on the dilatation of the so-called transtrophoblastic channels, cotyledons of 32 term human placentas have been perfused in vitro. Periods of perfusion with isotonic Ringer solution under various arterial and venous hydrostatic pressures were followed by perfusion fixation. In some experiments, lanthanum hydroxide as an extracellular marker was added to the fixative. Distention of the fetal vascular system, stromal edema and continuity, as well as trophoblastic vacuolization were studied via electron microscopy with subsequent morphometry. The findings suggest that arterial hydrostatic pressures in the perfusion system of about 80 cm H2O are needed to guarantee homogeneous perfusion of the fetal vascular system. To avoid stromal edema and trophoblastic vacuolization, venous hydrostatic pressures of 4 cm H2O and arterial hydrostatic pressures of 80 cm H2O should not be exceeded. It is concluded that the trophoblastic vacuoles are dilated segments of the so-called transtrophoblastic channels. The functional importance of in vivo variations of fetal intravascular hydrostatic pressure for the dilatation of transtrophoblastic channels and for fetal water balance is discussed.

Classification of human placental stem villi: review of structural and functional aspects.

The stem villi of the human placenta represent the central branches of the villous trees. They are characterized by a condensed fibrous stroma in which the fetal arteries and veins as well as the arterioles and venules are embedded. Functionally they are accepted as the mechanically supporting structures of the villous trees, and they are supposed to control fetal blood flow to the maternofetal exchange area, which is located in the peripheral villi. To obtain further insights into the functions of the stem villi, the recent literature has been reviewed, and some immunohistochemical, ultrastructural, and reconstruction studies have been added. These new studies were aimed at identifying immunohistochemically different subtypes of stem villi, their branching patterns, the distribution of macrophages, the stromal proliferation patterns, and the differentiation of extravascular stromal cells. Our findings demonstrate that the stem villi and their precursors, the immature intermediate villi, can selectively be identified by anti-gamma-smooth muscle (sm) actin staining. Furthermore, the existence of three different subtypes of stem villi is shown; these differ regarding the presence and distribution of gamma-sm actin-positive cells. These cells were immunohistochemically and ultrastructurally identified as smooth muscle cells and myofibroblasts. Increasingly complex coexpression patterns of cytoskeletal proteins reflect a clearly defined differentiation gradient of extravascular stromal cells, which covers the whole range of an undifferentiated germinative layer beneath the trophoblast to highly differentiated myofibroblasts surrounding the medias of the stem vessels. Possible functions of the extravascular contractile system include the regulation of villous turgor and the control of intervillous blood flow impedance.

Location of insulin receptors in the placenta and its progenitor tissues.

The insulin receptor gene is constitutively expressed, so the presence of insulin receptor proteins might be expected on all mammalian tissues, with the plasma membrane as the predominant site of receptor location. Results reviewed here indicate that insulin receptors are also present in all placental tissues and the placenta's progenitor tissues and cells, i.e., oocytes, spermatozoa, and preimplantation embryos, in most of the species studied. Receptor densities, however, vary among individual cells and cell types and at various developmental stages. Three aspects deserve emphasis. 1) In human placenta, the insulin receptor distribution pattern is characterized by a spatiotemporal change between first trimester and term. At the beginning of pregnancy, insulin receptors are found predominantly on the maternal side (apical membrane of syncytiotrophoblast, low density on cytotrophoblast); at term, however, they are on the fetal side (lining the fetal vessels). This suggests that, in the first trimester, maternal insulin regulates insulin-dependent processes, whereas, at term, it must be fetal insulin mainly controlling these processes. 2) The majority of insulin receptors is expressed on structures that are currently assumed to drive placental growth, i.e., syncytial sprouts and mesenchymal villi in first-trimester placentas and fetal endothelium at term. Therefore, we hypothesize a growth-promoting function, among others, of insulin on the placenta. 3) At present, no histologic evidence is available to demonstrate insulin receptors in structures commonly associated with receptor-mediated endocytosis. Whether placental insulin receptors are internalized, therefore, awaits clarification.

Increased fetoplacental angiogenesis during first trimester in anaemic women.

BACKGROUND: Epidemiological studies describe an association between relative size of the placenta at delivery and cardiovascular morbidity and mortality during adult life. Some determinants of placental size, such as maternal anaemia, have been acknowledged, but no plausible mechanism has been advanced to explain the initiation of postnatal disease. METHODS: Placental villous vascularisation in anaemic women (Hb<90 g/L) was assessed in the first and third trimesters of pregnancy by immunohistochemical identification of villous capillaries and compared with that of gestational age-matched groups of women with normal (Hb>110 g/L; control group) concentrations of haemoglobin, and an intermediate group (Hb 90-110 g/L). FINDINGS: Anaemia, especially in the first trimester, was associated with increased numbers of capillaries per villous cross section (mean 11.70 [SE 0.35] vs 4.14 [0.27]) located mainly in the outer third of the stroma beneath the trophoblast (94% [1.15] vs 67% [1.82]) and with increased numbers of villous macrophages and of proliferating MIB-1-positive cells compared with the control group. INTERPRETATION: Maternal anaemia in early pregnancy seems to influence the pattern of placental vascularisation. Such changes might alter placental vascular impedance during early fetal life, thereby exerting important effects on cardiovascular development.

The impairment of mobility and development in freshwater snails (Physa fontinalis and Lymnaea stagnalis) caused by herbicides.

1. The pulmonate freshwater snails Physa and Lymnaea and the earthworms Eisenia and Lumbricus can take up and concentrate a carbamate herbicide (chlorpropham). 2. The mobility of freshwater snails was diminished in the presence of the herbicides chlorpropham (carbamate), cycloate (thiocarbamate), pentanochlor (amide) and chloroxurone (urea derivate). 3. The egg-assemblies of Lymnaea stagnalis turned out to be suitable objects for testing the influences of herbicides upon embryonic development. 4. In the presence of chlorpropham, chloroxurone, cycloate, propanil, simazine and terbutryne, all applied in concentrations lower than in practical use, the period of egg-maturing was delayed and the total number of dead embryos increased. 5. Developing snail eggs were very sensitive towards triazine herbicides (simazine, terbutryne) reflecting in an ED50-value lower than 10(-7) M. 6. Similar herbicidal-induced effects suggested that the developing stages of snail embryos may be suitable models for the ecologically more important but experimentally less accessible earthworms.

Villous sprouting: fundamental mechanisms of human placental development.

There is increasing evidence that maldevelopment of the placental villous tree can play an important role in the pathogenesis of various pregnancy diseases. In this review we present the most recent advances of cellular and molecular mechanisms involved in the early formation of chorionic villi. In particular we focus our attention on the structural events during early villous sprouting leading to the formation of the mesenchymal villi which are the forerunners of all other villous types, i.e. immature intermediate villi, stem villi, mature intermediate villi and terminal villi. Early villous sprouting starts as 'hot spots' which are circumscribed areas consisting of highly proliferating cytotrophoblastic and stromal cells. The post-proliferative cytotrophoblastic cells fuse with the overlying syncytium leading to the formation of the trophoblastic sprouts. When villous mesenchyme invades the trophoblastic sprouts, the latter are transformed into villous sprouts. The vascularization of the villous sprouts leads to the formation of the mesenchymal villi, the most basic villous type. This process is repeated throughout pregnancy. We analyse the influence of various extracellular matrix molecules, e.g. tenascin and hyaluronic acid, on the formation of hot spots and mesenchymal villi as well as the transformation of the latter in other villous types. We present a critical survey on the data on vessel formation related to villous sprouting and morphogenesis of mesenchymal villi as well as the expression of various angiogenic factors and their receptors.