Three-dimensional Arrangement of the Capillary Bed and Its Relationship to Microrheology in the Terminal Villi of Normal Term Placenta

Abstract

Spatial arrangement of the capillary bed, manifestations of its growth and symmetry of capillary branching were studied in peripheral villi of normal human placenta at term using confocal microscopy and image analysis. Unlike the model that has been accepted so far, it was shown that the arrangement of the capillary bed in terminal villi varied from simple, U-like loops to a richly branched network. Three different categories of terminal villi (TV) were recognised:

• TV developing and protruding from an existing terminal villus, whose capillaries supply developing villi;

• TV protruding from the mature intermediate villus in pairs or more numerous groups in which the capillary bed is formed by capillaries connecting both the mature intermediate villus vascular bed and the capillary bed of the neighbouring terminal villus;

• separate TV with a capillary bed leading directly off and joining the vessels of the mature intermediate villus.

Signs of capillary elongation and sprouting were observed in the villous capillary bed. Based on the assessment of the mean cross-sectional areas of capillaries constituting simple, Y-like capillary bifurcation in terminal villi, the capillary branching was found to be asymmetric. Therefore, we conclude that the conditions for the “plasma skimming” effect are met in human placenta.

Introduction

Knowledge of the spatial organisation of the microvascular bed is of great importance in understanding its function in different organs. Due to its role in transport between the mother and fetus, the placental microcirculation is a decisive factor in the appropriate intrauterine development of each individual. It is therefore intensively studied in both normal and pathological placenta.

As commonly accepted, the vast majority of the microvascular bed of the human term placenta is located in the mature intermediate and terminal villi. The mature intermediate villi encompass arterioles, thin capillaries and venules, from which capillaries of terminal villi arise. They run in a tight relationship with the trophoblast, together forming a placental barrier that separates maternal and fetal blood and, at the same time, transports nutrients and metabolites. In order to meet fetal requirements, the area of the trophoblast enlarges due to the mitotic division of cytotrophoblast cells and their subsequent fusion with syncytiotrophoblast, whereas the capillary wall area enlarges by angiogenesis. Signs of those processes are observed in conventional microscopic sections, but no information is available on the spatial organisation of the villous capillary bed and its changes.

Few studies on the three-dimensional (3D) arrangement of the villous capillary bed have been carried out in the last five decades. Due to different methodical approaches (injection of contrast medium and conventional light microscopy, corrosion casts, scanning electron microscopy, physical wax reconstruction) and also because the different stages of placental samples were examined, the models that were designed were barely comparable. Crawford [1] interpreted the villous capillary bed as being a long, non-branched, wavy capillary running garland-like throughout the grape-like arranged terminal villi of term placenta. On the other hand, in terminal villi of immature placenta, Boe [2] demonstrated capillary beds consisting of capillaries parallel with the villous axis, mutually connected by dense, short segments mainly in the villous tips. According to Arts's scheme [3], longitudinally oriented capillaries of terminal villi are mutually connected by short capillary segments, arise directly from arteries and run into veins. Scanning electron microscopy of corrosion casts [4], [5] showed more precisely some variability in the capillary beds in terminal villi and their relationships to vessels in mature intermediate villi.

The authors of the latest concept used two methodical approaches, namely SEM analysis of corrosion preparations, and physical 3D reconstruction based on photomicrographs of serial histological sections [6], [7], [8]. In their scheme, arterioles and paravascular capillaries of intermediate villi continue as coiled and sinusoidally dilated capillaries of the terminal villi. They are 3000–5000 μm long and run as predominantly hairpin-like capillaries throughout several successive terminal villi, in some places forming protrusions or new villi by bulging against the trophoblast. Capillary sprouts with blind ends were described and interpreted for the first time in that paper as being the initial stage of newly formed capillaries [6], [9].

Unlike the findings described above, our analysis, based on 3D images of capillaries acquired by confocal microscopy, has shown that only 44% of the terminal villi contain a simple, hairpin- or U-like capillary loop in normal placenta. In 32% of villi, the capillary bed takes the form of a simple bifurcation (Y); other villi have multi-branched capillaries [10]. A high proportion of villi with branched capillaries leads us to the question: how could such an arrangement be useful for placental function?

As observed many years ago and repeatedly studied in detail later on, at the asymmetric branching of microvessels with diameter less than about 35 μm, asymmetric red cell distribution and separation of blood plasma take place. It results in the heterogeneity of the haematocrit in the daughter branches. This phenomenon, called “plasma skimming”, influences local circulation [11], [12], [13]. The studies dealing with plasma skimming and other microrheological effects were carried out in vivo predominantly on easily accessible animal organs, e.g. frog skin [11], animal mesentery [13] or muscle [14], and quite exceptionally in humans, on the nerve root [15]. As for placenta, to our knowledge, the only reference to plasma skimming in the fetoplacental microvascular bed is represented by a short speculative paragraph based on Boe's model of villous circulation [16]. The question of whether this effect occurs in human placenta has not yet been answered, most likely due to the absence of appropriate methods of examination of the human placental microvascular bed.

Thanks to the recent technical progress it is possible to analyse placental microvasculature using sophisticated methods, e.g. by microcomputed tomography; however, these techniques have serious limits: it is very difficult, if even possible, to achieve completely homogenous filling of all vessels by the contrast medium, this procedure can cause collapse of the villous capillaries [17], and its resolution is still too low to resolve vessels with diameters smaller than 30 μm, which completely excludes analysis of the capillary bed [18]. Therefore, microscopic techniques are still necessary for the examination of placental capillaries. Here, confocal microscopy in particular is a useful tool, enabling 3D images to be acquired with sufficient resolution as well as good detection of cell and tissue compartments stained by relevant methods [19].

In this paper our aim was to demonstrate the spatial arrangement of the villous placental capillaries and to assess the symmetry of their bifurcation in order to judge if conditions required for the “plasma skimming” effect are met.