Morphomics: An integral part of systems biology of the human placenta.

INTRODUCTION: The placenta is a transient organ the functioning of which has health consequences far beyond the embryo/fetus. Understanding the biology of any system (organ, organism, single cell, etc) requires a comprehensive and inclusive approach which embraces all the biomedical disciplines and 'omic' technologies and then integrates information obtained from all of them. Among the latest 'omics' is morphomics. The terms morphome and morphomics have been applied incoherently in biology and biomedicine but, recently, they have been given clear and widescale definitions. METHODS: Morphomics is placed in the context of other 'omics' and its pertinent technologies and tools for sampling and quantitation are reviewed. Emphasis is accorded to the importance of random sampling principles in systems biology and the value of combining 3D quantification with alternative imaging techniques to advance knowledge and understanding of the human placental morphome. RESULTS AND CONCLUSIONS: By analogy to other 'omes', the morphome is the totality of morphological features within a system and morphomics is the systematic study of those structures. Information about structure is required at multiple levels of resolution in order to understand better the processes by which a given system alters with time, experimental treatment or environmental insult. Therefore, morphomics research includes all imaging techniques at all levels of achievable resolution from gross anatomy and medical imaging, via optical and electron microscopy, to molecular characterisation. Quantification is an important element of all 'omics' studies and, because biological systems exist and operate in 3-dimensional (3D) space, precise descriptions of form, content and spatial relationships require the quantification of structure in 3D. These considerations are relevant to future study contributions to the Human Placenta Project.

Turnover of human villous trophoblast in normal pregnancy: what do we know and what do we need to know?

How the turnover of villous trophoblast is regulated is important for understanding normal and complicated pregnancies. There is considerable accord that syncytiotrophoblast (STB) grows and is refreshed by recruiting post-mitotic cells from the deeper cytotrophoblast (CTB). Nuclei in STB exhibit a spectrum of morphologies and packing densities and, until recently, there seemed to be a consensus that this variation reflected a transition from an early undifferentiated CTB-like phenotype to a long pre-apoptotic and brief apoptotic phase. In these later phases, nuclei are sequestered in clusters (syncytial knots) prior to extrusion as part of normal epithelial turnover. Early in gestation, nuclear clustering and formation of protrusions (syncytial sprouts) also occurs as a preliminary to villous sprouting. Nuclei in these clusters have a CTB-like phenotype and some sprouts may also detach from STB and pass into the uteroplacental circulation. However, this apparent consensus has been challenged and new interpretations of events in the proliferative (CTB), terminal differentiation (STB) and deportation compartments have emerged. Several different types of STB fragment are deported in normal pregnancy: larger multinucleate STB fragments, smaller uninucleate elements with CTB-like morphology, anucleate cytoplasmic fragments, microparticles and nanovesicles. This review identifies points of agreement and disagreement and offers possible avenues of future research. An obvious need is to standardise best practice in several areas including choosing appropriate references for cell cycle phase labelling indices and combining immunolabeling of cell cycle and apoptosis markers (at LM or TEM levels) with design-based stereological estimates of absolute numbers of cells and nuclei in different compartments throughout normal gestation. This would also provide a surer foundation for interpreting results from different research groups and changes in normal and complicated pregnancies.

Cross-sectional data on soft tissue morphometry of the growing hand and fingers of dextral individuals 5-65 years old.

This study examines both hands of right-handed (dextral) subjects 5-65 years old in order to define the separate growth trajectories of digit lengths (2D-5D) and hand widths; to assess how 2D : 4D and other digit ratios also vary with age; and to test whether lengths are influenced by gender dimorphism and lateral (right/left) asymmetry. Calliper measurements were made from hand photocopies. Growth patterns were analysed by linear regression and correlation, main and interaction effects of age and gender were resolved by analysis of variance, and lateral asymmetries were identified by paired tests. All digits, and hand width, grew in a biphasic pattern in both hands, and inflection points between phases showed gender dimorphism. In the early fast-growing phase, male digits grew over a longer period than those in females, before switching to a slower growth phase during which gender dimorphism became more exaggerated. In right hands, age differences in digit ratios were confined to 2D : 4D and, except for 4D : 5D, females tended to show larger ratios than males. In left hands, all ratios (except 3D : 5D) varied with age and gender influenced only 2D : 4D, 2D : 5D and 3D : 5D. Again, ratios were greater in females. In females, 2D was longer in the right hand of older subjects, whilst 3D, 4D and 5D tended to be shorter in the right hand of younger subjects. No asymmetries were seen in 2D, 3D or 4D in males, but 5D tended to be shorter on the right in the group 9-12 years old. Finally, hand width tended to be greater in females on the right at 9-65 years old, and in males on the right at 18-23 years old. A further novel finding was that certain relationships (inflection points, correlation coefficients and gender differences in digit lengths) seemed to follow gradients running from 2D to 5D. It is tempting to speculate that these are manifestations of the antero-posterior gradients established by signalling events that control digit development and patterning in utero.

A quantitative analysis of transcriptionally active syncytiotrophoblast nuclei across human gestation.

The syncytiotrophoblast (STB) epithelial covering of the human placenta is a unique terminally differentiated, multi-nucleated syncytium. No mitotic bodies are observed in the STB, which is sustained by continuous fusion of underlying cytotrophoblast cells (CTB). As a result, STB nuclei are of different ages. Morphologically, they display varying degrees of chromatin compaction, suggesting progressive maturational changes. Until recently, it was thought that STB nuclei were transcriptionally inactive, with all the mRNAs required by the syncytium being incorporated upon fusion of CTB. However, recent research has shown the presence of the active form of RNA polymerase II (RNA Pol II) in some STB nuclei. In this study, we confirm the presence of transcriptional activity in STB nuclei by demonstrating immunoreactivity for a transcription factor and an RNA polymerase I (RNA Pol I) co-factor, phospho-cAMP response element-binding protein and phospho-upstream binding factor, respectively. We also show, through immunoco-localisation studies, that a proportion of STB nuclei are both RNA Pol I and II transcriptionally active. Finally, we quantify the numerical densities of nuclei immunopositive and immunonegative for RNA Pol II in the STB of normal placentas of 11-39 weeks gestational age using an unbiased stereological counting tool, the physical disector. These data were combined with estimates of the volume of trophoblast to calculate total numbers of both types of nuclei at each gestational age. We found no correlation between gestational age and the numerical density of RNA Pol II-positive nuclei in the villous trophoblast (r = 0.39, P > 0.05). As the number of STB nuclei increases exponentially during gestation, we conclude that the number of transcriptionally active nuclei increases in proportion to trophoblast volume. The ratio of active to inactive nuclei remains constant at 3.9:1. These findings confirm that the majority of STB nuclei have intrinsic transcriptional activity, and that the STB is not dependent on CTB fusion for the provision of transcripts.

Volumes and numbers of intervillous pores and villous domains in placentas associated with intrauterine growth restriction and/or pre-eclampsia.

The intrauterine environment has an important influence on placental development. In pre-eclampsia (PE) and intrauterine growth restriction (IUGR), early remodelling of spiral arteries has repercussions for uteroplacental blood flow. The IUGR placenta exhibits compromised growth of villous trees, a smaller intervillous space and a lower diffusive conductance. Here, we test whether or not term placentas associated with PE or IUGR also exhibit changes in structural quantities (notably, sizes and numbers of intervillous pores and villous domains) pertinent to uteroplacental haemodynamics. Paraffin wax sections were sampled at random locations and orientations and structural quantities obtained by combining design-based stereological estimates of total and star volumes with model-based estimates of pore hydraulic diameters. Total volumes of intervillous pores and villi were estimated by point counting, total villous surface by intersection counting and star volumes by measuring point-sampled intercept lengths. Other quantities were derived secondarily and group estimates compared by two-way analysis of variance. We found significant main effects of IUGR but no main or interaction effects involving PE. In IUGR, there were fewer intervillous pores and these had larger hydraulic diameters. IUGR also produced fewer villous domains but these were constant in star volume and villi had a constant mean diameter and volume fraction. We concluded that IUGR compromises placental development by producing intervillous pores and villous trees different in size and shape from those in control and PE pregnancies. Calculations suggest that Darcian conductances in the intervillous space improve in IUGR but, in reality, placental performance is compromised by other physiological and structural constraints including the known decline in diffusive conductances.

Quantifying immunogold localization patterns on electron microscopic thin sections of placenta: recent developments.

In recent years, there have been important advances in the quantification of high-resolution (electron microscopical) images of tissue sections on which colloidal gold-labelled probes serve to identify and localize interesting target antigens. With these new methods, the distributions of gold particle counts across volume-occupying and/or surface-occupying compartments can be compared within or between experimental groups of cells, tissues or organs. Method I (for within-group comparisons) tests whether or not there is preferential labelling of compartments by comparing observed and expected gold labelling distributions using Chi-squared (chi2) analyses. To this end, estimators of gold labelling intensity (labelling density, LD, and/or relative labelling index, RLI) are used to analyse one category of compartment (volume or surface occupiers) or a mixture of categories (volume and surface occupiers). This involves estimating compartment size taking into account specimen magnification and, in its most efficient form, is achieved simply by counting chance events after superimposing random test probes (lattices of points and/or lines). RLI=1 indicates random labelling but higher RLI values indicate the degree to which a compartment departs from random labelling. Method II (drawing between-group comparisons) does not require information about compartment size or specimen magnification. Comparisons are drawn using the observed gold particle counts in different groups and by combining chi2 analysis with contingency table analysis. Both methods rely on multistage systematic uniform random sampling of specimens and unbiased counting of gold particles associated with different compartments. Statistical degrees of freedom are determined by the numbers of compartments and experimental groups. Together with RLI values, compartmental values of chi2 which contribute substantially to total chi2 identify the principal sites of within- and between-group differences. The methods are illustrated using data taken from studies aimed at localising protein antigens (caveolin-1 and GLUT1) in specimens of term human placenta.

No correlation between ultrasound placental grading at 31-34 weeks of gestation and a surrogate estimate of organ function at term obtained by stereological analysis.

We test the experimental hypothesis that early changes in the ultrasound appearance of the placenta reflect poor or reduced placental function. The sonographic (Grannum) grade of placental maturity was compared to placental function as expressed by the morphometric oxygen diffusive conductance of the villous membrane. Ultrasonography was used to assess the Grannum grade of 32 placentas at 31-34 weeks of gestation. Indications for the scans included a history of previous fetal abnormalities, previous fetal growth problems or suspicion of IUGR. Placentas were classified from grade 0 (most immature) to grade III (most mature). We did not exclude smokers or complicated pregnancies as we aimed to correlate the early appearance of mature placentas with placental function. After delivery, microscopical fields on formalin-fixed, trichrome-stained histological sections of each placenta were obtained by multistage systematic uniform random sampling. Using design-based stereological methods, the exchange surface areas of peripheral (terminal and intermediate) villi and their fetal capillaries and the arithmetic and harmonic mean thicknesses of the villous membrane (maternal surface of villous trophoblast to adluminal surface of vascular endothelium) were estimated. An index of the variability in thickness of this membrane, and an estimate of its oxygen diffusive conductance, were derived secondarily as were estimates of the mean diameters and total lengths of villi and fetal capillaries. Group comparisons were drawn using analysis of variance. We found no significant differences in placental volume or composition or in the dimensions or diffusive conductances of the villous membrane. Subsequent exclusion of smokers did not alter these main findings. Grannum grades at 31-34 weeks of gestation appear not to provide reliable predictors of the functional capacity of the term placenta as expressed by the surrogate measure, morphometric diffusive conductance.

Human 2D (index) and 4D (ring) finger lengths and ratios: cross-sectional data on linear growth patterns, sexual dimorphism and lateral asymmetry from 4 to 60 years of age.

Human 2D : 4D ratios (measures of the relative lengths of index and ring fingers) attract considerable research interest because they exhibit sexual dimorphism and are associated with various morphological, physiological and behavioural traits as well as sporting abilities and medical conditions. In an attempt to identify potential confounding factors in such studies, we have examined how relative and absolute digit lengths vary with gender and tested whether they are influenced by age, right-left asymmetry and hand preference. Participants between 4 and 60 years of age were recruited from local educational sites. Hand photocopies and calliper measurement were used to obtain digit lengths. We employed linear regression analysis to examine the growth trajectories of individual digits, analyses of variance to isolate main and interaction effects of age, gender and hand preference, and paired t-tests to identify lateral asymmetries. Both digits exhibited biphasic growth with an early growth phase followed by a stable length phase. Digits in females attained their maximum length about 2.2 years (dextral subjects) or 5.1 years (sinistral subjects) earlier than those in males. Sexual dimorphism in 2D : 4D ratios was apparent by 4 years of age and age changes in ratios depended on gender, side and hand preference. Relative and absolute lengths displayed age, gender, hand-preference and age x gender interaction effects. Lengths tended to be greater in females in younger subjects and greater in males in older subjects. Ratios tended to be greater in sinistral subjects. In dextral subjects, significant lateral asymmetries in 2D lengths were seen at all ages but asymmetries in males and 4D lengths seemed to be age-dependent. We conclude that age, lateral asymmetry and hand preference are potential confounding factors and that future study designs should take account of these as well as other known confounders such as ethnicity, birth order, menstrual cycle phase and sexual preference.

Maternal asthma and placental morphometry: effects of severity, treatment and fetal sex.

Asthma is the most common respiratory disease to complicate pregnancy. Although adverse effects on the fetus have been documented, there is a paucity of information regarding the effects of asthma, and its treatment, on placental morphology. The aim of this study was to test for volumetric differences in placental composition between non-asthmatic pregnancies and those associated with maternal asthma grouped according to asthma severity and glucocorticoid (GC) treatment. Each placenta was weighed and random samples of tissue were fixed in formalin-saline, embedded in wax and analysed by design-based stereology. Volume densities of parenchymal compartments (peripheral villi and maternal intervillous space) and residual non-parenchyma were estimated by test point counting and converted to absolute volumes by taking into account placental size. Relative and absolute lengths of villi and capillaries were also estimated and used to derive secondary quantities related to villous capillarization and maturation. Between-group comparisons were drawn by two-way analysis of variance with group and fetal sex as the principal factors. Compared to non-asthmatic controls, asthmatics had reduced absolute volumes of fetal capillaries which was most marked in those with moderate/severe asthma and those using low and high doses of inhaled GCs. Changes in the total length and mean cross-sectional area of capillaries and peripheral villi were also observed. Lengths were greater in mild asthmatics and lowest in those with high GC usage. Calibre areas were lower in mild asthmatics and villous calibres in the high GC group were greater than those in asthmatics not taking GCs. Those making greatest use of inhaled GCs also had villi which were hypovascularized in terms of capillary:villus length ratios. The findings suggest that the morphometric differences in fetoplacental vascularity are likely to be due to the effects of asthma and use of inhaled GCs rather than the effects of maternal or fetal hypoxic stress.

A re-appraisal of the morphophenotype and basal lamina coverage of cytotrophoblasts in human term placenta.

A recent study of human placental villi [Mori et al., The cytotrophoblast layer of human chorionic villi becomes thinner but maintains its structural integrity during gestation, Biol Reprod 76 (2007) 164-172] concluded that cytotrophoblast (CT) cells occupy 80% of the basal lamina (BL) surface at term and that syncytiotrophoblast (ST) does not make direct contact with the BL. Based on SPINT-1 localisation using immunofluorescence on cryosections, these conclusions run counter to previous light and electron microscopic data suggesting that term CT cells cover no more than about 24% of the BL surface. To resolve these discrepancies, we have undertaken a stereological study of term placenta using transmission electron microscopy (TEM) and a novel immunofluorescence approach. Test line lattices were randomly superimposed on TEM images of villous trophoblast from 13 normal term placentae. Intersections with the test lines were counted to assess the fractional surface of BL occupied by CT cells. After trypsin-mediated removal of syncytium, cells in whole-mounted term and first trimester villi were stained with cytokeratin 7 to identify CT and then visualised by confocal microscopy. CT formed an almost continuous layer in the first trimester. In contrast, term CT cells and their processes were found to cover only 44% (SD 14%) of the BL surface with intervening regions occupied by ST. TEM and confocal images were consistent with the concept of a network of 'octopoid' CT cells with fine processes extending from a central cell body. Our estimates of CT coverage are lower than the recent immunofluorescence estimate but greater than earlier TEM estimates. The former may have been biased by overprojection (section thickness) effects whilst the latter may be underestimates due to failure to include the fine CT cell processes. We conclude that CT cells transform from a cuboidal phenotype early in gestation to flattened cells with multiple interconnecting processes. The CT layer thins but maintains a functional network within which cells intercommunicate without compromising substance transfer via the syncytium.

Taking tissue samples from the placenta: an illustration of principles and strategies.

Tissue samples are removed from placentas for a variety of reasons associated with a host of investigative techniques, including chorionic villus sampling, villus explant culture, cell culture, proteomic analysis, gene expression profiling, microscopy and morphometry. Apart from the latter, especially stereological analysis, many studies provide extremely limited information on how the samples were selected. At worst, we learn little more than the placenta was sampled. Sometimes, studies provide sufficient detail to reveal flaws in sampling, e.g. the selection of placentomes based on size rather than mere presence. Occasionally, the reader is informed, without further explanation, that representative samples were taken or that samples from placentas in different study groups were taken from standard or similar sites. Such statements raise doubts about the unbiasedness of the sampling process, leave the reader in ignorance of the quality of the final sample, thwart attempts at achieving study repeatability and compromise interpretations of the validity of study outcomes. And yet study outcomes depend critically on the selection process because sampling influences study errors, notably precision (random error) and bias (systematic error). This article aims to review the basic principles and virtues of random sampling in general and the practical utilities of variants of it. For many functional and structural studies, it suffices to randomise the positions of tissue samples but, in certain structural studies, orientation must also be randomised. Therefore, sampling tools for stereological estimation of membrane surface areas, tubule lengths and layer thicknesses are mentioned. Although emphasis is accorded to the placenta, the principles apply equally well to other organs and to lower levels of organisation including the subcellular. It is hoped that this review will inform future study designs, encourage greater transparency and facilitate sampling improvements.

Human 2D (index) and 4D (ring) digit lengths: their variation and relationships during the menstrual cycle.

It is known that there are sexually dimorphic differences in relative and absolute lengths of the index (2nd) and ring (4th) fingers and that the sizes of laterally-paired soft tissues (e.g. ears and fingers) show changes across the menstrual cycle. The aim of the present study was to determine whether cyclical changes in the digit lengths of the index and ring fingers also occur and, if so, to what extent these are related to changing patterns of circulating sex steroids. Digit lengths were assessed over two cycles in groups of right-handed females (19-21 years of age) who were divided on the basis of whether or not they were taking oral contraceptive pills (n = 13 and n = 6 respectively). Using callipers, finger lengths were measured on photocopy images of both hands taken at 4-day intervals for a total of 56 days. We tested the following null hypotheses: (1) digit length measurements do not exhibit fluctuations across the menstrual cycle; (2) there is no evidence of lateral asymmetry between measurements made on both hands; (3) the lengths of digits 2 and 4 do not differ in either hand. Null hypotheses were tested using Page's L trends test for related samples (cyclical fluctuations) and paired Student's t tests (left-right asymmetries and within-hand digital differences). In those not taking oral contraceptives, finger lengths and 2D:4D digit ratios fluctuated across the cycle with values tending to increase in the pre-ovulatory period and decline thereafter. Left-right asymmetries varied in a similar fashion with lengths generally being larger, and lateral asymmetries smaller, in the dominant hand. Although sample sizes were smaller, some of these patterns were retained but others were perturbed in those practising oral contraception. We conclude that finger lengths are cycle-dependent and that account should be taken of this, and of oral contraceptive usage, in future studies on female digit lengths and their ratios.

Histological changes in intestine of Atlantic salmon (Salmo salar L.) following in vitro exposure to pathogenic and probiotic bacterial strains.

Furunculosis and vibriosis are diseases that cause severe economic losses in the fish-farming industry. The foregut of the Atlantic salmon (Salmo salar L.) was exposed in vitro to two fish pathogens, Aeromonas salmonicida (causative agent of furunculosis) and Vibrio anguillarum (causative agent of vibriosis), and to one probiotic strain, Carnobacterium divergens, at 6 x 10(4) or 6 x 10(6) viable bacteria per milliliter. Histological changes following bacterial exposure were assessed by light and electron microscopy. Control samples (foregut exposed to Ringer's solution only) and samples exposed only to C. divergens had a similar appearance to intact intestinal mucosal epithelium, with no signs of damage. However, exposure of the foregut to the pathogenic bacteria resulted in damaged epithelial cells, cell debris in the lumen, and disorganization of the microvilli. Co-incubation of the foregut with a pathogen and C. divergens did not reverse the damaging effects caused by the pathogen, although these were alleviated when probiotic bacteria were used. Based on these results, we suggest that the probiotic bacterium, C. divergens, is able to prevent, to some extent, pathogen-induced damage in the Atlantic salmon foregut.

The placenta in pre-eclampsia and intrauterine growth restriction: studies on exchange surface areas, diffusion distances and villous membrane diffusive conductances.

We test the null hypothesis that the morphometric diffusive conductance of the placental villous membrane does not alter in pregnancies complicated by intrauterine growth restriction (IUGR) or pre-eclampsia (PE). Placentas were collected from cases of normotensive IUGR, pure PE, PE+IUGR and from control pregnancies. Microscopical fields on formalin-fixed, trichrome-stained histological sections were randomly sampled for location and orientation. Using stereological methods, the exchange surface areas of peripheral (terminal and intermediate) villi and their fetal capillaries and the arithmetic and harmonic mean thicknesses of the villous membrane (maternal aspect of trophoblast to luminal aspect of vascular endothelium) were estimated. An index of the variability in thickness of this membrane, and an estimate of its oxygen diffusive conductance, was derived secondarily. Group comparisons were drawn using two-way analysis of variance to identify main effects (of PE or IUGR) and interaction effects (between PE and IUGR). PE did not have significant effects on placental morphology and there were no significant effects of PE or IUGR on membrane thickness or its variability. In contrast, IUGR (with or without PE) was associated with reduced surface areas and this was the principal factor leading to a smaller membrane diffusive conductance in these placentas. When account was taken of fetal mass, specific conductance showed no effects of PE or IUGR despite the mass-specific conductance in pure IUGR placentas appearing to be smaller than that in controls. The decline in total conductances is indicative of perturbations operating at the levels of villous trophoblast and fetal vasculature and these may contribute to fetal hypoxic stress.

Stereology and the placenta: where's the point? -- a review.

The value of stereology for describing the three-dimensional (3D) structural composition and spatial arrangement of biological specimens from essentially two-dimensional (2D) thin sections is indisputable. By allowing economical quantitation of microscopical sections, stereology facilitates interpretations of normal and perturbed structure from the whole organ down to the subcellular levels. It incorporates the essential features of all sound study designs: (i) randomised sampling which is efficient and unbiased, and (ii) estimation tools which are simple, precise and unbiased or minimally biased. With these sampling and estimation tools, stereology offers a sure and safe option for characterising the total volumes, surfaces and lengths of placental compartments, the total numbers and mean sizes of nuclei or cells, and the spatial arrangements of tissues or intracellular ingredients. This review identifies the basic features of the stereological approach before indicating several ways in which stereology has been used to aid the description and interpretation of placental functional morphology from the whole organ to the molecular level. Examples include diffusive transport, villous growth, fetoplacental angiogenesis, trophoblast turnover, arterial vascular remodelling and high-resolution immuno-localization studies.

Effects of natural winter pasture and commercial pellet on the ultrastructure of small intestinal epithelium in reindeer.

Segments of small intestine (duodenum, jejunum and ileum) from slaughtered reindeer (Rangifer tarandus tarandus) grazing natural winter pastures (n=3) and reindeer fed commercially available pellets (RF-80) in winter (n=5) were collected and immediately fixed in McDowell's fixative. Transmission electron microscopy was employed to investigate the ultrastructural features of the epithelium and lamina propria along the small intestine and to relate these to the different diets. Major differences in ultrastructural features were observed between the small intestinal enterocytes of reindeer fed the two diets. Enterocytes in reindeer fed the natural diet displayed a normal appearance with a dense cytoplasm and distinct microvilli. In contrast, reindeer fed the commercial diet showed damaged enterocytes amongst the normal cells. Abnormal changes included disintegration and loss of microvilli, cytoplasmic swelling, loss of membrane integrity and increases in the width of intercellular spaces, especially in the jejunum.

Morphometric evidence that villous development and fetoplacental angiogenesis are compromised by intrauterine growth restriction but not by pre-eclampsia.

The aim of this study was to compare morphometric measures of villous development, villous capillarization, fetoplacental angiogenesis and capillary lumen remodelling in normal pregnancies with those complicated by intrauterine growth restriction (IUGR) with and without preeclampsia (PE). To this end, term placentas from control pregnancies (n = 9) and cases of IUGR alone (n = 5), PE alone (n = 5) and IUGR with PE (n = 5) provided random samples of tissue. These were fixed in formalin and Masson trichrome-stained wax sections were analysed stereologically. Overall growth of peripheral villi and fetal capillaries was assessed by estimating total volumes, surface areas and lengths. Villous capillarization was monitored using volume, surface and length densities and capillary:villus surface and length ratios. Measures of villous maturation and capillary lumen remodelling comprised mean cross-sectional areas, perimeters and shapes (perimeter(2)/area). Between-group comparisons were drawn using two-way analysis of variance. IUGR was associated with abnormal growth of villi and fetal capillaries. Reduced villous growth was not accompanied by changes in measures of villous capillarization or maturation and reduced capillary growth was not accompanied by changes in lumen calibre or shape. In contrast, PE was not associated with any main or interaction effects on placental morphometry. It is concluded that IUGR, but not PE, is associated with impoverished villous development and fetoplacental angiogenesis. The latter is due to production of fewer and/or shorter capillary segments (rather than a decrease in capillary calibre), does not affect villous capillarization and is not accompanied by luminal remodelling.

Vascular endothelial cadherin and beta-catenin in human fetoplacental vessels of pregnancies complicated by Type 1 diabetes: associations with angiogenesis and perturbed barrier function.

AIMS/HYPOTHESIS: Increased angiogenesis of fetoplacental vessels is a feature of pregnancies complicated by Type 1 diabetes mellitus, but the underlying molecular mechanisms are unknown. This investigation tests whether the diabetic maternal environment alters the phenotypic expression of placental vascular endothelial cadherin and beta-catenin, which have been implicated as key molecules in barrier formation and angiogenesis in the endothelium. METHODS: Term placental microvessels from normal pregnancies (n=8) and from those complicated by Type 1 diabetes (n=8) were perfused with 76-Mr dextran tracers (1 mg/ml) and subjected to immunocytochemistry, immunoblotting and microscopy. Junctional integrity, localisation and phosphorylation were investigated along with total protein levels of vascular endothelial cadherin, beta-catenin and vascular endothelial growth factor. Stereological sampling and estimation tools were used to quantify aspects of angiogenesis and endothelial proliferation. RESULTS: In the Type 1 diabetic placentae, junctional localisations of vascular endothelial cadherin and beta-catenin altered significantly, with more than 50% of microvessels showing complete loss of immunoreactivity and with no overall loss of total protein. Tracer leakage was associated with these vessels. There was a two- to three-fold increase in vessels showing junctional phospho-tyrosine immunoreactivity and hyperphosphorylated beta-catenin. Vascular endothelial growth factor levels were higher in these placentae. A four-fold increase in endothelial proliferation was observed, along with an increase in total length of capillaries without any change in luminal diameter. CONCLUSIONS/INTERPRETATION: Molecular perturbations of vascular endothelial cadherin and beta-catenin occur in fetoplacental vessels of pregnancies complicated by Type 1 diabetes. Phosphorylation and loss of these molecules from the adherens junctional domains may be influenced in part by the elevated levels of vascular endothelial growth factor in the placenta. Perturbations of the junctional proteins may explain the observed breach in barrier integrity and may contribute to the mechanisms that drive proliferation and increases in capillary length.

A simple method for comparing immunogold distributions in two or more experimental groups illustrated using GLUT1 labelling of isolated trophoblast cells.

Colloidal gold-labelling, combined with transmission electron microscopy, is a valuable technique for high-resolution immunolocalization of identified antigens in different subcellular compartments. Whilst the technique has been applied to placental tissues, few quantitative studies have been made. Subcellular compartments exist in three main categories (viz. organelles, membranes, filaments/tubules) and this affects the possibilities for quantification. Generally, gold particles are counted in order to compare either (a) compartments within an experimental group or (b) compartmental labelling distributions between groups. For the former, recent developments make it possible to test whether or not there is differential (nonrandom) labelling of compartments. The methods (relative labelling index and labelling density) are ideally suited to analysing label in one category of compartment (organelle or membrane or filament) but may be adapted to deal with a mixture of categories. They also require information about compartment size (e.g. profile area or trace length). Here, a simple and efficient method for drawing between-group comparisons of labelling distributions is presented. The method does not require information about compartment size or specimen magnification. It relies on multistage random sampling of specimens and unbiased counting of gold particles associated with different compartments. Distributions of observed gold counts in different experimental groups are compared by contingency table analysis with degrees of freedom for chi-squared (chi(2)) values being determined by the numbers of compartments and experimental groups. Compartmental values of chi(2)which contribute substantially to total chi(2)identify the principal subcellular sites of between-group differences. The method is illustrated using datasets from immunolabelling studies on the localization of GLUT1 glucose transporters in cultured human trophoblast cells exposed to different treatments.