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.

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.

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.

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.

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.

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.

Accurate prediction of Purkinje cell number from cerebellar weight can be achieved with the fractionator.

Purkinje cell nucleoli are used as counting units in order to obtain unbiased (fractionator) estimates of the number, N, of Purkinje neurons in adult mammalian cerebella of known weight, W. Regression analysis is then employed to establish the nature of the relationship between logN and logW. The linear regression equation defines an allometric relation that is employed to predict number in cerebella of known weight from other mammals. Predicted numbers are tested against empirical estimates. For 19 cerebella ranging in weight from 0.2 g (rat) to 113 g (human), the allometric relation between Purkinje cell number and organ weight was determined. By using this relation, the mean complement in three rabbit cerebella (average weight, 0.87 g) is predicted to be 0.63 million. This figure is confirmed by fractionator estimates made on the same three brains. The cat cerebellum should contain about 1.5-2.0 million Purkinje cells. An estimate of 1.2-1.3 million cells is to be found in the literature. Including rabbit cerebella in a refined equation yields the following relation: N = 686000W(0.695). With this refined equation, further predictions are made about the numbers likely to be found in the cerebella of the dog, goat, pig, ox, and horse. The numbers predicted for these animals must await experimental verification, but they are entirely consistent with previous suggestions that neuronal packing densities decrease with increasing brain size.

On the number of Purkinje cells in the human cerebellum: unbiased estimates obtained by using the "fractionator".

Stereological estimates of the numbers of Purkinje cell nucleoli in human cerebellar cortex have been obtained from systematic random samples of tissue by using the fractionator. The estimates are unbiased by fixation, section thickness, or sampling errors and are independent of any assumptions about cell shape, size, or spatial orientation. Twelve brains from aged subjects of both sexes were examined. The average complement of nucleoli in four female brains (age range 71-93 years) amounted to 14.8 millions (with an observed coefficient of variation between subjects of 29%). For three male brains (76-91 years), the corresponding estimates were 15.7 millions (10%). No significant sex differences were found for these small samples. Five brains of unknown sex and age yielded values of 15.8 millions (18%). For the twelve brains examined, the total number of Purkinje cell nucleoli per cerebellum was found to be 15.4 millions (19%). Estimated numbers showed a significant positive correlation with cerebellar weights. The number of nucleoli in an individual cerebellum was obtained with high precision in as short a time as 4 hours.

Methodological problems in placental morphometry: apologia for the use of stereology based on sound sampling practice.

Several problem areas in morphometry of human and animal placentae are reviewed. Attention is given to methods of tissue processing (handling, mode of fixation, embedding, shrinkage) and sampling (of organs, tissue blocks, sections, micrographs). Principal sources of bias and sampling variability are identified and the crucial importance of randomized sampling is emphasized. Methods for obtaining structural quantities from sections are compared. The case is made for estimating absolute values (volumes, surface areas, lengths, numbers, thicknesses) using stereological principles rather than relying on planar data (profile areas, perimeter lengths, numbers, apparent thicknesses). Absolute values may be obtained simply and efficiently without resort to expensive measuring devices. Finally, morphological descriptors suitable for correlating with functional data or for comparing normal and diseased organs are surveyed.

Placental morphogenesis and the star volumes of villous trees and intervillous pores.

In 27 human placentae collected at 13-39 weeks, the growth and morphogenesis of villi and of the maternal intervillous space were assessed using a design-based stereological approach for estimating star volume. The purpose was to quantify changes occurring during these processes and to derive parameters pertinent to physiological performance (notably transport capability and haemodynamics). Placentae were sampled by cutting paraffin sections at random locations and orientations. Estimates of the global volumes of peripheral (terminal and intermediate) villi and intervillous space were derived from placental volumes via point counting. Total villous surface was estimated by intersection counting and length by transection counting. Treating villi like the branches of tree-like networks and the intervillous space as a system of confluent pores, estimates of the star volumes of these compartments were also made. These volumes were calculated by measuring point-sampled intercept lengths. The total volumes of villi and intervillous space increased steadily throughout gestation. After the second trimester, the increase in volume of villi was accompanied by a decrease in villous star volume. This can be explained by the continued elaboration and maturation of terminal villi, the combined length of which increased whilst mean diameter declined. The star volume of the intervillous space also declined and this, too, is consistent with villous growth and proliferation.

Quantitative evidence for the spatial dispersal of trophoblast nuclei in human placental villi during gestation.

A common assertion in the literature is that Langhans cells in placental villi decline in number during gestation but this is a misinterpretation which may be caused by the greater growth of villous surface area compared with trophoblast volume. To test this possibility, human placentae were collected at 12-41 weeks of gestation for a cross-sectional study on the packing density of nuclei within villous trophoblast. Numbers of nuclei in the cyto- and syncytiotrophoblast were estimated using a design-based stereological device, the physical disector (parallel pairs of sections). Surface areas were estimated in order to assess the overall growth of villous arborizations. Packing densities of nuclei were calculated and expressed as numbers/1000 microns 2 of villous surface. Densities decreased during gestation and this can be explained by expansion of villous surface area and thinning of trophoblast. The biggest drop in packing density of cytotrophoblast nuclei (30 per cent) occurred between 17-21 and 22-26 weeks and this period coincided with the largest changes in villous surface area (62 per cent increase) and trophoblast thickness (30 per cent decrease). Results are consistent with the notion of an epithelial proliferative unit of constant volume and comprising about nine syncytiotrophoblast nuclei per Langhans cell.

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.

Villous trophoblast: morphometric perspectives on growth, differentiation, turnover and deposition of fibrin-type fibrinoid during gestation.

Villous trophoblast growth and deposition of perivillous fibrin-type fibrinoid were examined in human placentas from 10-41 weeks of gestation. The main aims were: (1) to study growth of different trophoblast domains implicated in epithelial turnover (proliferation, differentiation, extrusion, denudation); (2) to test predictions about relationships between fibrinoid deposits and intervillous volume or villous surface area; and (3) to derive baseline data for future studies on complicated pregnancies. Microscopical fields on trichrome-stained paraffin sections were selected by systematic random sampling. Volumes were estimated stereologically by point counting and surface areas by intersection counting. Apparent differences were tested by analyses of variance and relationships by regression and contingency table analyses. All compartments increased in absolute volume and/or surface area although not at the same rates. Relative volumes of cytotrophoblast were greater at earlier stages (10-20 weeks) but, due to differential growth, syncytiotrophoblast nuclear aggregation sites (syncytial knots and 'bridges') occupied greater proportions of trophoblast volume and surface near term (37-41 weeks). Fibrinoid volume correlated positively with intervillous volume and villous surface area but, relative to intervillous volume, seemed to increase near term. Findings confirm that the incidence of syncytial knots increases during gestation and contributes to trophoblast thickness variability. Greater relative volumes and surfaces of syncytial 'bridges' are consistent with increased incidences of true intervillous bridges and/or villous branching points. These findings support the notion that fibrinoid deposition during normal gestation is influenced by the quality of vascular perfusion but also emphasize that the villous surface is another important factor. Haemostatic events operate at the maternal surface of trophoblastic epithelium and influence the steady state between coagulation and fibrinolysis. Fibrinoid is deposited at sites of trophoblast de-epithelialization and these arise following trauma (e.g. abruption of intervillous bridges) or during the extrusion phase of normal epithelial turnover. Like knots and bridges, sites of de-epithelialization also expand at a faster rate than overall villous surface area. These and other events in villous development can be interpreted in terms of a coherent concept of epithelial turnover in which proliferation early in gestation is mainly for growth whilst that at later stages is mainly for renewal and repair.

Maternal diabetes mellitus is associated with altered deposition of fibrin-type fibrinoid at the villous surface in term placentae.

Placentae from control and diabetic patients were used to test three null hypothesis: (1) there are no significant group differences in the volumes of villous syncytiotrophoblast compartments or intervillous fibrin-type fibrinoid, (2) perivillous fibrin-type fibrinoid is deposited randomly at the surface of trophoblast, and (3) amounts and deposition patterns of perivillous fibrin-type fibrinoid do not vary between groups. Term placentae were collected from non-diabetic subjects and five groups of diabetic women classified according to duration, severity and insulin dependence. Tissue specimens and sections were obtained by uniform random sampling. Volumes and surface areas of fibrin-type fibrinoid and trophoblast compartments (thin, syncytial knot, syncytial bridge and denuded regions) were estimated stereologically and compared using variance, chi-squared and contingency table analyses. As to null hypothesis (1), no group differences in volumes of trophoblast compartments were found but volumes of intervillous fibrin-type fibrinoid were greater in the non-insulin-dependent diabetic group. As to null hypothesis (2), regardless of group, fibrin-type fibrinoid was deposited preferentially at sites of denudation in every placenta examined. As to null hypothesis (3), villous surface areas occupied by perivillous fibrin-type fibrinoid were greater in type 1 (insulin-dependent) diabetics with complications (diabetic nephropathy or retinopathy). The surfaces of trophoblast occupied by fibrin-type fibrinoid were also notably larger in non-insulin-dependent diabetics and type 1 diabetics with complications. Except for the surface of denudation sites (which also increased in diabetes), there were no differences in the surfaces of trophoblast regions. These results confirm that the haemostatic steady state is perturbed in the diabetic placenta, that perivillous fibrin-type fibrinoid is deposited preferentially at sites of epithelial loss/damage, and that some diabetic groups are affected differentially.

Quantitative studies on the villi, trophoblast and intervillous pores of placentae from women with well-controlled diabetes mellitus.

Human placentae from well-controlled diabetic women were collected after 37 weeks of gestation and divided into three groups according to the duration and severity of diabetes mellitus established by White classification criteria. A fourth group of subjects served as matched controls. Various morphometric variables not estimated hitherto (including the star volumes of villous 'domains' and intervillous 'pores' and trophoblast surface denudation) were assessed stereologically. The aims were to test whether or not (1) control values of these structural quantities are preserved in well-controlled diabetes mellitus, and (2) differences occurred between alternative diabetic groups. Placental specimens were obtained by systematic random sampling procedures and paraffin sections were cut at random positions and orientations. Volume densities of peripheral (terminal+intermediate) villi and intervillous spaces were estimated by test point counting and multiplied by placental volumes in order to convert them into absolute volumes. Volume estimates were also obtained for trophoblast, syncytiotrophoblast nuclei and intervillous fibrin-type fibrinoid. Villous surface areas were estimated by intersection counting and the star volumes of villi and intervillous pores were obtained by measuring the lengths of point-sampled intercepts. Calculations were also made of the theoretical numbers of villous domains and intervillous pores and of the numbers of syncytiotrophoblast nuclei. No significant differences were detected between control and diabetic placentae, or between White classes, for any of the estimated quantities. It is concluded that normal values are preserved by good glycaemic control regardless of diabetic grouping.

A stereological method for testing whether or not there is random deposition of perivillous fibrin-type fibrinoid at the villous surface: description and pilot applications to term placentae.

We present a stereological method for testing whether or not there is random deposition of fibrin-type fibrinoid (FTF) at the villous surface of human placenta. The method requires random sampling of tissue with test lattice lines superimposed on microscopic fields at random positions and orientations. Test lines are used to generate chance intersections with specified sub-domains of the villous surface. At least three sub-domains are distinguishable: non-syncytial knots (nonSK), syncytial knots (SK) and areas of trophoblast de-epithelialization (DEP). Other sub-domains may be included to suit individual circumstances and project aims. The relative numbers of intersections with sub-domains provide the basis for an 'expected' distribution. Subsequently, this is compared with an 'observed' distribution which can be calculated from empirical estimates of the numbers of intersections with sub-domains associated with perivillous FTF (e.g. nonSK+FTF, SK+FTF and DEP+FTF). Expected and observed distributions can be compared by a chi-squared analysis. If the null hypothesis (no difference) is rejected, chi-squared values for individual sub-domains can be analysed in order to localize and interpret sites of preferential deposition. Comparisons may be drawn for individual placentae as well as a group of placentae, thereby permitting assessment of inter-placental variability. Finally, between-group comparisons may be drawn in order to test whether or not FTF deposition patterns differ in control and other pregnancies. Worked examples of the statistical procedures are provided. Preliminary results of applications to placentae from normal and complicated (hypobaric hypoxia) pregnancies are presented. They show that FTF deposition is non-random and preferentially located at sites of de-epithelialization. De-epithelialization may be a consequence of syncytial degeneration but also, at least in part, of continuous trophoblast turnover in which syncytial fragments rich in (pre-) apoptotic nuclei detach from the epithelium and are deported from the maternal intervillous space. The nascent detachment site is immediately covered by FTF prior to repair by re-epithelialization.

Hypobaric hypoxia and villous trophoblast: evidence that human pregnancy at high altitude (3600 m) perturbs epithelial turnover and coagulation-fibrinolysis in the intervillous space.

Spatial relationships between fibrin-type fibrinoid and regions of villous trophoblast were examined in order to address two main questions: [1] is high-altitude pregnancy accompanied by changes in the sizes of trophoblast compartments (cytotrophoblast, syncytiotrophoblast, denudation sites)?, and [2] do highland placentae differ in the amounts and distribution patterns of perivillous fibrin-type fibrinoid? Placentae were collected from two ethnic groups completing term pregnancies at low (400 m above sea level; n=25) and high (3600 m; n=45) altitude in Bolivia. Masson trichrome-stained sections were sampled randomly and analysed stereologically to estimate compartment volumes and surfaces. Comparisons were drawn using variance, Chi-squared and contingency table analyses. At high altitude, birthweights were 265 g lower and placentas had a larger intervillous space (270 cf 181 cm(3)), less fibrin-type fibrinoid (4.1 cf 8.4 cm(3) by volume; 2570 cf 4430 cm(2) by surface area), less villous trophoblast (50 cf 73 cm(3)) and a smaller villous surface (5.6 cf 7.0 m(2)). Volumes were reduced in all syncytiotrophoblast compartments (with and without nuclear aggregations). Cytotrophoblast was maintained and its relative volume increased significantly (from 2.7 to 3.6 per cent of trophoblast volume). Decreases in villous surface area affected primarily thinner (nuclear aggregate-free) regions of syncytium. Regardless of altitude, fibrin-type fibrinoid was deposited non-randomly: it was preferentially located at sites of trophoblast denudation. Although no altitudinal differences in fibrin-type fibrinoid patterns were detected, absolute surfaces were diminished on denuded and thinner regions of trophoblast but not on syncytial knots or bridges. Ethnic differences at low altitude (relatively greater deposits on denudations in Amerindians) were minimized at high altitude. We conclude that pregnancy at high altitude alters the epithelial steady state (towards cytotrophoblast and away from syncytiotrophoblast) and the coagulation-fibrinolysis steady state in the intervillous space (to favour fibrinolysis over coagulation). Thinner regions of syncytiotrophoblast may be the main sites of greater fibrinolytic or anticoagulatory activity. The findings are partly consistent with results from in vitro studies which indicate that hypoxia stimulates proliferation of cytotrophoblast but impairs fusion into syncytium.