A comparison of two sampling procedures for stereological analysis of cell pellets.
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Animals↗
Biomedical subjects
T M Mayhew
Publications and source records attributed to T M Mayhew.
Stereology and its impact on our understanding of human placental functional morphology.
The new stereology permits the objective, quantitative description of morphology by efficient and design-based methods. Applications to placentas in normal and abnormal pregnancies have proved of great value for challenging earlier misconceptions and interpreting better the processes of growth, morphogenesis, adaptation, and functioning at the whole-organ level. This contribution reviews the essential features of the stereological approach, identifies useful structural quantities, and provides examples of their application in various experiments of nature. We focus particularly on normal gestation and the effects of pregnancies associated with high altitude, maternal diabetes mellitus, preeclampsia, and maternal smoking.
Altitude↗
Morphometric studies on villi in human term placentae and the effects of altitude, ethnic grouping and sex of newborn.
Stereological methods were used to test whether or not altitudinal, ethnic and sex differences in birthweight in Bolivia are associated with differences in placental morphology. Estimates of the volumes, surface areas, lengths and diameters of villi and of fetal capillaries were obtained by analysing paraffin sections of tissue. The average highland placenta contains villi of smaller overall volume, surface area and length. These villi possess a less voluminous fetal vascular space, and the capillaries are smaller in mean diameter. The average Amerindian placenta has longer but thinner capillaries than the average placenta delivered by non-indigenous women. Altitude and ethnic grouping interact to have an effect on total villous length, whilst ethnic grouping and sex interact to influence fetal capillary length. These structural differences are discussed in the context of their likely impact on placental functioning, notably on exchange by passive diffusion.
Acclimatization↗
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.
Delivery, Obstetric↗
On the factors which contribute to thinning of the villous membrane in human placentae at high altitude. I. Thinning and regional variation in thickness of trophoblast.
Sections of human term placentae delivered at low and high altitude to indigenous and non-indigenous populations in Bolivia were analysed by stereological methods. Estimates were obtained of the arithmetic and harmonic mean thicknesses of villous membrane and of trophoblast. These values were then used to derive indices of thickness variation. No significant ethnic, sex or interaction effects were found. At high altitude, the villous membrane was significantly more variable in thickness owing to a lower harmonic mean but the same arithmetic mean thickness. Alterations in the arithmetic and harmonic mean thicknesses of the trophoblast contributed to the thinning of the villous membrane. The trophoblast was thinner and more irregular in thickness at high altitude. These findings are discussed in the context of placental diffusing capacity and the possible mechanisms for formation of vasculosyncytial membranes.
Altitude↗
On the factors which contribute to thinning of the villous membrane in human placentae at high altitude. II. An increase in the degree of peripheralization of fetal capillaries.
The contribution made by fetal capillary peripheralization to the thinning of the villous membrane seen in human placentae from high-altitude pregnancies is examined by stereological methods. Variables characterizing the shape of the villous core and the spatial relationships between trophoblast and capillaries are quantified. They shed light on the relative importance of dynamic versus mechanistic processes of villous membrane attenuation. Highland villi differ from lowland villi in several ways. On average, they possess a thinner barrier due to closer approximation of capillaries to overlying trophoblast; in consequence, the villous core is more irregular in outline and its surface (that of the inner aspect of the trophoblast) exceeds in area that of the outer aspect of the trophoblast. These results suggest that the dynamic process (protoplasmic streaming within syncytiotrophoblast) cannot alone explain thinning of the villous membrane. A mechanistic process (capillary peripheralization and obtrusion into the trophoblastic epithelium) is sufficient to account for the differences observed, although the possibility that both processes operate concurrently cannot be discounted. This report completes a study into factors contributing to villous membrane thinning at high altitude.
Altitude↗
Stereological re-examination of the effects of varying oxygen tensions on human placental villi maintained in organ culture for up to 12 h.
The effects of exposure to various oxygen tensions on villi in organ culture are re-examined. Villi from ten normal mature placentae were cultured under hypoxic (6 per cent oxygen) and hyperoxic (40 per cent oxygen) conditions for 6 or 12 h. Control tissue (zero time in culture) was also taken. Pieces of tissue were fixed by immersion and embedded in resin for semithin sectioning. Systematically sampled microscopical fields were analysed stereologically to estimate harmonic and arithmetic mean thicknesses for the trophoblast and for the villous membrane and to assess the volumetric composition and mean diameter of villi. Trophoblast thicknesses were influenced significantly by oxygen tension, being smaller in hypoxic and greater in hyperoxic media. No significant interaction terms or effects of time in culture were detected. Villous membrane thicknesses altered in a similar fashion to trophoblast thicknesses. No significant differences in the composition of villi were detected but villi tended to be greater in diameter during hyperoxia. Findings are discussed in the context of previously reported adaptations in vivo, in different regions of the placental lobule and during chronic maternal hypoxic stress at high altitude. We conclude that there is insufficient evidence to support the claim that villi can adapt in vitro to varying ambient oxygen tensions.
Chorionic Villi↗
From 13 weeks to term, the trophoblast of human placenta grows by the continuous recruitment of new proliferative units: a study of nuclear number using the disector.
A method is presented for obtaining assumption-free estimates of the number of nuclei in the trophoblast of the human placenta and for defining the size of the trophoblast proliferative unit (TPU). The method relies on the disector, a stereological device for counting arbitrary particles in 3-dimensional space using pairs of parallel sections separated by a known distance. It is applied to investigate factors which contribute to trophoblast growth from 13 weeks of gestation to term. Physical disectors were sampled systematically using adjacent 4-4.6 microns thick paraffin sections. Nuclei in the trophoblast (syncytial and cellular) were counted if they appeared in an unbiased counting frame on one section but were absent from the adjacent section. Nuclear packing densities were converted to absolute numbers of nuclei by using placental volume as the reference space. At 37-39 weeks, the average placenta contained 6.4 x 10(10) trophoblast nuclei of which 90 per cent were located within the syncytium and the remainder in the cytotrophoblast. From a knowledge of total trophoblast volume, it was found that each nucleus is associated with 970 microns3 of trophoblast and each cytotrophoblast cell with 11,000 microns3. The latter may be regarded as the volume of a TPU. From 13 weeks of gestation to term, there was a ninefold increase in nuclear number but the trophoblast volumes associated with nuclei, including the size of the TPU, remained constant. Growth of trophoblast is purely hyperplastic and occurs by recruitment of new TPUs.
Cell Division↗
Quantitative description of the elaboration and maturation of villi from 10 weeks of gestation to term.
Stereological methods have been applied to a cross-sectional sample of human placentae collected at 10-41 weeks of gestation in order to provide a quantitative description of the growth and maturation of villi. Random tissue sections were analysed to derive volumes, surface areas, lengths, diameters and membrane thicknesses for villi and their fetal capillaries. Expansion of the total volume and surface area of villi can be explained by a dramatic linear growth of terminal villi which begins at about the middle of the second trimester. Growth of intermediate villi also occurs but to a more limited extent. Linear growth is accompanied by villous maturation which involves increases in the relative volume of capillaries and in villous capillarization coupled with decreases in villous diameter, capillary diameter and harmonic thickness of the villous membrane. These findings confirm that placental growth and development depend greatly on growth and maturation of terminal villi. They do not confirm sinusoidal dilation of fetal vessels as a generalized phenomenon. They also support the contention that changes in effective diffusion distances across the villous membrane have real adaptive significance.
Analysis of Variance↗
Aspects of human fetoplacental vasculogenesis and angiogenesis. I. Molecular regulation.
Patterns of fetoplacental angiogenesis vary during gestation and in association with certain pregnancy pathologies. In a set of three linked reviews, we provide a survey of current knowledge about the molecular regulation, cellular players, qualitative and quantitative morphological features of the vascularization of human placental villi. Here, an account is given of the role played by hypoxia-inducible factor in mediating the effects of oxygen on production of growth factor ligands and receptors which regulate angiogenesis and vessel maturation. However, it should be noted that, for the human placenta early in gestation, the normal (i.e. physiological) partial pressure of O(2)is low but this does not mean that the tissue is hypoxic. Thus, the mechanisms of regulating angiogenic growth factor production may differ at this time in comparison to those found later in gestation or in other tissues or organs. The vasculature in the placenta is plastic and changes markedly as gestation progresses. This is controlled by the complex interplay between physical factors and chemical factors including oxygen, growth factors and growth inhibitors. The companion reviews describe morphological features of normal and pathological development of the human placenta in the context of the factors discussed here.
Adult↗
Aspects of human fetoplacental vasculogenesis and angiogenesis. II. Changes during normal pregnancy.
In this second review, we describe the main morphological events which accompany the development of the fetoplacental vascular system throughout normal human pregnancy and summarize findings on the expression of angiogenic growth factors and their receptors. Fetoplacental vasculogenesis starts at day 21 after conception by formation of haemangioblastic cords. In the following phase of branching angiogenesis (day 32 to week 25 post conception), haemangioblastic cords develop into a richly branched villous capillary bed with low fetoplacental blood flow impedance. This period is characterized by high placental levels of VEGF but moderate PlGF expression. In week 15, large centrally located villi show regression of peripheral capillary nets. In parallel, some remaining central capillaries acquire a tunica media and transform into arteries and veins. Beginning at about week 25 in the newly formed peripheral villi, angiogenesis switches from branching to non-branching and this period is accompanied by a steep drop in VEGF and a slower decline in PlGF expression. As a consequence of this switch, long poorly branched capillary loops are formed in the periphery of the fetoplacental vascular trees. These increase fetoplacental impedance but blood flow still increases due to rising fetal blood pressure. The possible interactions between (a). the biphasic development of intraplacental oxygen tensions, (b). changes in VEGF and PlGF levels and (c). developing vascular geometry are discussed. Special attention is given to the obvious discrepancy between sudden elevation of intervillous oxygen tensions which is not coincident with the appearance of angiogenic growth factor peaks and the switch from branching to non-branching angiogenesis. Finally, we deal with methods of quantifying aspects of angiogenesis in the villous vascular system and summarize the main findings during uncomplicated human pregnancy.
Adult↗
Aspects of human fetoplacental vasculogenesis and angiogenesis. III. Changes in complicated pregnancies.
Patterns of fetoplacental angiogenesis vary not only during the course of a normal pregnancy but also in certain pregnancy pathologies. Here, we review some of the molecular and morphological events which occur in complicated pregnancies. The pregnancy complications are chosen in an attempt to represent the possible different origins (preplacental, uteroplacental, postplacental) of fetal hypoxia. Molecular events focus on reported changes in hypoxia-inducible factors, angiopoietins and the vascular endothelial, basic fibroblast and placenta growth factors and their receptors. Morphological changes focus on patterns of angiogenesis (branching and non-branching) and a consistent set of morphometric descriptors (covering measures of total capillary growth, villous capillarization and capillary size and shape in transverse section). Apart from some uncertainties due to lack of information, or failure to resolve fully the effects of intrauterine growth restriction and pre-eclampsia, alterations in the angiogenic growth factors and morphologies of capillaries and villi in different complicated pregnancies seem to conform reasonably well to those predicted by the fetal hypoxia paradigm. However, it is clear that future studies on the effects of different origins of fetal hypoxia should exercise more care in the choice and interpretation of relevant descriptors and take more account of the parallel effects of possible confounders. In addition, rather than comparing uncomplicated and complicated pregnancies only at term, more information about molecular and morphological events that occur throughout gestation would be extremely valuable. This includes further studies on changes in growth factor receptors, the less-well-documented angiogenic factors (e.g. angiogenin, angiostatin, endostatin) and the associations between endothelial cells and pericytes. A more integrated approach involving also parallel analysis of the effects of erythropoietin and other potential vasoactive factors on the behaviour and morphology of fetal vessels would be beneficial.
Adult↗
Oxygen diffusive conductances of human placentae from term pregnancies at low and high altitudes.
A morphometric model of oxygen diffusion is employed to calculate the partial, total and specific conductances of human placentae from low- and high-altitude term pregnancies. Placentae were obtained from indigenous and non-indigenous populations and the diffusion pathway dissected into six tissue compartments. Birthweights were reduced at high altitude but were greater in natives versus non-natives. The altitudinal differences were associated with changes in placental diffusive conductances. The partial conductance of the villous trophoblast was conserved but the conductance on the maternal side was increased as was the conductance of the villous stroma. Fetal conductances were conserved (plasma) or diminished (erythrocytes). We conclude that birthweights are reduced at high altitude despite the attempts to increase the total placental diffusive conductance for oxygen. The mechanisms underlying these adaptations are discussed.
Adaptation, Physiological↗
Changes in oxygen diffusive conductances of human placentae during gestation (10-41 weeks) are commensurate with the gain in fetal weight.
Evidence is adduced that the oxygen diffusive conductance of the human placenta increases during gestation. Results are based on placentae from 10 weeks to term. The diffusion pathway is analysed as six discrete tissue compartments arranged in series. Stereological and physicochemical data are used to determine partial, total and specific diffusive conductances for each organ and for its tissue compartments. During gestation, maternal and fetal vascular spaces became more voluminous and exchange surface areas of villi and capillaries expanded. Both were manifestations of placental growth and development. Maturation of villi was evident as decreases in effective diffusion distances across the trophoblast and villous stroma. These alterations contributed to improvements in partial and total conductances which continued towards term. Changes within the trophoblast and stroma were particularly influential in determining total conductance. Those on maternal and fetal sides of the diffusion pathway were less important. The rise in total conductance was related directly to the rise in fetal weight. Specific diffusive conductance was constant throughout gestation.
Diffusion↗
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.
Female↗
Stereological studies on the true thickness of the villous membrane in human term placentae: a study of placentae from high-altitude pregnancies.
Stereological principles were used to calculate functionally significant dimensions of the human villous membrane, its arithmetic mean thickness (Ta) and its harmonic mean thickness (Th). The former is proportional to tissue mass and oxygen consumption, the latter to diffusional resistance. For a group of 15 term placentae from uncomplicated pregnancies at high altitude, the average values were Ta = 4.44 micron, Th = 3.56 micron and Ta/Th = 1.26. The latter figure provides a useful quantitative expression for the efficiency of the membrane in gas and metabolite diffusion. It implies that vasculosyncytial membranes and syncytial knots decrease resistance to diffusion by 26 per cent, compared with that of a membrane with uniform thickness throughout. The methods are simple to apply and provide better estimates of true thickness than do measurements confined to thin sections.
Altitude↗
Microscopical morphology of the human placenta and its effects on oxygen diffusion: a morphometric model.
A combination of stereological and physicochemical data was used to estimate the oxygen-diffusing capacity of the human term placenta. The effects on this morphometric diffusing capacity of isolated changes in placental structure were investigated by permitting volumes, surface areas and harmonic mean diffusion distances to deviate from normal baseline (term) values. Diffusion performance could be monitored effectively by estimating only three structural quantities. The most influential variable was the harmonic mean thickness of the villous membrane followed by the surface areas of villi and of fetal capillaries. Blood space volumes and plasma diffusion distances had negligible effects on diffusing capacity. Conclusions are discussed in the context of published findings on changes in placental anatomy which occur during gestation, abnormal pregnancies and pregnancies at high altitude.
Chorionic Villi↗
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.
Adult↗