J. Anat. (2009) 215, pp77–90 doi: 10.1111/j.1469-7580.2008.00994.

REVIEW
Blackwell Publishing Ltd

A stereological perspective on placental morphology in

normal and complicated pregnancies
Terry M. Mayhew
Centre for Integrated Systems Biology & Medicine, School of Biomedical Sciences, Queen’s Medical Centre, University of
Nottingham, UK

Abstract
Stereology applied to randomly-generated thin sections allows minimally-biased and economical quantitation of
the 3D structure of the placenta from molecular to whole-organ levels. With these sampling and estimation tools, it
is possible to derive global quantities (tissue volumes, interface surface areas, tubule lengths and particle numbers),
average values (e.g. mean cell size or membrane thickness), spatial relationships (e.g. between compartments and
immunoprobes) and functional potential (e.g. diffusive conductance). This review indicates ways in which stereology
has been used to interpret the morphology of human and murine placentas including the processes of villous
growth, trophoblast differentiation, vascular morphogenesis and diffusive transport. In human placenta, global
quantities have shown that villous maturation involves differential growth of fetal capillaries and increases in
endothelial cell number. Villous trophoblast is a continuously renewing epithelium and, through much of gestation,
exhibits a steady state between increasing numbers of nuclei in cytotrophoblast (CT) and syncytiotrophoblast (ST).
The epithelium gradually becomes thinner because its surface expands at a faster rate than its volume. These changes
help to ensure that placental diffusing capacity matches the growth in fetal mass. Comparable events occur in the
murine placenta. Some of these processes are perturbed in complicated pregnancies: 1) fetoplacental vascular
growth is compromised in pregnancies accompanied by maternal asthma, 2) changes in trophoblast turnover occur
in pre-eclampsia and intrauterine growth restriction, and 3) uteroplacental vascular development is impoverished,
but diffusive transport increases, in pregnant mice exposed to particulate urban air pollution. Finally, quantitative
immunoelectron microscopy now permits more rigorous analysis of the spatial distributions of interesting molecules
between subcellular compartments or shifts in distributions following experimental manipulation.
Key words complicated pregnancies; functional morphology; placenta; random sampling; slice images; stereology;
uncomplicated pregnancy.

including the positions and directions of slices through them,

Introduction
Revealing the internal structure of the placenta often
involves viewing sectional images on physical, optical or
medical slices. Whilst slicing permits visualization of
substructure (including localization of interesting molecules)
at adequate resolution, it also causes loss of dimensional
information, which, if ignored, has the potential to confuse
interpretations of 3D spatial composition and organization.
Fortunately, by randomizing the sampling of placentas,
Correspondence
T. M. Mayhew, Centre for Integrated Systems Biology & Medicine,
School of Biomedical Sciences, Floor E, Queen’s Medical Centre,
University of Nottingham, Nottingham NG7 2UH, UK.
T: + 44 115 8230132; F: + 44 115 8230142;
E: terry.mayhew@nottingham.ac.uk
Accepted for publication 5 September 2008
Article published online 2 January 2009
the 3D properties of individual components (e.g. the average
CT cell), sets of components (e.g. the terminal villi) or the
average organ can be estimated efficiently and with minimal
bias. Sampling methods, including those suitable for murine
and human placentas, have been reviewed elsewhere (Coan
et al. 2004; Mayhew, 2008; Veras et al. 2008).
Stereology (Howard & Reed, 2005; Mayhew, 2006a)
combines random sampling with estimation tools to derive
global quantities (total volumes, surfaces, lengths, numbers),
average sizes (e.g. mean cell volume, mean layer thickness),
spatial relationships (e.g. between immunogold markers and
subcellular structures) or functional indices (e.g. diffusive
conductance). Generating quantitative data in this way
facilitates the interpretation of 3D functional morphology
during development, complicated pregnancy and experi-
mental manipulation.
The aim here is to emphasize the practical value of
stereology in microscopical studies based on thin sections

© 2009 The Author

Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
78 Stereology and the placenta, T. M. Mayhew
of human and animal placentas. Findings from recent studies
illustrate how spatial quantities can be used to describe
normal development of the placenta and the ways in which
morphology is altered in complicated pregnancies. Topics
include 1) fetoplacental vascular growth in pregnancies
accompanied by maternal asthma, 2) trophoblast turnover
in pre-eclampsia (PE) and intrauterine growth restriction
(IUGR), 3) maternal vascular development and diffusive
transport in placentas from mice exposed to polluted air,
and 4) quantifying the spatial distributions of interesting
molecules using high-resolution immunocytochemistry.
Morphometric descriptors of relevant
processes
Growth and development
The growth of villi, fetoplacental vessels and maternal vascular
spaces can be expressed in terms of volumes, surface areas,
lengths or numbers. Volume is the measure of size in 3D and
placental compartments are 3D spaces constructed from
lower levels of organization (organelles, cells, extracellular
matrices, tissues). The volumes of these spaces provide
measures of their mass or bulk and so can be used not only
to monitor growth but also to indicate the likely demands
for nutrients and respiratory gases. Volume may also provide
an index of supply, e.g. the volume of a vascular space is
related to the transport of gases, nutrients or heat.
The surfaces of villi, capillaries and intervascular barriers
act as interfaces between compartments and are involved
in passive transport (e.g. diffusion of respiratory gases) and
incorporate molecules active in transport and recognition
(carriers, ion channels, receptors), cell-cell and cell-matrix
adhesion (tight junctions, adhaerens junctions, desmosomes),
communication (gap junctions) and metabolism (membrane-
bound enzymes). Like volume, surface area can be employed
to monitor growth.
Length, another measure of growth, describes the linear
extent of arborizations (e.g. villi) and tubular structures
(e.g. capillaries). The linear features may be straight or curved,
continuous or discontinuous, branched or unbranched.
Particle number provides biologically useful information
in two main contexts: (1) genesis, growth and transformation,
and (2) communication and connectivity.
Genesis, growth and transformation
Growth may occur by proliferation (cell division), hypertrophy
(cell growth) or accretion (interstitial or extracellular growth).
For instance, an increase in placental size may depend on the
number of cells, the sizes of a fixed set of cells or syncytium
and the quantity of extracellular matrix.
Communication and connectivity
Vascular endothelial and smooth muscle cells communicate
via gap-junctions. Where communication is due to molecular
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
interactions, usually a marker or label will indicate a site of
enzymic or other activity or the location of a specific pro-
tein (e.g. connexins). In immunoelectron microscopy, the
markers are gold particles which are counted to monitor
shifts in localization between study groups or to quantify
labelling intensities of different compartments within a
cell or syncytium.
The maturation status of villi might be expressed in terms
of their calibre, which in human placenta declines as one
passes along the topological sequence from stem villi to
terminal villi. A convenient and simple way of estimating
this is to divide total volume by total length. For linear
structures, this equates to mean cross-sectional area and
offers a less biased estimator of calibre than mean diameter
(Kaufmann et al. 2004). Other useful measures of maturation
are the vascularization or capillarization indices, which
include volume, surface or length densities and capillary : villus
surface or length ratios (Kaufmann et al. 2004).
Angiogenesis
Angiogenic changes may be quantified effectively by
estimating nett growth (total capillary volume, surface,
length or endothelial cell number) and indices of villous
capillarization and capillary calibre (Kaufmann et al. 2004).
Again, the latter can be estimated as mean cross-sectional
area. To assess the contributions made by branching,
estimates are required of the numerical density of branch
sites and this information can be combined with length data
to estimate segment lengths (Gambino et al. 2002). However,
microvascular arrangements and branching patterns can
be analysed directly in 3D by scanning electron microscopy
or confocal microscopy (e.g. Jirkovska et al. 2002).
Villous trophoblast recruitment and loss
Trophoblast presents a number of different compartments
which reflect its phases of differentiation (Mayhew, 2001).
The volumes of these compartments (including CT and ST)
can be estimated, together with their surface areas or
complements of nuclei. It is possible also to subdivide the
ST compartment to account for areas of thinning (vasculo-
syncytial membranes) or thickening (syncytial knotting)
or loss of epithelial integrity due to damage (Mayhew &
Barker, 2001). By determining relevant ratios (numerical
or surface), the epithelial steady state between CT and ST,
and the association between trophoblast compartments
and perivillous fibrin-type fibrinoid, can be evaluated.
Passive diffusion
As it grows, the fetus requires more oxygen and nutrients
from the mother. Oxygen crosses the placenta by passive
diffusion as do certain nutrients and other gases. For the
intervascular barrier, diffusive transport is partly determined
© 2009 The Author

by its physical dimensions, including its surface areas and
effective diffusion distances. The total volume of a barrier,
divided by surface area, can be used to express an arithmetic
mean thickness, e.g. the mean depth of the trophoblastic
epithelium. Barrier thickness is inversely proportional to
diffusive conductance but, in this context, it is preferable
to monitor harmonic rather than arithmetic mean thickness
because harmonic means deal more effectively with barriers
of varying local thicknesses.
The capacity of the human placenta for passive diffusion
is expressed as a total conductance in cm3 min–1 kPa–1
(Mayhew et al. 1990, 1993a,b; Ansari et al. 2003). In the
human placenta, oxygen dissociates from haemoglobin
in maternal erythrocytes and crosses maternal plasma,
trophoblast, fetal endothelium and plasma before binding
to haemoglobin in fetal erythrocytes. Consequently, at
least six tissue compartments offer resistances to diffusion
and these can be summed to derive a total resistance, the
reciprocal of which is total conductance. Serial resistances
can be calculated from vascular space volumes and oxygen–
haemoglobin chemical reaction rates, surface areas,
harmonic mean thicknesses and permeability coefficients.
The intervascular barrier of the murine placenta differs
in composition from that of the human placenta and
estimates of its diffusive conductance have been confined
to the intervascular tissue rather than the placenta as a
whole (Coan et al. 2004; Veras et al. 2008). This intervascular
barrier comprises a layer of CT, two layers of ST and a layer
of fetal vascular endothelium.
In reality, several factors (e.g. vascular shunts and local
perfusion : diffusion ratio inequalities) reduce the effective-
ness of the placenta for passive diffusion. Consequently,
this approach provides the maximal diffusive conductances
achievable under optimal conditions. Nevertheless, estimates
have real comparative worth. Moreover, mass-specific con-
ductances can be calculated by relating total conductances
to fetal weight (Mayhew et al. 1993a,b; Coan et al. 2004).
Spatial shifts in molecular localization
Molecules detected by high-resolution immunocytochem-
istry localize in different ultrastructural compartments which
may be volumes (e.g. nucleus, mitochondria, cytosol) or
surfaces (e.g. plasma membrane, cristae membranes).
Their presence in different compartments can be assessed
by counting immunogold particles. The conventional way
of evaluating intensity of labelling has been to relate
numbers of gold particles to compartment profile areas
or trace lengths, an index known as labelling density, LD
(Griffiths, 1993). Recently, more efficient ways of estimating
LD have been devised and these can be used to express
preferential labelling of compartments as a relative
labelling index. This provides a measure of the degree
to which a compartment is labelled in comparison with
random labelling (when all compartments are expected
© 2009 The Author
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Stereology and the placenta, T. M. Mayhew 79
to show the same LD). By combining these approaches with
appropriate statistical tests, it is now possible to compare
labelling patterns between compartments in a cell or to
follow shifts in patterns between groups of cells (Mayhew
& Lucocq, 2008b). Between-group comparisons rely simply
on counts of gold particles associated with compartments
and do not require estimates of compartment sizes.
The following examples illustrate the use of these
measures to study the placenta.
Morphological features of normal placental
development
Human placenta
Villous trees are established in two phases (Benirschke &
Kaufmann, 2000; Kaufmann et al. 2004): an early phase
provides the main branches (stem and immature inter-
mediate villi) and, starting around mid-gestation, a later
phase establishes a multitude of fine peripheral branches
(mature intermediate and terminal villi). Terminal villi
have an extensive surface (>10 m2 at term) and small calibre
(40–100 μm) and are crucially important for transplacental
exchanges. Their trophoblast comprises inner proliferative
CT cells and an outer differentiating ST which surround a
mesodermal stroma containing fetoplacental capillaries.
In the third trimester, the main tissue layers intervening
between the maternal and fetoplacental circulations of
these villi are the ST, scattered CT cells, epithelial basal
laminae and variable amounts of stroma, and vascular
endothelium. Local thinning of trophoblast, coupled with
peripheralization of capillaries, reduces effective diffusion
distances by creating vasculosyncytial membranes (Benirschke
& Kaufmann, 2000). The incompleteness of the CT layer later
in gestation contributes to this thinning and explains why the
intervascular barrier is described as haemomonochorial. In
fact, CT cells maintain contact via contiguous processes which
radiate from the cell body and seem to form a functional
continuum (Jones et al. 2008).
Villous growth is influenced by fetoplacental angio-
genesis which is also biphasic and involves changes in the
number and dimensions of vessel segments: an initial phase
of capillary branching is followed by one of greater non-
branching angiogenesis (Benirschke & Kaufmann, 2000;
Mayhew, 2002; Kaufmann et al. 2004). In a vessel segment,
resistance to blood flow is proportional to length and inversely
proportional to the square of cross-sectional area. For a set of
segments, total resistance depends on whether the arrange-
ment is parallel or serial. If parallel, total resistance is less
than the partial resistance of any individual segment but,
for a serial arrangement, it is equal to the sum of partial
resistances. Consequently, parallel arrangements (produced
by branching angiogenesis) tend to be better than serial
arrangements (produced by non-branching angiogenesis)
because they offer smaller overall impedance.
80 Stereology and the placenta, T. M. Mayhew
Branching angiogenesis occurs in two main ways (Djonov
et al. 2003; Charnock-Jones et al. 2004). Sprouting creates
side branches whilst intussusception involves creating two
lumina by means of endothelial ingrowth. By contrast, non-
branching angiogenesis involves elongation of existing
vessel segments and this may be driven by either or both of
two processes: endothelial cell proliferation and intercalation
of endothelial progenitor cells (Charnock-Jones et al. 2004).
During the first trimester, large-calibre immature inter-
mediate villi (with a complex capillary network surrounding
larger central vessels) are common. In the third trimester,
there is a preponderance of slender terminal villi containing
one to two peripheral capillary loops (Leach et al. 2002).
Phased changes in indices of villous capillarization, notably
capillary : villus length ratios (Mayhew, 2002), are consistent
with the notion that angiogenesis influences villous
differentiation. Reductions in the mean calibres of fetal
capillaries have been reported in some studies (Kaufmann
et al. 2004).
Mouse placenta
The definitive placenta of the mouse is haemotrichorial and
divisible into three main zones: the labyrinth, junctional
zone and decidua basalis. The labyrinth lies closest to
the chorionic plate and contains the irregularly shaped
maternal vascular spaces and fetoplacental capillaries.
Separating the two circulations is the intervascular barrier,
which comprises a superficial layer of CT cells and two
layers of ST. Recent studies from embryonic days E12.5
to E18.5 (Coan et al. 2004) have shown that the placenta
reaches its maximum size by E16.5, with the labyrinth
continuing to expand thereafter and at a faster rate than
the other two zones. Within the labyrinth, the volumes
and surfaces of the maternal vascular spaces expand
rapidly until E16.5, but the growth of fetal capillaries
continues from E12.5 to E18.5. The changes in fetal
capillary volumes are associated with increases in total length
and reductions in mean cross-sectional area.
Trophoblast volume increases between E12.5 and E16.5
and, this, together with the growth in maternal and fetal
exchange surface areas, leads to a significant reduction in
thickness of the intervascular barrier between E14.5 and
E16.5 (Coan et al. 2004). This is mainly due to thinning of
the CT and vascular endothelium components (Coan et al.
2005).
During human and murine pregnancies, oxygen diffusive
conductances are matched to fetal weight despite the fact
that tissue compartments experience different growth
trajectories (Mayhew et al. 1993a; Coan et al. 2004; Mayhew,
2006b). In such circumstances, the regression line for a
log-log plot of conductance against weight is expected
to have a slope equal to 1. In fact, the regression line of log
Dvm against log fetal weight (where D vm signifies the
conductance of the villous membrane), has a slope of 1.04
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
and this value is not significantly different from 1 (Mayhew,
2006b). Studies on murine placentas (Coan et al. 2004) also
suggest that the intervascular conductance is commensurate
with changes in fetal weight (slope 0.93). When combined
with human findings, the results further suggest that mass-
specific conductances may be similar across species, implying
that different types of placenta may vary in absolute
efficiency whilst still meeting the needs of their fetuses.
The effects of maternal asthma on villous
growth and fetoplacental angiogenesis
Vascular patterns and villous development alter in various
complicated pregnancies and are associated with differences
in the expression of angiogenic growth factors and their
receptors (Mayhew et al. 2004a). Changes can occur in the
total and relative volumes, surface areas and lengths of
villi and capillaries, as illustrated by recent findings on the
effects of maternal asthma.
Asthma prevalence is increasing and adverse pregnancy
outcomes include reduced birth weight, the origins of
which are unresolved but might be attributable to asthma
severity, drug treatment, placental vascular function or fetal
hypoxia (Clifton et al. 2001; Bracken et al. 2003; Schatz et al.
2006). Drug treatment includes the use of glucocorticoids
and, recently, we have examined the effects of asthma severity
and glucocorticoid treatment on placental morphology
(for details, see Mayhew et al. 2008).
Pregnant women (n = 60 asthmatics, n = 15 non-asthmatics)
were recruited in the first trimester and measurements
made of age, body weight, haematocrit, vital capacity and
forced expiratory volume. Pregnancy complications other
than asthma were excluded. Asthmatics were classified on
the basis of asthma severity or glucocorticoid treatment.
Here, findings are presented for four asthmatic groups:
those not receiving steroids (n = 28) and those receiving
antenatal glucocorticoids at low (n = 10), moderate (n = 15)
or high (n = 7) levels of usage.
Immediately after delivery, umbilical cords were clamped
close to their placental insertion. Birth and trimmed
placental weights were recorded before full-depth columns
of tissue were sampled systematically, diced and immersed
in 10% phosphate-buffered formalin-saline. Tissue cubes
were allowed to settle haphazardly in paraffin wax to
randomize the positions and orientations of encounters
between tissues and section planes. Microscopical fields
on stained sections were selected by systematic uniform
random (SUR) sampling and analysed stereologically to
estimate the volumes of placental compartments and total
lengths of villi and fetal capillaries. Mean cross-sectional
areas of peripheral villi and capillaries, together with measures
of villous capillarization (capillary volume densities and
capillary : villus length ratios), were derived from global
volumes and lengths. Groups were compared by analysis
of variance and post-hoc testing (Mayhew et al. 2008).
© 2009 The Author
 Stereology and the placenta, T. M. Mayhew 81

Table 1 Maternal, neonatal and placental characteristics in non-asthmatic controls and in asthmatics grouped by glucocorticoid (GC) use. Values are
group means (CVs expressed as % of means). Data based on Mayhew et al. (2008)

Controls No GCs Low GCs Moderate GCs High GCs

Variable (n = 15) (n = 28) (n = 10) (n = 15) (n = 7)

*‡Age, years 29.4 (15%) 24.3 (19%)§ 29.5 (20%) 26.2 (20%) 26.6 (14%)
†Weight 36 weeks, kg 82.5 (13%) 88.7 (20%) 86.3 (28%) 85.0 (27%) 86.0 (38%)
Gestation, weeks 39.9 (3%) 40.2 (4%) 39.3 (3%) 39.8 (3%) 40.1 (3%)
Birth weight, kg 3.55 (13%) 3.65 (12%) 3.37 (17%) 3.42 (17%) 3.30 (17%)
Placental volume, mL 652 (21%) 609 (16%) 569 (19%) 615 (20%) 519 (11%)
HCTmat, % 36 (8%) 35 (8%) 36 (4%) 35 (8%) 36 (5%)
HCTf, % 46 (13%) 48 (15%) 46 (12%) 48 (15%) 50 (9%)

HCTmat and HCTf refer to maternal and fetal haematocrits, respectively. Results of 2-way ANOVA and post hoc testing: *significant group
effect; †significant sex effect; ‡significant interaction (group × sex) effect; §significantly different from non-asthmatic controls.

Table 2 Morphometric indices of placental composition, villous capillarization and the mean cross-sectional areas of peripheral villi and capillaries in
non-asthmatic controls and in asthmatics grouped by glucocorticoid (GC) use. Values are group means (CVs expressed as % of means). Data based
on Mayhew et al. (2008)

Controls No GCs Low GCs Moderate GCs High GCs

Variable (n = 15) (n = 28) (n = 10) (n = 15) (n = 7)

Intervillous space, mL 213 (16%) 192 (18%) 183 (23%) 200 (23%) 163 (22%)
Stem villi, mL 71.4 (68%) 57.5 (41%) 72.3 (52%) 81.9 (71%) 66.0 (56%)
Peripheral villi, mL 326 (27%) 308 (20%) 268 (24%) 289 (30%) 241 (15%)
Trophoblast, mL 95.5 (29%) 89.3 (18%) 83.9 (26%) 85.2 (35%) 74.5 (14%)
Stroma, mL 184 (32%) 174 (25%) 154 (27%) 160 (31%) 143 (16%)
*Fetal capillaries, mL 46.9 (50%) 44.1 (42%) 30.5 (24%)‡ 43.4 (43%) 23.7 (35%)‡§
Non-parenchyma, mL 41.5 (39%) 52.5 (43%) 44.9 (32%) 44.7 (48%) 49.8 (30%)
*†Peripheral villi, km 89.2 (23%) 103 (22%) 86.5 (33%) 86.6 (36%) 68.6 (19%)‡§
*Fetal capillaries, km 310 (37%) 382 (40%) 259 (39%) 359 (43%) 169 (44%)‡§
TS area villi, μm2 3700 (21%) 3020 (17%)‡ 3360 (37%) 3530 (26%) 3560 (12%)¶
TS area capillary, μm2 150 (27%) 119 (26%)‡ 126 (25%) 130 (41%) 149 (36%)
Capillaries, mL mL−1 0.147 (47%) 0.144 (33%) 0.116 (19%) 0.151 (33%) 0.098 (35%)
*Length ratio, km km−1 3.6 (38%) 3.7 (30%) 3.0 (26%) 4.3 (33%) 2.4 (31%)‡,§

Results of 2-way ANOVA and post-hoc testing: *significant group effect; †significant sex effect; ‡significantly different from
non-asthmatic controls; §significantly different from no GC and moderate GC groups; ¶significantly different from no GC group.

Findings are summarized in Tables 1 and 2. Most of
the differences associated with asthma severity were also
detected when groups were classified according to gluco-
corticoid usage (Mayhew et al. 2008). We found significant
between-group differences in placental composition, the
main changes involving fetoplacental capillaries in peripheral
villi. Compared with non-asthmatic controls, significant effects
on villous maturity were detected, notably decreases in
volumes of fetal capillaries and lengths of villi and fetal
capillaries in the group with high steroid use. Capillary
volumes were also reduced in the group with low steroid
use. Smaller mean cross-sectional areas of villi and capillaries
were found in the group not receiving glucocorticoid treat-
ment (Table 2). In addition, there were group differences
in capillary : villus length ratios, which were lower than controls
in those with high steroid use. Changes in villus and capillary
calibres were confined to asthmatics not receiving steroid
treatment.
Decreases in capillary volumes might be attributable to
poor asthma control leading to fetal hypoxia, asthma severity,
treatment by inhaled glucocorticoids, or other factors (Schatz
et al. 2006). Because glucocorticoid status is related to, and
not isolated from, asthma severity, resolution of these
possibilities requires further studies. In some instances, the
different origins of fetal hypoxia can be distinguished by
studying patterns of villous development and changes in
angiogenic growth factors (Kingdom & Kaufmann, 1997;
Charnock-Jones et al. 2004; Mayhew et al. 2004a). While
the morphological changes seen in asthmatic pregnancies
superficially resemble postplacental hypoxia, we did not
detect any increases in fetal haematocrits, although these
have been reported previously (Littner et al. 2003).

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Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
82 Stereology and the placenta, T. M. Mayhew
The association between hypocapillarization of villi and
steroid status raises the possibility that glucocorticoids
compromise vascular morphogenesis. The effects may be
mediated by hypoxia-inducible transcription factor-1 (HIF-1),
vascular endothelial growth factor-A (VEGF) and other
angiogenic factors. Administration of oral glucocorticoids
to rat dams leads to reduced VEGF expression and fetopla-
cental capillary growth (Hewitt et al. 2006). At other tissue
sites, glucocorticoids have been shown to impair HIF-1 function
and affect angiogenesis via VEGF and angiopoietin-1
(Jotko et al. 2007; Kim et al. 2008; Wagner et al. 2008).
Antenatal administration to rat and sheep dams also
reduces fetal and placental weights and these effects may
be mediated by decreased expression of genes involved in
cell proliferation and trophoblast apoptosis (Kerzner et al.
2002; Baisden et al. 2007).
Short-term infusion of glucocorticoids into the fetopla-
cental vasculature leads to arterial dilation, whilst the effects
of intramuscular betamethasone on IUGR fetuses with
absent or reversed flow in umbilical arteries include responses
which increase the risk of perinatal mortality (Clifton et al.
2001, 2002; Simchen et al. 2004). In asthmatics, including
those on high-dosage glucocorticoids, Doppler flow-velocity
waveforms in umbilical arteries were reduced at 18 weeks
of gestation but not at 30 weeks. These temporal differences
are interesting because mid-gestation is critical in terms of
villous growth, fetoplacental angiogenesis, and changes in
oxygen tensions (Mayhew, 2002; Charnock-Jones et al. 2004;
Kaufmann et al. 2004). Whilst inflammatory cytokines may
also affect vascular morphogenesis as inhibitors or activators
of angiogenesis (Clifton & Murphy, 2004; Romagnani et al.
2004; Salvucci et al. 2004), resolution of this possibility
requires further investigation.
Human villous trophoblast and the effects of
PE and IUGR
PE and IUGR are pregnancy complications responsible for
significant perinatal morbidity and mortality. They may
occur singly or together and both are associated with changes
in placental morphology. Although poor implantation
occurs in both types of pregnancy, stereological studies
have shown that differences in placental morphology,
particularly at the villous membrane, distinguish PE from
IUGR (Teasdale, 1984, 1985, 1987; Egbor et al. 2006; Mayhew
et al. 2007). The changes are associated with differences in
diffusive conductances (Mayhew et al. 2007). In IUGR, with
or without PE, total diffusive conductances are reduced in
comparison with control or PE subjects.
Villous trophoblast exhibits phases of proliferation, recruit-
ment, differentiation and loss (Huppertz et al. 1998; Mayhew
et al. 1999). Following CT mitosis, some cells fuse into the
overlying ST and it has been shown that, whilst total
numbers of nuclei in CT and ST increase to term, ST : CT
numbers are maintained at about 10 : 1 from at least 13
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
weeks of gestation to term (Simpson et al. 1992; Mayhew
et al. 1994). Once CT cells fuse into the ST, nuclei follow a
terminal differentiation pathway which culminates in
apoptosis. A protracted pre-apoptotic phase is followed by
a shorter execution phase (Mayhew et al. 1999; Benirschke
& Kaufmann, 2000) and many apoptotic nuclei congregate
in syncytial knots which are extruded into the intervillous
space as part of normal epithelial turnover (Huppertz
et al. 1998; Mayhew et al. 1999). The ST : CT ratio provides
a convenient index of the epithelial steady state between
CT proliferation and recruitment vs. ST differentiation and
extrusion (Mayhew et al. 1999).
Perturbations of the ST : CT steady state occur in PE and
IUGR. Apoptosis increases in PE and trophoblast fragments
are shed into the maternal blood in greater numbers
(Johansen et al. 1999; Ishihara et al. 2002). Cultured primary
villous CT cells from cases of PE, IUGR and PE + IUGR differ
in their secretory profiles and ability to form syncytia
(Newhouse et al. 2007). Culturing explanted villi at different
oxygen tensions has shown that hypoxia favours necrotic
over apoptotic shedding (Huppertz et al. 2003) and can
compromise fusion of CT into ST and completion of the
late apoptotic phase within ST. The result is a shift from
apoptosis to aponecrosis (a mixture of apoptosis and
secondary necrosis), and aponecrotic shedding is a feature
of PE (Huppertz & Kingdom, 2004; Huppertz & Herrler,
2005).
Published findings on villous growth and development
and the turnover of trophoblastic epithelium indicate that
morphological features distinguish term placentas in PE
and IUGR (Table 3). Whilst the total volumes and surfaces
of trophoblast in peripheral villi do not alter in PE, they
decrease in IUGR and PE + IUGR. It is likely that the differ-
ences are attributable to changes in total numbers of nuclei
(Teasdale, 1984, 1985, 1987), which are better descriptors of
total proliferation and loss than proliferation and apoptosis
indices (Mayhew et al. 2003b, 2007). The total complement
of trophoblast nuclei is similar in PE and control placentas
(Teasdale, 1985) but numbers decline in IUGR and PE + IUGR
(Teasdale, 1984, 1987). The findings are consistent with observa-
tions on relative rates of trophoblast recruitment and loss:
rates of differentiation and loss are accelerated in PE and
IUGR (Smith et al. 1997; Axt et al. 1999; Johansen et al. 1999;
Allaire et al. 2000; Erel et al. 2001; Ishihara et al. 2002) but
rates of CT proliferation are greater in PE than in IUGR
(Jones & Fox, 1980; Smith et al. 1998).
The comparisons in Fig. 1 are based on the morphometric
literature summarized in Table 3 together with findings
on trophoblast loss in terms of syncytial fragments (whether
apoptotic or aponecrotic) and microparticles (MPs, either
ST microvillous fragments or assorted cell-free debris).
Figure 1 incorporates the following important elements:
1) the surface area of peripheral villi is maintained in PE
but declines in IUGR and PE + IUGR; 2) trophoblast growth
is determined mainly by increased numbers of nuclei and
© 2009 The Author
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
© 2009 The Author

Table 3 Summary of published findings on morphological changes in the villous membrane of peripheral villi found in cases of PE with and without IUGR. Changes are with respect to placentas from control
(uncomplicated) pregnancies

Measures Case Types Natures of change References Comments

Surface area of capillaries IUGR and PE + IUGR Decrease 2,5,6,8,9,16,18,22,23, present Some supposedly PE may be PE + IUGR
PE NS 4,16,18,22, present
Volume of trophoblast IUGR and PE + IUGR Decrease 2,5,6,8,18
PE NS 18
Surface of trophoblast IUGR and PE + IUGR Decrease 2,3,5,6,8,9,16,18,22,23, present Some supposedly PE may be PE + IUGR
PE NS
Number of CT nuclei IUGR and PE + IUGR Decrease 2,5
PE NS 4
Number of ST nuclei IUGR and PE + IUGR Decrease 2,5
PE NS 4
Thickness of trophoblast IUGR and PE + IUGR Increase or NS 18 Arithmetic mean
Thickness of basal lamina IUGR and PE + IUGR Increase 1,10,17,20 Worse when IUGR is with ARED
Thickness of villous membrane IUGR and PE + IUGR NS or decrease 16,18 Arithmetic and harmonic means
PE NS present Arithmetic and harmonic mean
CT proliferation rate IUGR and PE + IUGR Increase or NS or decrease 1,7,10,12,14,17 Some IUGR with ARED.
Nature of change influenced by
method and way in which rate is expressed
PE Increase or NS 7,21
ST differentiation/loss rates IUGR and PE + IUGR Increase or NS 1,11,13,15,19,24,25,26 Methods and choice of rate vary
PE Increase 19,21,24,26,27 Increase may vary between early and late onset

Stereology and the placenta, T. M. Mayhew 83

NS indicates no significant change; ARED is absent or reversed end-diastolic flow in umbilical arteries.
References: 1. Jones & Fox (1980); 2. Teasdale (1984); 3. Boyd & Scott (1985); 4. Teasdale (1985); 5. Teasdale (1987); 6. Pivalizza et al. (1990); 7. Arnholdt et al. (1991); 8. Jackson et al. (1995);
9. Burton et al. (1996); 10. Macara et al. (1996); 11. Smith et al. (1997); 12. Smith et al. (1998); 13. Erel et al. (2001); 14. Chen et al. (2002); 15. Ishihara et al. (2002); 16. Ansari et al. (2003); 17.
Madazli et al. (2003); 18. Mayhew et al. (2003a); 19. Austgulen et al. (2004); 20. Battistelli et al. (2004); 21. Huppertz & Kingdom (2004); 22. Mayhew et al. (2004c); 23. Egbor et al. (2006); 24.
Goswami et al. (2006); 25. Axt et al. (1999); 26. Allaire et al. (2000); 27. Johansen et al. (1999).
84 Stereology and the placenta, T. M. Mayhew

Fig. 1 Villous membrane morphology in PE and IUGR. In each schematic, height represents the arithmetic thickness of the membrane (from luminal
aspect of vascular endothelium to maternal aspect of villous trophoblast). Width is proportional to total villous surface area. CT cells lie on basal lamina
and some post-mitotic cells are recruited (small arrows) into ST where terminal differentiation occurs. Turnover involves loss (large arrows) of
membrane-bound ST fragments (SFs) and microparticles (MPs). Membrane thickness depends on the steady state between recruitment and loss and
on the proximity of fetal capillaries to overlying trophoblast. In uncomplicated term pregnancies, ST differentiation leads to apoptosis, SFs contain
multiple apoptotic nuclei and there is minimal loss of MPs. In PE, proliferation and loss increase and are associated with aponecrosis. Surface areas and
thicknesses are maintained by the steady state. In IUGR and PE + IUGR, surface areas decline and overall thickness is preserved. However, in PE + IUGR,
MP loss is greater than in PE or IUGR alone and rates of loss in IUGR are comparable to those in controls. See also Huppertz & Herrler (2005) and
Goswami et al. (2006).

the total number per placenta is maintained in PE but
declines in PE and PE + IUGR; 3) trophoblast thickness is
determined by spatiotemporal changes in the steady state
and contributes to villous membrane thickness which is
affected also by the proximity of subjacent capillaries
(related to their calibre, degree of peripheralization and
extent of villous capillarization); 4) CT proliferation and ST
apoptosis rates are not absolute measures and their
impacts depend heavily on the total complements of CT
and ST nuclei; 5) in uncomplicated pregnancy, terminal
differentiation involves apoptosis but minimal necrotic
damage or MP loss. Consequently, trophoblast extruded
into the maternal intervillous space normally comprises
membrane-bound syncytial fragments containing multiple
apoptotic or pre-apoptotic nuclei; 6) PE involves accelerated
turnover and failure to complete apoptosis so that ST shows
increased aponecrosis and releases greater numbers of
syncytial fragments and MPs.
It is likely that steady states alter when total villous
surfaces are reduced, as in IUGR and PE + IUGR (Fig. 1). For
given rates of trophoblast recruitment and loss, and a
given steady state between them, greater absolutes amount
of shedding would be expected from larger surfaces and
volumes. In PE, trophoblast shedding is enhanced and
associated with aponecrosis, MPs and membrane-bound
fragments (Huppertz & Kingdom, 2004; Goswami et al. 2006).
Losses are matched by recruitment because trophoblast
volumes, surfaces and numbers are maintained. However,
in IUGR, there are reduced volumes and surfaces and
shedding levels. If the arithmetic mean thickness of
trophoblast increases (Mayhew et al. 2003a), this might
be due to greater recruitment vs. loss or shedding of smaller
fragments or recruitment of more or larger CT cells (Mayhew
et al. 2007). Whatever the mechanism, a constant arithmetic
mean thickness of the villous membrane overall (Mayhew
et al. 2007) suggests greater peripheralization of capillaries

© 2009 The Author

Journal compilation © 2009 Anatomical Society of Great Britain and Ireland

Table 4 Litter sizes, fetal weights and placental volumes in filtered and non-filtered groups of mice. Values are group means (CVs expressed as % of
means). Data based on Veras et al. (2008)
Variable Filtered (n = 6)
Fetal weight, g 0.846 (17%)
Litter weight, g 6.64 (27%)
Placenta volume, mm3 95.4 (14%)
Decidua volume, mm3 16.6 (55%)
Chorionic volume, mm3 9.3 (26%)
Junctional zone volume, mm3 20.1 (13%)
Labyrinth volume, mm3 49.4 (12%)
Trophoblast volume, mm3 25.4 (18%)
Maternal blood volume, mm3 15.7 (12%)
Fetal capillary volume, mm3 8.3 (27%)
within villi. In PE + IUGR, trophoblast and villous membrane
thicknesses are preserved despite reduced villous surfaces.
Goswami et al. (2006) monitored MP concentrations in
blood from maternal peripheral veins in age-matched
controls and cases of PE and IUGR. Compared with controls,
values in early-onset PE were almost 160% higher and
those in all cases of PE (early- and late-onset) over 120%
higher. However, values in IUGR did not alter significantly.
Taking into account the reported changes in trophoblast
surface areas, and other things being equal, we can
deduce that IUGR produces MPs in amounts roughly
commensurate with the loss in surface area (i.e. the rate
of production per unit of surface is essentially ‘normal’). In
contrast, the reduced villous surface in early-onset PE
(equivalent to PE + IUGR) produces higher concentrations
of MPs and so represents a disproportionately greater rate
of loss than either late-onset PE or IUGR.
These findings support the notion of differences between
PE and IUGR, re-emphasizing the need to monitor fetal
growth in PE and to resolve pure PE from pure IUGR and
from PE + IUGR. They also suggest differences between
IUGR and PE + IUGR and these may be related to stimulated
release of MPs associated with endoplasmic reticulum stress
(Yung et al. 2008).
Placental diffusive conductance and the effects
of urban air pollution
The mass-specific conductance response seen in normal
human and murine pregnancies may not obtain in all
pregnancies as shown by an experimental model of the
effects of air pollution on murine placenta. Air pollutants
may be man-made, biological, industrial or geological and
the main pollutants affecting human health are carbon
monoxide, nitrogen dioxide, sulphur dioxide, ozone, lead,
hydrocarbons and particulate matter (PM).
The impact of air pollutants on human health and preg-
nancy outcomes is of growing concern and is associated
© 2009 The Author
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Stereology and the placenta, T. M. Mayhew 85
Non-filtered (n = 6) P value
0.693 (16%) < 0.05
4.61 (37%) < 0.05
97.1 (19%) NS
21.0 (62%) NS
11.5 (34%) NS
20.5 (23%) NS
44.1 (23%) NS
24.0 (19%) NS
10.3 (43%) < 0.05
9.6 (33%) NS
with prematurity and IUGR (Bobak, 2000; Djemek et al.
2000; Rogers & Dunlop, 2000; Lee et al. 2003; 9rám et al.
2005; Wang & Pinkerton, 2007). Even moderate levels of
air pollution can affect the reproductive health of mice
(Mohallem et al. 2005; Lichtenfels et al. 2007) and, recently,
we have shown that mice exposed to particulate air pollution
not only produce smaller offspring but also show changes
in placental functional morphology (Veras et al. 2008).
Two groups of second-generation Balb C mice were
mated inside paired exposure chambers (see Veras et al.
2008) which produced differences in levels of particulate
matter by filtering air sampled close to a busy traffic inter-
section. In one chamber, the air was not filtered (group
NF) and in its partner chamber, it was filtered (group F).
Animals were kept at ambient conditions of temperature,
humidity and air pressure and were exposed to the same
levels of gaseous pollution. In group F chambers, serial
filters removed larger particles so that only particulate
matter smaller than 2.5 μm in diameter (PM2.5) remained.
After exposure, all pregnant females were killed on E18
and fetuses and placentas were removed and weighed.
Placentas were immersion-fixed in buffered 4% formalin
and sampled using a multistage SUR design (Mayhew,
2006a, 2008; Veras et al. 2008). Each organ was cut into
slices orthogonal to the chorionic plate to generate sets of
vertical sections for estimating exchange surface areas and
tissue volumes. One set of slices was embedded in paraffin
wax (to estimate volumes of different zones) and the other
was embedded in glycolmethacrylate resin (to analyse
volumes and surfaces within the labyrinth). Other quantities
(vessel calibres, total oxygen diffusive conductance and
mass-specific conductance of the intervascular barrier)
were derived from the primary measures. Here, statistical
comparisons between F and NF groups are drawn using
the Mann–Whitney U-test.
Findings are summarized in Tables 4 and 5. In the NF
group, fetal weight declined by about 18% and total
litter weight by about 31%. There were no significant
86 Stereology and the placenta, T. M. Mayhew
Table 5 Exchange surface areas (cm2), vessel calibres (equivalent diameter, μm), barrier thicknesses (μm) and total and mass-specific oxygen diffusive
conductances (cm3 min−1 kPa−1 and cm3 min−1 kPa−1 g−1) of placentas in filtered and non-filtered groups. Values are group means (CVs expressed as %
of means). Data based on Veras et al. (2008)
Variable Filtered (n = 6)
Maternal blood surface 19.0 (13%)
Fetal capillary surface 17.1 (8%)
Maternal space diameter 33 (13%)
Fetal capillary diameter 19 (25%)
Diffusive conductance 0.0022 (11%)
Mass-specific conductance 0.0027 (27%)
differences in total placental volume or in the volumes of
the decidua basalis, chorionic plate, junctional zone or
labyrinth (Table 4). However, within the labyrinth, maternal
blood volume was about 34% smaller. Apparent differences
in the volumes of fetal capillaries and trophoblast were
not significant. In group F, the exchange surface areas of
the maternal blood spaces and fetal capillaries were 19 cm2
and 17 cm2, respectively (Table 5). Although the roughly
52% increase in capillary surface area was significant,
there was no change in maternal surface area. Differences
in vascular volumes and surfaces were accompanied by
changes in the apparent mean diameter of maternal blood
spaces (from 33 μm to 23 μm) but not in mean diameters
of fetal capillaries (Table 5).
In group F, the total oxygen diffusive conductance of the
intervascular barrier amounted to 0.0022 cm3 min–1 kPa–1
and this value increased significantly in the NF group.
Moreover, mass-specific conductances more than doubled
in the NF group (0.0054 vs. 0.0027 cm3 min–1 kPa–1 g–1).
These estimates are comparable with those obtained
by others (Coan et al. 2004; Rutland et al. 2005, 2007).
Agreement is best for compartment volumes and vascular
surfaces and any discrepancies are probably attributable
to strain differences. However, larger discrepancies exist
for values of arithmetic mean thickness, vessel calibres
and diffusive conductances and it is likely that these are
influenced additionally by differences in methods of
estimation.
The mix of changes seen after exposure to non-filtered
air is surprising in that some seem to be adaptive and
others deleterious. Thus, the greater surface area of fetal
capillaries, total diffusive conductance and mass-specific
conductance of the intervascular barrier may be interpreted
as adaptations aimed at maintaining or expanding oxygen
and nutrient delivery to the fetus. The fact that fetal weight
declines despite these adaptations implies that other factors
exert more influential effects, notably on maternal blood
space volumes and calibres. Changes at this site suggest
compromised delivery of maternal blood to the placenta
and an increase in resistance to its flow. Indeed, PM2.5 has
been shown to produce significant vasoconstriction in
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Non-filtered (n = 6) P value
17.8 (17%) NS
25.9 (21%) < 0.01
23 (29%) < 0.01
15 (32%) NS
0.0037 (40%) < 0.05
0.0054 (41%) < 0.05
small arteries of the hearts and lungs of rats (Rivero et al.
2005). Amongst other factors which might influence birth
outcomes are systemic alterations in haematocrit, erythrocyte
deformability, blood viscosity and coagulability (Baskurt
et al. 1990; Peters et al. 1997; Pekkanen et al. 2000; Rivero
et al. 2005). Increases in such factors, exacerbating the
effects of decreases in vessel calibres, could have marked
effects on maternal blood rheology.
Quantitative immunoelectron microscopy and
molecular localization
In immunoelectron microscopy, gold particles are used to
label defined antigens in different intracellular compart-
ments (organelles or membranes) and labelling patterns
are compared by counting particles (Griffiths, 1993). The
technique has been applied to localize interesting molecules
within placentas but, surprisingly, there have been few
attempts to quantify immunolocalization (see Mayhew &
Desoye, 2004).
When quantifying the labelling intensity of compart-
ments, LD is usually calculated and relates gold particle
counts to the profile areas (golds μm−2) or trace lengths
(golds μm−1) of the sectional images of compartments.
Recently, we developed simpler alternatives for counting
immunogolds and statistically evaluating and comparing
gold-labelling patterns. The methods address two basic
questions: are some compartments preferentially labelled?
(method 1), and do labelling patterns vary in different
groups of cells? (method 2).
With method 1, numbers of gold particles lying on defined
organelles or membranes are used to generate an observed
frequency distribution. By randomly superimposing a lattice
of test points on the same cell profiles, the numerical
frequencies with which points overlie different organelles
are determined and provided the expected distribution
because random points hit compartments on sections with
probabilities determined by profile areas. By replacing
points with test lines, and counting sites of intersection
with membrane traces, analogous procedures provide
labelling distributions for different categories of membrane
© 2009 The Author

Table 6 Immunolocalization of gold-labelled GLUT1 in three groups of human trophoblast cells. Values are observed (expected) numbers of gold
particles in each compartment. For details, see Mayhew & Desoye (2004)
Compartments Euglycaemia Hyperglycaemia
Apical membranes 112 (101.3) 77 (101.3)
Basal membranes 34 (34.0) 33 (34.0)
Cell interior 54 (64.7) 90 (64.7)
The distributions are significantly different: total χ2 = 23.9, df = 4, P < 0.001. The main sources of difference are a shift towards fewer gold
particles on apical membranes and more at the cell interior (hyperglycaemia group) and fewer golds than expected at the cell interior
(osmotic control group).
(Mayhew et al. 2002, 2003c). By treating membranes (surface-
occupying compartments) as organelles (volume-occupying
compartments), it is possible to deal with target proteins
that translocate between membranes and organelles
(Mayhew & Lucocq, 2008a,b).
Observed and expected distributions provide relative
labelling indices (RLI = observed golds/expected golds) for
each identified compartment and, for random labelling,
the predicted value is RLI = 1. Observed and expected
distributions are compared using Chi-squared analysis. If
the observed distribution of gold particles proves to be
non-random, RLI values and partial Chi-squared values
identify compartments which are preferentially labelled.
In fact, two criteria must be satisfied: first, RLI must be > 1 and,
secondly, the partial Chi-squared must make a substantial
contribution (at least 10%) to total Chi-squared. This
approach may be used in tandem with LD values and
has been applied to study gold particle distributions in
various biological contexts (see Mayhew & Lucocq, 2008b).
With method 2, a simpler protocol is followed and raw
gold counts in different groups of cells are compared
directly by contingency table analysis (Mayhew & Desoye,
2004; Mayhew et al. 2004b). This method has been used to
compare localization patterns of the glucose transporter,
GLUT1, in cultured human trophoblast cells grown in
euglycaemic (medium containing 5.5 mmol L−1 glucose),
hyperglycaemic (25 mmol L−1 glucose) and osmotic control
(19.5 mmol L−1 D-mannitol + 5.5 mmol L−1 glucose) groups.
Results (summarized in Table 6) showed a shift in labelling
away from the plasma membrane and into the cell interior
in cells exposed to hyperglycaemia and this shift seemed to
be specific to glucose and not a response to osmotic stress
(Mayhew & Desoye, 2004).
Concluding remarks
Design-based stereology has been used to interpret the
morphology of human and murine placentas by providing
precise and minimally biased estimates of functionally
relevant structural quantities. Its sampling and estimation
tools have been used to describe the processes of villous
growth, trophoblast differentiation, vascular morphogenesis
© 2009 The Author
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Stereology and the placenta, T. M. Mayhew 87
Osmotic-control Chi-squared values
115 (101.3) 1.12, 5.84, 1.84
35 (34.0) 0.00, 0.03, 0.03
50 (64.7) 1.76, 9.92, 3.33
and diffusive transport in normal and compromised preg-
nancies. By way of illustration, changes in these processes
during normal placental development have been summarized
and it has been shown how fetoplacental vascular growth
is compromised in cases of maternal asthma, that trophoblast
turnover is affected differently in pre-eclampsia and intra-
uterine growth restriction, and that particulate urban
air pollution affects maternal vasculature and diffusive
transport in the murine placenta. Finally, new and efficient
methods for quantitative immunoelectron microscopy
have been emphasized because they now permit more
rigorous analysis of the subcellular distributions of inter-
esting molecules between compartments or in different
experimental groups of cells or organs.
Acknowledgements
I am grateful to The Anatomical Society of Great Britain & Ireland,
BBSRC and MRC for recent research grant funding and thank all
those collaborators who have contributed their talents to these
researches. In particular, I thank Vicki Clifton (Newcastle and
Adelaide), Gernot Desoye (Graz), Moira Jackson (Aberdeen and
Gainesville) and Mariana Veras and her colleagues (Saõ Paulo).
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