Placenta (2004), 25, 114–126

doi:10.1016/j.placenta.2003.10.009

CURRENT TOPIC

Aspects of Human Fetoplacental Vasculogenesis and Angiogenesis.

II. Changes During Normal Pregnancy

P. Kaufmann a, T. M. Mayhew b,* and D. S. Charnock-Jones c

a
Department of Anatomy II, University Hospital, RWTH-Aachen, Germany; b Centre for Integrated Systems Biology and
Medicine, School of Biomedical Sciences, E Floor, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH,
UK; c Reproductive Molecular Research Group, Departments of Pathology and Obstetrics & Gynaecology, The Rosie Hospital,
University of Cambridge, UK
Paper accepted 5 August 2003

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.
Placenta (2004), 25, 114–126 ! 2003 Elsevier Ltd. All rights reserved.

INTRODUCTION developing fetus (the ultimate consumer). Based on this

Current understanding of the development of the human
fetoplacental vascular system is based, almost exclusively, on
descriptive structural and immunohistochemical studies.
Meaningful in vitro approaches (which would help to elucidate
the peculiarities of human fetoplacental angiogenesis) are
under-represented due to a series of problems inherent to the
placenta.
In placental villi, angiogenesis takes place in the presence of
an oxygenation and nutrition gradient which extends from the
maternal circulation (the supplier), via the villous trophoblast
(an important consumer) and developing fetal circulation
(which extracts oxygen and nutrients from the villi) to the
*
To whom correspondence should be addressed. Tel.: +44-(0)115-
970-9414; Fax: +44-(0)115-970-9259; E-mail: terry.mayhew@
nottingham.ac.uk
0143-4004/04/$–see front matter
situation, increasing development of the fetoplacental vascu-
lature diminishes rather than increases its own nutrition [1]. So
far, it has been impossible to mimic this unusual situation in
vitro, either in villous explant culture or by in vitro perfusion
of the organ.
Cell culture experiments with human placental microvascu-
lar endothelium are practicable since suitable endothelial cell
lines are available [2] and human endothelial cells of different
microvascular origins, and showing placenta-specific pheno-
types, have been isolated [3]. However, it has not been possible
to-date to establish co-culture systems which are sophisticated
enough (a) to maintain the placenta-specific phenotypes and
(b) to mimic the specific microenvironmental conditions of a
developing placenta.
Animal experiments designed to study the influences of
various factors on chorioallantoic or fetoplacental vasculo-
genesis and angiogenesis are easy to perform (e.g. [4–7]) but
! 2003 Elsevier Ltd. All rights reserved.
Kaufmann et al.: Fetoplacental Vasculature in Normal Pregnancy
the data are difficult to extrapolate to the human condition due
to substantial interspecies differences regarding pregnancy
length, development of the oxygenation gradient and the final
architecture of the fetoplacental vascular bed. Interspecies
differences also make it difficult to transfer descriptive data
obtained in animals to the human placenta. Exceptions are
provided by the placentae of higher primates (e.g. rhesus
monkey [8]) which display a villous structure, haemodynamic
conditions similar to those of the human placenta and a
pregnancy length in the same range as that in humans.
Because of these peculiar local conditions of the placenta,
fetoplacental vasculogenesis and angiogenesis also differ from
vessel formation in other human vascular beds. The connec-
tion between developing placental capillaries and the embry-
onic circulatory system is established via the connective stalk
(forerunner of the umbilical cord) around day 32 post concep-
tion (for review, see [9]). From this early stage onwards, the
highly immature intravillous vascular bed has to perform all
placental transport functions. In doing so, it never attains
a stable state but rather expands continuously to meet the
oxygen and nutritional needs of the growing embryo/fetus
(e.g., see [10,11]).
Development takes place in an environment of reduced
oxygen tensions relative to maternal tissues but, during the
course of pregnancy, intervillous oxygen tensions increase [12].
In contrast to most other developing organs, in which oxygen-
ation improves with advancing vascularization, tissue oxy-
genation in placental villi appears to be inversely related to the
numerical density of fetal capillaries since, rather than merely
delivering oxygen, the latter extract it from the villi [1].
Consequently, a low numerical density of fetal capillaries
because of reduced oxygen extraction results in increasing
intraplacental oxygen levels [13,14] which, in turn, impact
further on angiogenesis ([15], this volume). In contrast, and
under otherwise constant conditions, high numerical densities
of capillaries and high oxygen extraction by the fetal circu-
lation would lower intraplacental oxygen tensions [13,14].
When exceeding certain limits, both situations may result in
vicious circles.
In any vascular bed, increased flow is achieved by a
combination of physiological adjustments (e.g. increased per-
fusion pressure, decreased vascular impedance) and changes in
vascular anatomy (e.g. increases in vessel calibre, decreases in
vessel length, formation of parallel rather than serial arrange-
ments of vessels). According to the Poiseuille equation, resist-
ance to flow is directly proportional to vessel length and
inversely proportional to the square of vessel cross-sectional
area. Consequently, the advantage of parallel arrangements is
that, other variables being constant, they generate multiple
interconnected vessel segments of reduced mean length and,
hence, reduced impedance. Thus, vascular anatomy influences
vascular impedance in two main ways: by the dimensions and
spatial arrangements of vessels. These principles are of par-
ticular importance in the fetoplacental vascular bed which has
to adapt continuously to the increasing requirements of the
growing embryo/fetus.
115
Doppler studies have shown that fetal vascular resistance
decreases during normal gestation and may be compromised in
complicated pregnancies [16]. The principal strategy for mini-
mizing lengths and resistances is branching angiogenesis. It is
now recognized [17,18] that new branches may be created by
sprouting angiogenesis (lateral sprouting from existing vessels)
or by intussusceptive angiogenesis (formation of transvascular
epithelial pillars which partition one lumen into two or more
lumina). Both types of branching strategy [15] are capable of
creating parallel vascular arrangements in which the individual
vessel segments are relatively short and numerous. In contrast,
non-branching angiogenesis (which is likely to increase resist-
ance to blood flow) involves elongation of existing vessel
segments by vascular endothelial cell proliferation, inter-
calation of endothelial progenitor cells or a combination of the
two [15].
Fetoplacental angiogenesis shapes villous development
[1,9,19–23]. In normal human pregnancy, capillary growth is
biphasic, involving an initial phase of branching angiogenesis
(formation of tightly looped capillaries) followed by one of
increased non-branching angiogenesis (formation of longer
capillaries). To some degree, villous morphology reflects the
underlying angiogenic processes (see also, Figures 4 and 5).
For example, during the first trimester, immature intermediate
villi are large calibre, bulbous structures covered by a thick
layer of trophoblast and containing a complex capillary network
surrounding central stem vessels. In contrast, villi in the third
trimester are overwhelmingly filiform structures associated
with a thin trophoblast and tightly looped capillaries. These
relationships between capillaries and villi suggest that the
villous trophoblast is a plastic layer which adapts in parallel
with, or in response to, the changing structure of the under-
lying vasculature. This view implies that the trophoblast does
not sculpt the vasculature but, instead, angiogenesis helps to
drive villous development and differentiation. The phasic
nature of gestational changes in morphometric indices of villous
capillarization, notably capillary : villus length ratios [23] are
consistent with this notion. Vascular patterns and villous shapes
also vary in complicated pregnancies (see [24], this volume).
Development of the fetal vasculature of the placenta de-
pends on the actions of angiogenic growth factors and their
receptors produced by cells and extracellular matrix ingredi-
ents lying in or near the fetal vessels. Interestingly, the vessels
lack autonomic innervation and so resistance to flow in the
maturing vasculature must also be regulated by local vasoactive
effectors (for reviews, see [25,26]). Therefore, it is the purpose
of this review to compare both structural aspects of human
fetoplacental development and some of the molecules, cell
players and other factors whose interplay sculpts vascular
anatomy.
VASCULOGENESIS (DAYS 21 TO 32 POST
CONCEPTION)
Fetal vascularization of the first generation of placental villi is
the result of local de novo formation of capillaries (vasculo-
116
Figure 1. Vasculogenesis and angiogenesis in human placental villi. Modified from Demir et al. [28], with permission.
genesis) rather than protrusion of embryonic vessels into the
placenta. Vasculogenesis starts at about 21 days post concep-
tion (21 dpc), in 4-somite embryos [27,28]. In the closely
related rhesus monkey (gestation 166 days), the onset of
vasculogenesis is around day 19 [8]. At this stage, in both
species, villous trees comprise primary (solid trophoblastic)
and secondary villi, the latter being characterized by invasion
of the villous core by a loose mesenchymal stroma derived
from the extra-embryonic coelomic cavity.
The first precursors of fetal endothelium in the villous
stroma, so-called haemangioblastic cell cords, can be demon-
strated as early as 15–21 dpc (Figure 1a–c), using the mono-
clonal antibody QBend10 which detects CD34 (Figure 3a).
Haemangioblastic cells form string-like aggregates of polygonal
cells (Figure 1a, b) which differ from their mesenchymal
precursors by the absence of cellular extensions and presence
Placenta (2004), Vol. 25
of fewer organelles. The narrow intercellular spaces between
these cells are bridged by either desmosomes or band-like
junctions resembling tight junctions (Figure 1c). Extensions of
surrounding mesenchymal cells are often integrated into these
clusters.
Basic fibroblast growth factor (FGF-2 or bFGF) is thought
to be involved in recruitment of haemangiogenic progenitor
cells and its expression in human placental villi has been
described repeatedly [29–31] but, to the best of our knowledge,
never at this early stage of pregnancy. Also, data on FGF
receptors (FGFR) are still missing. By contrast, it is well
known that vascular endothelial growth factor A (VEGF-A or
VEGF), responsible for commitment, growth and aggregation
of the endothelial precursors for the formation of the haeman-
giogenic cords, is highly expressed in early pregnancy [32–37].
According to in situ hybridization and immunohistochemical
Kaufmann et al.: Fetoplacental Vasculature in Normal Pregnancy
studies, villous trophoblast and villous stromal macrophages
are the main sources of this cytokine [32,33,37]. VEGF-A
secretion by human trophoblast was also demonstrated in vitro
by Shore et al. [36]. The assumption that macrophage-derived
angiogenic growth factors, such as VEGF-A, are involved in
vasculogenesis is supported by the finding that macrophages
differentiate locally in villous stroma even prior to the devel-
opment of haemangiogenic cords (Figure 1b and [28]). Our
unpublished immunohistochemical data show stromal im-
munoreactivities for both VEGF-A and its receptor VEGFR-2
(KDR/flk-1) by the time that secondary villi differentiate into
tertiary villi. In subsequent stages of pregnancy, VEGFR-2
was localized in human placental villous endothelium
[31,38,39]. Knock-out experiments in mice have also high-
lighted the importance of VEGFR-2 for specification and early
differentiation of haemangioblastic precursors of fetoplacental
capillaries [40].
In human placenta, formation of endothelial tubes out of
haemangiogenic cords starts at 21 dpc. This occurs by focal
enlargement of centrally located intercellular clefts which later
fuse to become a larger lumen (Figure 1c, d). In contrast to
other organs [41,42], we have never observed lumen formation
by fusion of intraendothelial vacuoles. Rather, the fetoplacental
capillary lumen always seems to form by acquisition of a
junctionally defined extracellular compartment within the hae-
mangioblastic cords (guinea pig [43]; rhesus monkey [8];
human [28]). The acquisition of such still-unconnected seg-
ments of capillaries (Figure 2a) defines the transition from
secondary to tertiary villi. Analysis of knock-out animals by
Fong et al. [44,45] has revealed that VEGFR-1 (flt-1) regulates
the action of VEGF-A on endothelial precursors. However,
deletion of the tyrosine kinase encoding portion of the
VEGFR-1 gene has no effect on vascular structures [46].
These findings suggest that it is the extracellular portion of the
molecule (i.e. the soluble VEGF antagonist, sflt-1) that is
required for placental vascular development. Immunohisto-
chemical studies have found that the receptor is expressed on
human villous endothelium [31,38,39] but also in villous
macrophages and trophoblast [33,38]. However, it should be
noted that the trophoblast (in human and murine placentae) is
a rich source of soluble VEGFR-1 [47,48].
By 28 dpc, the former haemangioblastic cords of most villi
show clearly defined, long, polygonal lumina with surrounding
endothelial cells becoming considerably flattened (Figure 1d,
e). Additional mesenchymal cells closely appose the endothelial
tubes, their extensions being integrated into both the mesen-
chymal network and the endothelial tubes (Figure 1e). These
‘juxta-haemangioblastic’ cells are characterized by richly de-
veloped rough endoplasmic reticulum and are considered to
become pericytes [8,49]. Moreover, their contribution to en-
largement of the pool of endothelial cells has been discussed,
since focal extension of these cells may even protrude between
endothelial cells [49,50].
During this phase, haematopoietic stem cells become visible
in the capillary lumen (Figure 1e). These cells are not yet
circulating since no anatomical connection exists via the cord
117
to the embryonic circulation. The latter is established a few
days later (between 32 and 35 dpc) by fusion of villous
capillaries with each other (Figure 2a, b and Figure 3b, c) and
with the larger, allantoic vessels [9]. The latter are formed by
vasculogenesis within the allantois [51] and thereafter spread in
both, embryonic and placental directions and finally establish
the connection between intraembryonic and placental vascular
beds.
FORMATION OF CAPILLARY NETWORKS
(DAY 32 TO WEEK 25 POST CONCEPTION)
The subsequent stages of angiogenesis can be divided into
three partly overlapping periods:
1. formation of capillary networks from 32 dpc to 25 weeks
post conception (25 wpc) by prevalence of branching
angiogenesis (Figure 2a–c);
2. regression of peripheral capillary webs and formation
of central stem vessels mainly through 15–32 wpc
(Figure 2d);
3. formation of terminal capillary loops by prevalence
of non-branching angiogenesis (25 wpc until term)
(Figure 2e).
From 32 dpc until the end of the first trimester, the
endothelial tube segments formed by vasculogenesis are trans-
formed into primitive capillary networks by the balanced
interaction of two parallel mechanisms; (a) elongation of
pre-existing tubes by non-branching angiogenesis, and (b)
ramification of these tubes by lateral sprouting (sprouting
angiogenesis) and, maybe also, intussusceptive microvascular
growth. Further work is required to assess the incidences of
these two forms of branching angiogenesis.
In the stroma of the youngest generation of small-calibre villi
(so-called mesenchymal villi), branching angiogenesis is less
frequent than non-branching angiogenesis and the resulting
capillary webs are only poorly developed (see Figure 2b and
Figure 3b, c). With advancing gestation and increasing diameter
of villi, branching angiogenesis is stimulated and the moderately
branched capillary loops transform into a dense two-
dimensional network which is located just below the villous
surface. This is particularly impressive in the immature inter-
mediate villi which develop from mesenchymal villi from week
9 post menstruation onwards (Figure 2c and Figure 3d, e).
In vitro experiments on the chorioallantoic membrane of
the chicken [4,5] have shown that binding of VEGF to both of
its receptors (VEGFR-1 and -2) stimulates branching angio-
genesis and consequently results in highly branched capillary
webs (‘branching angiogenesis’). Expression of VEGF-A and
VEGFR-2 are most intense in these early stages of pregnancy
and decline steeply as pregnancy advances [39,52–55]. By
contrast, expression of PlGF and the soluble form of
VEGFR-1 [31,47,48,55] have been found to increase towards
term when branching angiogenesis is increasingly replaced by
non-branching angiogenic mechanisms (see below and [9]).
118
Figure 2. Patterns of villous development in relation to gestational phases of fetal vascular development. Modified from Benirschke and Kaufmann [9], with
permission.
PlGF binds selectively to VEGFR-1 and, in some systems,
appears to suppress sprouting angiogenesis (chorioallantoic
membrane of the chicken, Wilting, personal communication).
However, in other systems (rabbit cornea and mouse skin),
PlGF stimulates formation of highly branched capillary net-
works [56,57]. Recent data show that PlGF is an essential
component of VEGF-driven angiogenesis and that activation
of VEGFR-1 leads to an important and distinctive cellular
response [58].
Around the capillary-like tubes, differentiation processes
occur. The endothelial tubes acquire an incomplete layer of
Placenta (2004), Vol. 25
precursor pericytes. A basal lamina begins to form around the
endothelial tubes and around the pericytes from about 6 wpc
but, so far, complete envelopment of capillaries by basal lamina
has been observed only in the last 10 weeks of pregnancy
(Figure 1f and [28]).
FORMATION OF STEM VESSELS (WEEK 15
UNTIL WEEK 32 POST CONCEPTION)
In the third month of pregnancy, some of the centrally located
endothelial tubes of immature intermediate villi achieve larger
Kaufmann et al.: Fetoplacental Vasculature in Normal Pregnancy 119

Figure 3. Comparison of different stages of vasculogenesis and angiogenesis seen using fetoplacental vascular casts (left) and immunohistochemical staining of
tissue sections with anti-CD34 (Qbend10). Magnifications (a) "330, (b) "500, (c, e, g) "180, (d) "400, (f) "700. Images (a) and (d) from Kaufmann and
Kingdom [79], with permission.

diameters of 100 µm and more (Figure 2d, e and Figure 3d, e). and by differentiation of precursor smooth muscle (sm) cells
Within a few weeks, they establish thin media- and adventitia- expressing !- and "-sm-actins in addition to vimentin and
like structures by concentric fibrosis in the surrounding stroma desmin. This is followed soon afterwards by the expression of
120 Placenta (2004), Vol. 25

Figure 4. Arrangement of fetal capillaries in a group of terminal villi branching from a central mature intermediate villus as reconstructed from serial tissue
sections. Note the complex loop formation of terminal capillaries. Formation of branches is usually followed by early refusion of the two capillary branches. An
erythrocyte must pass along the capillaries of several terminal villi arranged in series. From Benirschke and Kaufmann [9], with permission.

Figure 5. Fetal vascularization of terminal villi. (A) Cast of vessels of three terminal villi branching from a mature intermediate villus (upper right corner) "650.
(B, C, D, E) Corresponding semithin cross sections at various levels of this capillary convolution. Note focal sinusoidal dilation of the terminal capillaries, "800.
From Kaufmann et al. [20], with permission.

sm-myosin [59,60]. These vessels are forerunners of villous
arteries and veins. In larger, proximal immature intermediate
villi, the adventitia of the centrally located precursors of
arteries and veins fuse thereby forming a fibrosed stromal core
within the villus. Henceforth, this type of villus is called a stem
villus.
Establishment and maintenance of the outer parts of
vessel walls is thought to be controlled, at least in part, by
the balance of angiopoietin-1 (Ang-1), and angiopoietin-2
(Ang-2), interacting at the angiopoietin receptor Tie-2 (Tek)
[61]. Accordingly, Ang-1 and Ang-2 protein and mRNA
have been detected in perivascular cells of immature inter-
mediate villi [62] where development of arteries ands veins
takes place. Genetic studies suggest that the situation might
be more complicated since deficiency of Tie-2 results in
poorly developed capillary networks and constitutive acti-
vation of this receptor leads to venous malformations [63,64].
In human placental tissues, expression of Tie-2 has been
Kaufmann et al.: Fetoplacental Vasculature in Normal Pregnancy
detected by PCR and localized to vascular endothelium
[65,66].
REGRESSION OF PERIPHERAL CAPILLARY
NETS (WEEKS 15–32 POST CONCEPTION)
In the second half of pregnancy, the fibrotic process within the
stroma of the stem villus advances in a radial manner towards
the villous trophoblast. The superficial, subtrophoblastic cap-
illaries become transformed into a rarified paravascular capil-
lary network (Figure 2d, e). In tandem with the expanding
villous fibrosis in stem villi, and the transformation of central
capillaries into arteries and veins, the superficial capillary
networks gradually regress. By term, very few paravascular
capillaries remain in the larger stem villi [67,68]. The mech-
anisms by which capillary regression occurs in stem villi is not
yet known. Nor is it known whether Ang-2 and its receptor
Tie-2, as well as the absence of VEGF-A and its receptors, are
involved. Interestingly, regression of capillary nets in develop-
ing stem villi is contemporaneous with loss of trophoblast at
the villous surface, and reduction of macrophages in the
fibrosing stroma [9,60], both known to be rich sources of
VEGF-A [32,33,37].
By contrast to these differentiation and regression processes
in the more proximal parts of the villous trees, nascent villous
outgrowths (mesenchymal and immature intermediate villi)
with new capillary networks are formed in peripheral regions
by a balanced mixture of branching and non-branching angio-
genesis. Also, within a few weeks, these villi undergo the
vascular changes described above and, thereby, differentiate
into stem villi [69]. In this way, the villous trees and their fetal
vascular bed continue to expand peripherally.
PREVAILING NON-BRANCHING
ANGIOGENESIS (WEEKS 24–25 POST
CONCEPTION UNTIL TERM)
From 25 wpc until term, patterns of villous vascular growth
switch from prevailing branching angiogenesis to prevalence of
non-branching angiogenesis. This is due to the development of
new villous types, the mature intermediate villi, at the further-
most tips of existing villous trees. Mature intermediate villi are
slender (80–120 µm diameter), elongated villi (>1000 µm long
containing one or two long, poorly branched capillary loops.
Analysis of proliferation markers at this stage reveals a relative
reduction of trophoblast proliferation and an increase in
endothelial proliferation along the entire length of these
structures, resulting in non-sprouting angiogenesis (Figure 2e)
by proliferative elongation. Capillary growth by elongation
could also occur by intercalation, i.e. by recruitment into
existing vascular endothelium of circulating endothelial pro-
genitor cells but, currently, there is little direct evidence to
support this. The final length of these peripheral capillary
loops exceeds 4000 µm [19,20]. They grow at a rate which
121
exceeds that of the villi themselves, resulting in coiling of the
capillaries (Figure 3f, g). The looping capillaries bulge to-
wards, and obtrude into, the trophoblastic surface (Figure 2e
middle path) and thereby contribute to formation of the
terminal villi. Each of the latter is supplied by one or two
capillary coils and is covered by an extremely thin (<2 µm)
layer of trophoblast which contributes to the so-called vasculo-
syncytial membranes. These are the principal sites of diffu-
sional exchange of gases between mother and fetus. Normally,
the capillary loops of 5–10 such terminal villi are connected to
each other in series by the slender, elongated capillaries of the
central mature intermediate villus (Figures 4 and 5).
With advancing gestation, terminal capillaries focally dilate
forming large sinusoids >40 µm in diameter (Figure 5). These
sinusoids are thought to counterbalance the adverse effects
of the long, poorly branched capillary loops upon total feto-
placental vascular impedance [20].
Immunohistochemical studies on the expression patterns of
VEGF-A, PlGF and their receptors give hints as to their
importance in villous angiogenesis. Expression of VEGF-A
and VEGFR-2 are intense early in pregnancy and decline as
pregnancy advances [39,52–55]. By contrast, expression of
VEGFR-1 and PlGF increase towards term [31,38,55]. PlGF
is expressed in both villous syncytiotrophoblast [36,37] and the
media of larger stem vessels [70,71]. Immunolocalization of
these factors in other species also suggests that they are
involved in implantation and the development and remodelling
of placentae with different architectures [26,72–75]. Exper-
iments on the avian chorioallantoic membrane [4,5] have
shown that binding of VEGF-A to its receptors results in
sprouting angiogenesis and a highly branched capillary web.
In contrast, PlGF (which binds selectively to VEGFR-1) is
reported to suppress angiogenesis [76]. Furthermore, different
splice forms of PlGF and VEGF-A/PlGF heterodimers,
appear to have differing effects on the in vitro proliferation
of human umbilical vein endothelial cells with PlGF1 increas-
ing and PlGF2 decreasing uptake of tritiated thymidine
[71,76].
The role of PlGF is much more complex than originally
envisaged. Initially, it was thought that this factor had little
potency in vitro to stimulate endothelial cell proliferation.
Therefore, it was suggested that PlGF functioned either as a
weak stimulator or, more likely, as an antagonist of the
pro-angiogenic actions of VEGF-A [71,76]. However, Lang et
al. [3] showed that PlGF in vivo stimulates proliferation of
microvascular endothelial cells in human term placenta and
Ziche et al. [56] showed that PlGF in vivo is a potent
stimulator of angiogenesis. Data have indicated further that
PlGF plays an essential role in pathological angiogenesis [77].
Overexpression of PlGF in mouse skin leads to a substantial
increase in vessel growth and ischaemic tissues can revascular-
ize following PlGF treatment [57,78]. In addition, gene abla-
tion of PlGF indicates a potent synergistic interaction between
VEGF-A and PlGF which contributes to angiogenesis in
pathological conditions [77]. The notion that PlGF is not
merely an antagonist of VEGF-A is also supported by studies
122
on the angiogenic effects of combined VEGF and PlGF, or
VEGF/PlGF heterodimers, and of VEGF receptor hetero-
dimer cross-talk [58]. It is supported further by the obser-
vation that antibody-mediated blockade of the flt-1 receptor
(the site of action of PlGF) results in inhibition of angiogenesis
in tumours, arthritis and atherosclerosis [78].
Correlation of growth factor data with development of the
villous angioarchitecture [19,20,68,79] also suggests that the
final geometry of villous vascular bed is defined, to some
degree, by the balance of VEGF-A and PlGF together with
their receptors. Predominance of VEGF-A promotes establish-
ment of richly branched, low-resistance capillary beds within
mesenchymal and immature intermediate villi both of which
prevail during the first two trimesters of pregnancy. By
contrast, absence of complex capillary beds to the benefit of
poorly branched terminal capillary loops in the last trimester
[19] may be controlled by predominance of PlGF and its
receptor VEGFR-1.
Recent studies in the mink have shown shifts in expression
throughout pregnancy [80]. High expression levels of both
VEGFR-1 and VEGFR-2, the receptors of VEGF, were
followed by downregulation of VEGFR-2 whereas expression
of VEGFR-1, the sole receptor of PlGF, was maintained until
term. However, different from the findings in the human, in
situ hybridization in the sheep placenta revealed a continuous
increase of placental VEGF mRNA until term [81]. These
animal data underline our earlier notion that it is dangerous to
transfer findings from non-human to human placentae. Fur-
ther studies are required to test the contrasting roles of VEGF
and PlGF and their receptors on villous angiogenesis and to
analyse the complication of soluble VEGFR-1.
The balance between VEGF-A and PlGF secretion may be
regulated by oxygen partial pressures. As discussed above,
VEGF and its receptors in placental tissues, and in vitro in
related chorioallantoic tissues, are upregulated under con-
ditions of reduced oxygen [4,5,35,36,71]. The reverse appears
to be the case for PlGF [36,71]. These findings raise the
question of whether or not the switch (from VEGF-A domi-
nance in early pregnancy to PlGF dominance in the second
and third trimesters), together with the concomitant changes
in vascular geometry, result from increasing intraplacental
oxygenation due to the known exponential increase in utero-
placental blood flow. However, caution must be exercised since
placental tissues may respond differently to changes in pO2 at
different stages of gestation [66].
THE TIMING MISMATCH BETWEEN THE
OXYGEN SWITCH AND MORPHOLOGICAL
CHANGE
At early and late stages of gestation, it is necessary to protect
the fetus from the potentially harmful effects of high oxygen
tensions. Development and remodelling of the fetoplacental
vasculature may be part of this protection. It is clear that there
is a time lag of about two months between the rises in
Placenta (2004), Vol. 25
intervillous pO2 levels and perfusion rates on the one hand and
the morphological changes which are evident qualitatively (a
transition from mainly branching to predominantly non-
branching angiogenesis) and quantitatively (accelerated growth
of villi and fetal capillaries) on the other. Oxygen levels rise
after week 12 [12] whereas effective changes in peripheral
vascularization become apparent around 20–25 weeks [79].
The anatomical changes are considerably more protracted than
the rapid in vitro effects of altered oxygen tensions on cultured
cells and reflect the difference between the acute (short-term
and rapid-onset) and chronic (long-term and delayed-onset)
responses to oxygen [82]. Clearly, sculpting the fetoplacental
vascular network takes time and, if the placenta as a whole is
to function in a coordinated manner, this must be integrated
with changes in other villous compartments, especially the
trophoblast.
It seems likely that the fetal demand for oxygen in the
second trimester is met comfortably since the intervillous pO2
seems to be at its highest during this period. However, as
pregnancy advances, fetal demand rises further and the villous
vasculature may need to be remodelled in order to extract more
oxygen from the maternal vasculature. As fetal oxygen extrac-
tion increases, one might predict that the intervillous pO2
would drop. In fact, the peak pO2 is about 60 mmHg at 16
weeks and 45 mmHg at term [12,83,84].
Downregulation of VEGF-A, and upregulation of PlGF,
may occur almost contemporaneously with the oxygen switch
although there is no direct evidence for this at present. In
contrast, stimulated vascularization takes time since sculpting
is the net outcome of neoformation and pruning. The time
course of these events is not known in the placenta but, in the
eye and in tumours, these events can occur rapidly, within just
a few days [85,86]. Regression of capillaries in immature
intermediate villi, together with establishment of a tunica
media around maturing vessels in developing stem villi, may
begin at about the time of the oxygen switch. However, the
transition from branching angiogenesis (in mesenchymal and
immature intermediate villi) to non-branching angiogenesis (in
mature intermediate and terminal villi) involves different types
and generations of villi. Once the key (mature intermediate)
types of villi are generated, persistently higher pO2 levels may
combine with low VEGF-A and high PlGF levels (and changes
in other, as yet unidentified, factors) to facilitate non-
branching angiogenesis. Clearly, some time is required before
an alteration in the overall pattern of the fetoplacental vascu-
lature (produced by non-branching angiogenesis) becomes
apparent histologically together with an altered pattern of
villous development.
This scenario allows the possibility that angiogenic growth
factors are not the only factors which regulate and steer villous
development and angiogenesis. Perhaps the formation of the
first mature intermediate villi requires additional and different
signals than oxygen-dependent growth factors. At least for
terminal villi, there is evidence that their development is
influenced directly by oxygen levels, capillary elongation and
(probably) angiogenic growth factors.
Kaufmann et al.: Fetoplacental Vasculature in Normal Pregnancy
Table 1. Summary of reported findings on fetal capillary morphology during gestation in uncomplicated pregnancies
Variable
Total volume
Total surface
Total length
Volume density
Surface density
Length density
Length ratio
Diameter or area in TS
Shape in TS
Degree of branching
THE MATURE VILLOUS VASCULAR SYSTEM
As a consequence of the growth mechanisms described above,
the villous vascular system differs from that of most other
human organs in two principal respects. First, the arteries and
veins of this low pressure system have a rather thin tunica
media and, except for a few residual paravascular capillaries,
vasa vasorum are mostly absent [68]. In spite of their low
intraluminal pO2 (10 and 20 mmHg, respectively), adequate
supply of the vascular walls is achieved since the intervillous
space surrounding stem villi has a mean pO2 value exceeding
40 mmHg. Second, in contrast to capillary beds of extra-
placental organs, the peripheral capillary bed in terminal villi is
not represented by richly branched capillary nets but rather
composed of large numbers of elongated (>4000 µm), coiled,
moderately branched capillary loops (Figure 2e, Figure 3f, g,
Figure 4 and Figure 5; [19,20]).
QUANTIFYING THE VILLOUS VASCULATURE
DURING GESTATION
Most of the processes involved in angiogenesis can be assessed
by stereological quantification of the morphological features of
capillaries and villi evident on placental tissue sections [23,87–
90]. Nett growth of fetal capillaries within villi (which results
from the counteracting effects of angiogenesis and vascular
pruning) can be described by their total volume, surface area
and length. Endothelial cell proliferation can be assessed by
estimating the total number of cells or their nuclei. With
suitable cell markers, it may also be possible to distinguish
vascular endothelial cells from endothelial progenitor cells and,
by counting them separately, to assess the relative impacts
on capillary elongation of proliferation versus intercalation.
Degrees of villous capillarization or vascularization can be
expressed by relative data, e.g. by estimating capillary volume,
surface and length densities within villi and/or capillary :
villus surface and length ratios. Capillary volume density
represents the fractional volume of villi occupied by capillaries
whilst the surface and length densities are measures of the
packing of capillary surface and length in a fixed volume of
villi. Surface ratio reflects capillary surface area divided by
villous surface area and length ratio the total length of
123
Nature of change in specified variable
Increase [10,23,98]
Increase [10,98]
Increase [10,23]
Increase [99,100]; no change [98]
Decrease [99]
Decrease [99]
Increase [10]; biphasic increases [23]
Increase [99,103]; decrease [10,98,101,102]; no change [23]
Biphasic with increase then decrease [23]
Biphasic with increase then decrease [9]
capillaries divided by total length of villi. Calibre and shape
remodelling can be described by estimating cross-sectional
areas, perimeters and shape coefficients (perimeter2/area)
whilst cellular remodelling can be described by mean cell size,
e.g. squame volume, surface area or depth.
Branching and non-branching angiogenesis, and different
forms of branching angiogenesis, may be distinguished by
viewing 3D vascular casts [19,68,91] or by 3D reconstruction
of serial physical or optical slices [17,20,68,92,93]. For quan-
tification, 3D counts of capillary segments or branchpoints
(including intussusceptive endothelial pillars) can be combined
with estimated lengths to obtain mean capillary segment length
[94–96]. Other possibilities include relating the numbers of
intervillous connections on sections to the numbers of capillary
profiles. The latter ratio should be larger in instances of
branching angiogenesis (see Fig. 15.6 in [9]).
The third review of this series ([24], this volume) sum-
marizes some of the findings obtained by applying these
angiometric methods to placentae collected from various com-
plicated pregnancies. Together with studies during normal
gestation [23], they demonstrate the utility of these measures
of angiogenic activities. The gestational studies have confirmed
that the growth of villi and capillaries is biphasic with slower
growth occurring before mid-gestation (when branching
angiogenesis is the dominant pattern) and rapid growth there-
after (when there is a shift towards non-branching angio-
genesis). The phasic nature of changes emphasizes the need for
caution when drawing comparisons between fixed periods of
pregnancy (say, between first and third trimesters) and this is
particularly important when studying capillary : villus length
ratios and capillary shape coefficients which peak at mid-
gestation. Findings also show that capillary growth involves
increases in total length brought about principally by prolifer-
ation of vascular endothelial cells with some remodelling of
cells towards term [23]. Cellular remodelling is evident as
increases in mean squame area. There is some inconsistency
between studies in whether there are attendant changes in
capillary calibre and, if so, whether this involves an increase or
decrease. Most studies suggest that mean calibres (expressed as
mean diameters or cross-sectional areas) decrease during
gestation (Table 1) but this is at odds with the subjective
impression of increases in the incidence of sinusoidal
124
dilations within the capillary beds of terminal villi [9]. Possible
explanations for this apparent inconsistency have yet to be
tested.
Measures of overall growth
Between 10 weeks of pregnancy and term there is relatively
little change in the absolute volume of stem villi. However,
there is a roughly 4-fold increase in volume of intermediate
(mostly mature intermediate and mesenchymal) villi and a
dramatic (almost 40-fold) increase in volume of terminal villi
[10,23]. These finer branches eventually come to account for
over 90 per cent of total villous length [10,23,97]. Indeed
the intermediate and terminal villi grow by increasing their
total length and surface area whilst becoming more slender
(Table 1).
Within the mature intermediate and terminal villi there is
disproportionate growth in the total volume, length and
surface of capillaries [10,23,98]. The changes in total length
mainly result from endothelial hyperplasia although the true
impact of intercalation is unknown.
Measures of villous capillarization
Because of preferential growth (Table 1), the volume density
of capillaries within villi increases between 10 weeks and term
[23,99,100] although one study found no change after 28 weeks
[98]. Available data on surface and length densities suggest that
these decline towards term [99]. However, the rate of increase
of total capillary length is not commensurate with that of villi
because the capillary : villus length ratio varies. It peaks at
about mid-gestation and then drops sharply before gradually
being restored to peak values again by term [23,98]. The shifts
in this ratio may coincide with changes in the dominance of
branching and non-branching angiogenesis.
ACKNOWLEDGEMENTS
PK is grateful to the Deutsche Forschungsgemeinschaft and the European Union (Biomed 2) for financial support. TMM wishes to thank The Medical Research
Council (Development Grant Scheme, G9826907) and The Wellcome Trust for research funding. DSCJ is supported by The Medical Research Council
(Reproductive Angiogenesis Cooperative Group, G9722567).
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