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Abstract
9Uterine spiral arteries play a vital role in supplying
nutrients to the placenta and fetus, and for this purpose they are
remodelled into highlydilated vessels by the action of invading
trophoblast (physiological change). Knowledge of the mechanisms of
these changes is relevant for a bet-ter understanding of
pre-eclampsia and other pregnancy complications which show
incomplete spiral artery remodelling. Controversies stillabound
concerning different steps in these physiological changes, and
several of these disagreements are highlighted in this review,
therebysuggesting directions for further research. First, a better
definition of the degree of decidua- versus trophoblast-associated
remodelling mayhelp to devise a more adequate terminology. Other
contestable issues are the vascular plugging and its relation with
oxygen, trophoblast invasionfrom the outside or the inside of the
vessels (intravasation versus extravasation), the impact of
haemodynamics on endovascular migration, thereplacement of arterial
components by trophoblast, maternal tissue repair mechanisms and
the role of uterine natural killer (NK) cells. Several ofthese
features may be disturbed in complicated pregnancies, including the
early decidua-associated vascular remodelling, vascular pluggingand
haemodynamics. The hyperinflammatory condition of pre-eclampsia may
be responsible for vasculopathies such as acute atherosis,although
the overall impact of such lesions on placental function is far
from clear. Several features of the human placental bed are
mirroredby processes in other species with haemochorial
placentation, and studying such models may help to illuminate
poorly understood aspectsof human placentation. 2005 Elsevier Ltd.
All rights reserved.
Keywords: Spiral arteries; Trophoblast invasion; Vascular
remodelling; Human; Pre-eclampsia; Decidualization; Uterine NK
cells
1. Introduction
The interrelationship between maternal and fetal circula-tions
enabling physiological exchange within the placenta isstill not
fully understood. Probably the most significant break-through was
achieved in the 18th century when William andJohn Hunter
demonstrated by injection experiments thatmaternal and fetal
vascular structures remain separate, thusrefuting the previously
held opinion of a continuous mother-to-fetus circulation. This
might have been a chance observation,since injecting blood vessels
with coloured wax was becoming
time. Most probably it was the younger brother John, who
ac-tually did the dissections, who quickly grasped the
physiolog-ical implications of this observation, which in later
years led toa quarrel concerning priority [1,2]. Nevertheless, it
was Wil-liam who illustrated for the first time spiral arteries in
his fa-mous Anatomia uteri humani gravidi tabulis illustrata e
Theanatomy of the human gravid uterus exhibited in figures(1774)
[3], in a series of superb engravings based on carefuldissections
of several pregnant uteri. The spiral arteries werethen described
as arteriae convolutae/convoluted arterieswhich are passing between
the womb and placenta. TheseThe Uterine Spiral ArteriFacts and
C
R. Pijnenborg*, L. Ve
Department of Obstetrics & Gynaecology, Universitair
Ziekenhuis G
Accepted 20 D
Placenta 27 (2006)a routine procedure for anatomical
demonstrations at that
* Corresponding author. Tel.: 32 16 346190; fax: 32 16
344205.E-mail address: [email protected] (R.
Pijnenborg).
0143-4004/$ - see front matter 2005 Elsevier Ltd. All rights
reserved.doi:10.1016/j.placenta.2005.12.006s In Human
Pregnancy:ntroversies
cruysse, M. Hanssens
sthuisberg, Katholieke Universiteit Leuven, B3000 Leuven,
Belgium
cember 2005
39e958vessels were most clearly visible in drawings of the
insideof the uterus after separation of the placenta, which we
brokethrough in separating these two parts. This short passage
re-veals their ignorance about the factual arrangement of mater-nal
blood supply to the placenta. In spite of the ingenuity of
-
this point is considered to form the demarcation line
betweenradial and spiral arteries. Meanwhile observations on
placentalbed biopsies have confirmed the deep myometrial origin
ofbasal arteries, which nourish both the inner myometrium andbasal
endometrium. Therefore the designation of the inner my-ometrial
arteries as spiral arteries has subsequently been fol-lowed. A
precise terminology e although it may look likea rather
insignificant issue e is important regarding the depthover which
pregnancy-associated changes in spiral arteries arereported to
occur.
Studies on the endometrial spiral arteries of the non-pregnant
uterus indicated the remarkable sensitivity of thesevessels to
stimuli by hormones or growth factors, while thebasal arteries are
thought to be more stable non-hormone re-sponsive structures
[4,8,9]. The convoluted course of the spiralarteries obviously
results from arterial growth exceeding theincrease in endometrial
thickness during the cycle and duringpregnancy. There can be no
doubt that this spiral shape has
Fig. 1. Vascular anatomy of the pregnant uterus according to (A)
Ramsey and Dlocation of the branching points of basal arteries from
the main arteries, Ramsey
confirmation of deep basal artery branching points, spiral
arteries were recogncoiling of endometrial arteries is related to
decidualization ofthe endometrium which in primates is started off
spontane-ously during the luteal phase of each cycle. In
non-menstruat-ing animals such as rats, mice and hamsters, spiral
outgrowthof uteroplacental arteries definitely occurs during
pregnancyand is also associated with the decidualization process,
whichin these animals does not occur spontaneously during their
es-trous cycle, but is induced by blastocyst implantation [11].
2. Basic histological observations on spiral arteriesduring
pregnancy: a historical overview
The earliest histological observations on uterine spiral
ar-teries in human pregnancy were reported by German investiga-tors
in the last third of the 19th century. This early researchwas
summarized and extensively discussed by Grosser, andhis monograph
should be consulted for detailed references[12]. He quoted
Friedlander (1870) as the first to have
onner [8] and (B) Brosens et al. [7]. Because of initial
uncertainties about thethe Hunter brothers, they could not have
envisioned at thattime how the uteroplacental circulation is
established by theaction of invading cells from the placenta. Since
this reviewdeals a lot with placentaleuterine interactions, it may
be rele-vant that it was also William Hunter who introduced the
termdecidua for the thickened inner lining of the pregnant
uteruswhich is discarded at parturition. The term is
etymologicallysignificant since the Latin word decidere means to
falloff, which also applies to the expulsion of the
decidualizinguterine mucosa at menstruation.
After these pioneering observations it took more than a cen-tury
to clarify the detailed anatomy of the uterine vasculature[4e6].
The matter is basically settled now, although some con-fusion arose
concerning terminology, i.e. whether the arteriesin the inner
myometrium should be considered as spiral [7]or radial arteries [8]
(Fig. 1). Part of the argument was dueto a disagreement about the
level where the basal arteriesare branching off in the decidua or
in the myometrium, as
haemodynamical repercussions. John Hunter already notedthat The
intention of these spiral turns would appear to bethat of
diminishing the force of the circulation as it approachesthe spongy
substance of the placenta. where quick motion ofthe blood is not
wanted (quoted by Ramsey [6]). Their pecu-liar shape would not only
lead to a progressive decrease inblood pressure along their length,
but would also dampenthe pulse, which is important for maintaining
a steady bloodflow to the intervillous space of the placenta.
Ramsey foundthat the coiling of the spiral arteries still increased
in earlypregnancy, but that uncoiling started around
midpregnancy[6,10]. The reserve length of the convoluted vessels
obviouslyallows easy stretching during pregnancy-associated
uterinegrowth.
Reynolds [4] made the interesting point that coiled or
spiralarteries are not seen in the endometria of most
non-pregnantanimals, and therefore he speculated that their
peculiar shapein primates may be related to menstruation. In fact,
increased
940 R. Pijnenborg et al. / Placenta 27 (2006) 939e958considered
the whole myometrial segments as radial arteries (A). Following
later
ized as having myometrial as well as decidual segments (B).
-
described endovascular cells in maternal arteries at
midpreg-nancy, without making any statement as to their possible
ori-gin. Most investigators then thought that these cells
werederived from the decidua or endothelium, but Grosser anda few
others (Aschoff; Hinselman; Meyer; Schickele; allquoted by Grosser
[12]) favoured a trophoblastic origin(Fig. 2). Furthermore, they
made the association between in-vading trophoblastic cells and
fibrinoid changes in vesselwalls, thought to result from vascular
degeneration by fermen-tative action of a trophoblast-secreted
choriotrypsin. Theyconceived that in this way maternal tissues are
digested and re-placed by fetal cells, sometimes leading to
complete pluggingof the vessels. Also breakdown of the elastica and
the appear-ance of intimal cushions were described, although it was
ques-tioned whether the latter alterations were related to
thepregnancy rather than to maternal age. Finally Grosser madethe
astute remark that the described vascular changes were re-stricted
to the Placentarstelle (presumably the placental in-sertion site,
i.e. the placental bed), and were extended to thesuperficial
myometrium. It is therefore clear that at the begin-ning of the
20th century several features of spiral artery mod-ification had
already been recognized by several authors, yetmost observations
were anecdotal, performed on occasionalspecimens. In order to
elucidate the step-by-step alterationsin spiral artery conversion
in relation to trophoblast invasion,series of well-dated pregnant
uteri with the placenta in situhad to be studied in a systematic
fashion. Famous classic stud-ies of human placentation, which
included observations onspiral artery transformation, were those by
Boyd and Hamilton[13e16] and Harris and Ramsey [10,17].
Hamilton and Boyd [16] described the presence of intravas-cular
cells in spiral arteries, for which they favoured a
cytotro-phoblastic shell origin based on histological continuity,
ratherthan their being derived from surrounding maternal or
fetalcells. They noted that these cells may form intravascular
tro-phoblastic plugs which in their view could conceivably
R. Pijnenborg et al. / PlaFig. 2. Endovascular cells (ET) in
spiral arteries of early pregnancy, as illus-
trated by Grosser [12].damp down the arterial pressure, but
added that they com-pletely obliterate the vascular lumen only in
regions where thecirculation has been passed by a new perforation
of a spiralartery proximal to an already established opening.
Theyalso described associated degenerative changes in the
vesselwalls, but did not seem to consider that the endovascular
cellsthemselves might become embedded into the wall. This is
themore surprising since they noted their disappearance at the
endof pregnancy, seemingly without considering a possible wayhow
they might have been lost [9,14]. These authors did notconsider
interactions between spiral arteries and interstitialtrophoblast,
which they described as being overall multinu-clear. Indeed, they
were not able to distinguish morpho-logically interstitial
cytotrophoblast from decidual cells,immunohistochemical techniques
not yet being available.Elizabeth Ramseys group, on the other hand,
described howsome of the syncytial streamers (multinuclear
interstitialtrophoblast), come into contact with the outer walls of
spiralarteries, without finding evidence that these cells enter the
ar-terial lumina. They thought that these perivascular
tropho-blasts induced degenerative changes in the arterial
walls[17], foreshadowing later findings of a correlation between
in-terstitial trophoblast concentrations and degenerative changesin
spiral arteries preceding endovascular trophoblast invasion[18].
Harris and Ramsey also noted the appearance of largestellate cells
within the vessel walls, but could not ascertaintheir trophoblastic
origin. Apart from this interstitial to peri-vascular invasive
pathway, like Hamilton and Boyd [15,16],they observed
intra-arterial migration by cytotrophoblast thatpresumably
originated directly from the cytotrophoblasticshell, proceeding as
far as the myometrium. Their ultimatefate was unknown, and they
even wondered whether intravas-cular cells might ultimately be
washed out into the intervillousspace. They considered that
accumulating endovascular cellsmight cause cessation of circulation
which, according to theirhistological observations, would be
overcome by the forma-tion of multiple openings of spiral arteries
into the intervillousspace. Ramsey et al. did consider a
bidirectional invasion ofthe spiral arteries from the inside as
well as from the outsideof the vessels, the latter illustrated by
suggestive microphoto-graphs of perivascular trophoblast in contact
with the endothe-lium, seemingly pushing this layer towards the
lumen. Suchhistological appearances may of course reflect the
tendencyof endothelium to grow over anything placed within a
vessellumen [19]. The contribution of endovascular cells to
arterialwall changes was supported by Ramseys observations on
sim-ilar vascular changes in rhesus monkeys, where interstitial
tro-phoblast invasion is uncommon [10].
Both groups (Boyd and Hamilton, Ramsey and coworkers)regularly
referred to animal work in their publications to illus-trate the
probable trophoblastic nature of cells whose intravas-cular
migratory behaviour was regarded as an oddity. Asa support to the
reality of this observation, Billingtons 1966paper was frequently
quoted, in which he described a similarinvasive behaviour of
hamster trophoblast towards the arteries
941centa 27 (2006) 939e958after transplantation of ectoplacental
cone tissue into the testis[20]. Also Orsinis study on the golden
hamster [21] was often
-
mentioned, because it revealed a very intensive
intravascularinvasion by trophoblast. The same author developed a
unilater-ally pregnant deciduoma model, allowing direct
comparisonbetween invaded and noninvaded decidua within the same
an-imal, thus confirming the trophoblastic nature of
endovascularcells in uterine spiral arteries at implantation sites
[22].
The aforementioned studies on spiral arteries and their rolein
placental development and function were performed froma mainly
academical point of view. A change of perspectivewas provided by
the publication of a first report in 1953 ondefective placental
perfusion in hypertensive pregnancies byBrowne and Veall [23]. This
paper suddenly revealed a possi-ble clinical impact of such
studies, and stimulated a search forvasculopathies in spiral
arteries which might be responsiblefor disturbed placental flow in
pathological conditions. There-fore it was necessary to devise a
technique for obtainingappropriate tissue samples to be used as a
clinical tool. In1956 the first placental bed biopsy was collected
during a cae-sarean section by Dixon and Robertson, who
subsequentlyjoined forces with Renaer and Brosens who had
independentlybeen alerted by the same Browne and Veall paper [24].
Under-standing the histology of this unfamiliar term pregnancy
mate-rial was the first challenge, and even biopsies from
normalpregnancies presented unexpected difficulties. Instead of
thefamiliar spiral arteries known from endometrial tissue sam-ples,
big calibre vessel-like profiles were observed, lackingrecognizable
vascular smooth muscle, but showing insteadan amorphous fibrinoid
wall with embedded pale-lookingcells. Referring to the
comprehensive papers by Hamiltonand Boyd and Harris and Ramsey,
which were mainly con-cerned with early stages of placentation, did
not providemuch guidance at that time. By studying serial sections
ofwhole pregnant uteri with placentas in situ Brosens et al.
[7]proved that these dilated structures are indeed spiral
arterieswhich, by having lost their smooth muscle and elastica,
hadbecome physiologically changed to accommodate theincreasing
maternal blood supply to the placenta. They alsohypothesized that
the fibrinoid-embedded cells are trophoblastic,derived from the
endovascular cells which had been observedin the lumina of the
spiral arteries by previous investigators.
This pioneering groundwork was followed by the discoverythat in
pre-eclamptic women the normal physiological changesof spiral
arteries are restricted to the decidual segments [25]. Itwas then
felt that a better knowledge of the successive steps ofthe normal
vascular transformation would be essential forunderstanding the
defects in complicated pregnancies. As a re-sult, several lines of
study were started. Electronmicroscopicalobservations supported the
endovascular origin of the embed-ded mural cells in spiral arteries
by describing successivesteps of endothelial penetration, fibrinoid
secretion and muralincorporation [19,26]. A separate study on
complete hysterec-tomy specimens of early pregnancy with placentas
in situtraced the successive steps in the physiological change of
spi-ral arteries [18,27,28]. One of the questions asked was in
howfar trophoblast-associated physiological changes were pre-
942 R. Pijnenborg et al. / Placeded by structural alterations in
spiral arteries, as previouslyobserved in pregnant golden hamsters
[29,30]. By applyingOrsinis unilaterally pregnant deciduoma model,
early vascularchanges had been detected in this animal which were
closelyassociated with the decidualization process, thus
providingthe inspiration for investigating this feature in the
human.Brettner [31] had already reported the swelling of
vascularsmooth muscle in the decidualized human endometrium.
InDixons 8e18 weeks hysterectomy collection early
vascularalterations leading to media disruption were also observed
inmyometrial spiral arteries as a preliminary step preceding
en-dovascular trophoblast invasion. While some of the early
vas-cular changes (vacuolation) also occurred outside the
placentalbed and were therefore considered to be associated with
de-cidualization, the outstanding vascular smooth muscle
degen-eration preceding endovascular invasion was found to
becorrelated with the presence of interstitial trophoblast
aroundthe vessels (Fig. 3) [18]. In the latter paper it was also
hypoth-esized that the early loss of smooth muscle coherence leads
tovascular distension, which might provide a rheotropic triggerfor
endovascular migration.
During the last decennia technical developments have
sig-nificantly widened the research possibilities in this
excitingfield. A major advance was the introduction of
cytokeratinimmunohistochemistry [32e34], by which the
trophoblasticnature of the endovascular and intramural cells could
at lastbe definitely proven. In addition, the development of a
plethoraof immunohistochemical tools for detecting various
markermolecules, cell adhesion molecules, receptors, enzymes,
cyto-kines and growth factors, allowed more precise studies of
thecomplex interrelationships between trophoblast and
maternaltissue components. Another important development was
theimprovement of methods for maintaining trophoblastic cellsand
placental tissue fragments in culture. With regards to spi-ral
arteries, the latest development of arterial explant
culturespromises to be another valuable tool for analysing the
succes-sive steps in uteroplacental vascular remodelling and
function[35]. At the clinical side, improvement of Doppler
techniquesallowed studies of flow patterns in the uterus and
placenta asa functional in vivo correlate to vascular remodelling
in nor-mal as well as complicated pregnancies. Placental bed
studiesmaking use of all these different technologies will be
furtherdiscussed in the following sections of this review.
3. Trophoblast invasion and vascular remodelling:questions and
controversies
3.1. Decidualization of spiral arteries
andtrophoblast-independent changes
The aforementioned concept of decidual changes in spi-ral
arteries as a necessary prelude to trophoblast invasion,
at-tractive as it might be, still needs further substantiation.
Inhow far early vascular decidualization is a conditio sine quanon
for the later trophoblast-associated vascular remodellingis still
not known.
Several years after the first vascular decidualization
studies
centa 27 (2006) 939e958[18,29,31], the idea of
trophoblast-independent changes of spi-ral arteries was revived by
Craven et al. [36]. Unfortunately, in
-
their early presentations the changes observed in
uterinedecidual tissue of women with ectopic pregnancies were
referredto as physiologic changes [37], thereby infusing the
scien-tific community with the idea that pregnancy-associated
vas-cular remodelling of spiral arteries does not need
trophoblastat all. As a response King and Loke [38] looked for
physiolog-ical changes, as previously defined by Brosens [7], in a
similarseries of uterine deciduas of ectopic pregnancies and,
asexpected, refuted Cravens hypothesis. The importance of us-ing a
precise terminology is the principal lesson of the Cravenincident.
Indeed, terms such as physiological change, arte-rial conversion,
vascular remodelling are probably too
specific terminology, for which we tentatively propose
tro-phoblast-associated remodelling to replace the old term
ofphysiological change, and decidua-associated remodellingfor
referring to the early vascular changes which precede
thetrophoblast-associated remodelling. However, since theearly
remodelling partly depends on the presence of
interstitialtrophoblast, an interstitial trophoblast-associated
remodel-ling should be interposed, preceding the
endovasculartrophoblast-associated remodelling (Fig. 3).
Unfortunatelysuch rather wordy circumscriptions do not really offer
apalatable terminology. Moreover, these terms may suggesta
well-defined causal relationship which is by no means fully
Fig. 3. Diagram showing the different steps in uterine spiral
artery remodelling, starting from the non-pregnant condition. The
earliest stage in vascular remodelling
(stage 1) consists of endothelial vacuolation and some swelling
in individual muscle cells. Invasion of stromal and perivascular
tissues by interstitial trophoblast is
associated with further disorganization of the vascular smooth
muscle layer (stage 2). Only then endovascular trophoblast appears
(stage 3). Trophoblast becomes
embedded intramurally within a fibrinoid layer, which replaces
the original vascular smooth muscle (stage 4). Finally
re-endothelialization occurs, which may be
accompanied by the appearance of subintimal cushions containing
a-actin immunopositive myointimal cells (stage 5).R. Pijnenborg et
al. / Placgeneral and vague for designating the very specific
changesin placental bed spiral arteries. There is a need for a
more943enta 27 (2006) 939e958established. Another difficulty of
these terms is that they stillrefer to a complex of morphological
changes, some of which
-
care ill-defined such as medial disorganization.
Identificationof molecular markers for the different steps of early
vascularchanges would be helpful for working out better
definitions,but so far no such molecule has been identified.
The concept of decidualization of myometrial spiral ar-teries,
i.e. the idea that also trophoblast-independent vascularchanges in
the myometrium are associated with the decidual-ization process,
was recently taken up again by Brosens et al.[39], who considered
its possible relevance to pregnancycomplications and even
infertility. A cautionary note shouldbe added concerning the
application of the term deciduali-zation to myometrial tissue
because of the etymologicalroots of the term decidua (see Section
1). The term de-cidualization, as suggested from its first use,
indeed refersto the irreversible shedding of tissue and cell death,
whilethe decidual changes in the myometrium may bereversible.
3.2. Flow interruption by endovascular plugging
In our historical overview we mentioned the first
consider-ations of haemodynamic consequences of endovascular
plugs,but also how possible flow defects might be partially
overcomeby the formation of multiple openings [16,17]. Because of
theoblique course of spiral arteries with respect to the
placentalfloor, especially in lateral parts of the placental bed,
progres-sive decidual erosion by the basal plate may indeed
generateadditional openings. In our previous studies we
observedthat such openings into the intervillous space may appear
half-way down a decidual segment of a spiral artery, with the
moredistal portion eventually ending up within a necrotic mass
ofdecidua (Fig. 4) [27,40]. In how far complete plugging ofthe most
distal portions is related to this decidual necrosis isnot
known.
Fig. 4. Diagram based on histological sections of a 12 week
placental attach-
ment site, showing successive cross-sections of a decidual
spiral artery, open-
ing into the intervillous space through a gap created halfway
through the
decidual segment. The more distal portions of the artery are
located within
944 R. Pijnenborg et al. / Plaa necrotic mass of decidua
(reproduced from Ref. [40], with kind permission
of the editor of Trophoblast Research).The physiological
importance of plugging was deducedfrom the ultrasonographic and
hysteroscopic observations ofHustin and Schaaps [41], showing
absence of maternal redcells in the intervillous space of early
pregnancy. The earlierhistological observations on vascular
plugging fitted nicelywith the newer evidence that early placental
developmenttakes place in a low oxygen environment [42]. While
thisidea was received with a healthy scepticism at the
beginning[43e45], different lines of evidence e physiological,
bio-chemical, in vitro studies e clearly support a low flow
situa-tion in early pregnancy [46]. A revisit to the Boydcollection
revealed the presence of channels in the cytotropho-blastic shell
from 8 weeks onwards, whereas various degreesof plugging persisted
during the following weeks, and onlyfrom the 14th week clear
patency of spiral artery outlets intothe intervillous space was
observed [47]. Since the extent ofplugging tends to be more
complete in centrally located spiralarteries, the same
investigators proposed that maternal flowstarts at the periphery
rather than at the centre, and there ismorphological as well as
ultrasound evidence to support thisview [48]. It is not known in
how far the oblique course of spi-ral arteries and the formation of
additional openings could berelated to the onset of flow at the
periphery. In contrast, from17 weeks onwards Doppler observations
on individual spiralarteries indicated higher flow in central than
in peripheralspiral arteries, reflecting dissolution of the plugs
and morecomplete spiral artery remodelling at the centre than at
thesides [49].
Genbacev et al. [50] were the first to establish a
relationshipbetween oxygen levels and trophoblast differentiation,
in thesense that low oxygen favours proliferation, while high
oxy-gen levels are required for differentiation into the
invasivephenotype. The in vitro experiments by Caniggia et al.
[51]pointed to the same direction, and were extended by the
find-ing that the transcription factor HIF-1a plays a principal
reg-ulatory role. Although providing a neat cellular model
forexplaining trophoblast invasion there are, in our opinion,
stillgaps in the overall story. Trophoblastic plugging of spiral
ar-teries is considered to be the mechanism for creating a low
ox-ygen environment [41], but then the question arises how
thistrophoblast gets into this plugging position in the first
place.Is endovascular plugging to be regarded as a real invasive
phe-nomenon (which would require high oxygen according to
theexperimentalists), or does it merely occur by filling up
emptyspaces by proliferating trophoblast? During the 7e13
weeksperiod under low oxygen, extravillous trophoblast, whichwould
include the endovascular plugs, never shows prolifera-tion [52].
The expression of invasion markers (integrins, met-alloproteinases)
in endovascular plugs e if such markerswould persist in that
situation e would be an indication foran invasive phenotype. In
spite of extensive studies of suchmarkers in extravillous
trophoblast, there is a remarkablelack of reported information
concerning their expression in en-dovascular plugs. Only in the
rhesus monkey immunopositivestaining for MMP-1 in the trophoblast
of a plugged artery pro-
enta 27 (2006) 939e958vided unmistaken evidence for invasive
potential [53]. Thiswould indicate that the endovascular
trophoblasts have
-
acquired the invasive phenotype from the beginning,
notwith-standing the presumably hypoxic condition of early
pregnancy.To avoid a vicious circle, we might postulate that
arterial plug-ging as a result of trophoblast invasion from the
cytotropho-blastic shell, indeed occurs in a high oxygen
environment inaccordance to Caniggias experimental findings. The
shell isformed during the first 2e3 weeks, well preceding the
8weeks period of the earliest reported intrauterine
oxygenmeasurements [42,46]. In this connection one may refer tothe
observed hyperaemia of implantation sites in the human[54] as well
as in laboratory rodents, where endothelial leak-age provides the
earliest sign of implantation [55]. Blastocystimplantation may
therefore be associated with (temporary)high oxygen, allowing quick
invasion by trophoblast, followedby a period of relatively low
oxygen because of the partialsealing of supplying arteries which
would protect the develop-ing early embryo against oxidative
stress.
It is interesting to consider the occurrence of vascular
plug-ging in laboratory animals. The only rodent species in
whichvascular plugging has been well documented is the
goldenhamster [29]. In this case the plugging is effected by a
gi-ant-cell type of trophoblast, preceding deeper
endovascularmigration 1 or 2 days later. This is not the case in
the mouse,where vascular invasion in this species seems to proceed
viaa perivascular, rather than an endovascular, pathway [56e58]. In
the rat, in which arterial invasion clearly follows an
in-traluminal pathway, complete plugging of small side branchesof
spiral arteries may occasionally be seen (unpublished obser-vations
by Pijnenborg and Vercruysse). In none of these ani-mals
endovascular plugging occurs to the same extent as isseen in the
human, and possible events of oxidative stressmust therefore be met
in a different way. Moreover, one shouldalso take into account that
the chorioallantoic placenta is fullyfunctioning only relatively
late in these three rodent species,and that early embryonic
development depends mainly onthe yolk sac. The plugging of the
spiral arteries in the hamsterfor instance occurs during this
yolk-sac dependent period. In-teraction between invading
trophoblast and maternal vascula-ture in these animals shows
similarities with the human onlyduring the last third of their
pregnancy when haemochorialplacentation is fully established.
Vascular invasion then takesplace in the period after
organogenesis, when the developingembryo is no longer so sensitive
to oxidative stress.
3.3. The intravasation versus extravasation controversy
The relationship between interstitial and endovascular
tro-phoblast has been a topic of debate and disagreement formany
years. In a recent review Kaufmann et al. [59] discussedthe
possible origin of endovascular trophoblast, favouringa process of
intravasation, i.e. inward movement into thevessel of
(interstitial) trophoblast from the outside. Althoughit is
conceivable that intravasation indeed occurs in the super-ficial
decidua close to the placenta, it can be questioned
R. Pijnenborg et al. / Placwhether the same applies to the
deeper layers of the placentalbed, particularly to the
myometrium.In early pregnancy spiral arteries become surrounded
byinterstitial trophoblast, resulting in marked perivascular
clus-tering in the superficial decidua [27,60,61]. Our own
observa-tions were performed on 8e18 weeks complete
hysterectomyspecimens processed in toto, allowing to evaluate
gradientsof changes from decidua to myometrium. We found that
vol-ume density of interstitial trophoblast diminishes with
depth,while perivascular clusters of interstitial trophoblast seem
tofan out away from the vessels in the deeper regions[11,60]. We
think it highly probable that in the most superfi-cial decidua at
least some of the endovascular trophoblastsare indeed derived from
the numerous interstitial trophoblasticcells via intravasation.
This is the more likely since in theupper decidual areas the
arteries have a poorly defined muscu-lar wall lacking a clear
elastica, in contrast to the deeper, es-pecially myometrial, tissue
layers [62]. However, deeperdown in the myometrium increasing
scarcity of perivascularinterstitial trophoblast may lower the
opportunity for suchcells to invade a vessel wall, which at the
same time has be-come more muscular compared to the decidua. Hence
ourview that the intravascular presence of trophoblast in
myome-trial spiral arteries is more likely the result of
intraluminal mi-gration from the decidual segments after the
disintegration ofthe endovascular plugs (Fig. 5).
Considering the intramural trophoblasts, their presence inthe
vessel wall could theoretically be explained by different
Fig. 5. Diagram illustrating the interstitial invasion gradient
from decidua to
myometrium, indicating their possible contribution to
endovascular tropho-
blasts in spiral arteries. Deeper in the placental bed,
interstitial trophoblast
are less concentrated around the spiral arteries, and therefore
there is theoret-
945enta 27 (2006) 939e958ically a lower chance of perivascular
interstitial trophoblast to penetrate the
spiral artery wall.
-
eintravasation or extravasation scenarios (Fig. 6). The
simplestpossible invasion route would be either direct
extravasation(Fig. 6A) or direct intravasation (Fig. 6B). The
latter is defi-nitely too simplistic since it does not account for
the presenceand subsequent disappearance of intraluminal
trophoblast. Onecould speculate that the primary intravasation step
consists ofa complete crossing of the vessel wall to reach an
endovascu-lar position, followed by intramural incorporation
intofibrinoid, possibly after intraluminal migration (Fig. 6C).
Al-ternatively, intravasation of interstitial trophoblast may
indeedresult in direct intramural incorporation into fibrinoid, but
thelatter should then not be regarded as a permanent burial sitefor
trophoblast. After being released into the lumen, the tro-phoblast
may proceed its intraluminal migration until its finalincorporation
into the wall of a deeper vascular segment(Fig. 6D). The
theoretical possibility even exists that intramu-ral trophoblast
may escape back into the interstitial tissue(Fig. 6E). It is
virtually impossible to prove which of thesestories is correct
using in vivo collected tissue samples, sincedynamic events e
especially when a shuttling to and fro of
Fig. 6. Diagram of different possible scenarios of intramural
invasion of a spiral
artery by intravasation or extravasation by interstitial (blue)
or endovascular
(green) trophoblast. Arrows indicate the direction of cell
migration. (A) Shows
direct extravasation of endovascular trophoblast into the vessel
wall, while (B)
indicates direct intravasation of interstitial trophoblast into
the wall. Intramural
trophoblast is indicated by its incorporation within fibrinoid
(pink). (C) and
(D) indicate two different migratory intravasation pathways. (C)
Shows the
theoretical possibility of direct migration of interstitial
trophoblast through
the vessel wall, becoming temporarily endovascular and being
subsequently
embedded into the vessel wall by fibrinoid deposition. (D) Shows
the theoret-
ical possibility of interstitial trophoblast becoming intramural
by fibrinoid de-
position, being subsequently released from the fibrinoid to
become
endovascular followed by later re-embedding. (E) Shows the
theoretical pos-
sibility of interstitial release of previously
fibrinoid-embedded endovascular
trophoblast.
946 R. Pijnenborg et al. / Placcells would occur e are
notoriously difficult to reconstructfrom static histological
preparations. Explant cultures ofisolated spiral arteries
cocultured with purified extravilloustrophoblast seem to indicate
that trophoblast from outsidethe artery may cross the vessel wall
and end up in the lumen[35], and it would be interesting to look
for fibrinoid formationin such experimental conditions. In our
opinion, a combinationof scenarios A and C of Fig. 6 provides the
most likely story,but the impact of scenario C must depend on the
local densityof interstitial trophoblast. Considering the
correlation betweenthis density and the depth within the placental
bed, we stillthink that endovascular e i.e. intraluminal e
migration isthe principal route of vascular invasion, which
accounts forobservations of continuous streaks of endovascular
cellswithin myometrial spiral arteries showing almost no
interstitialtrophoblast in their immediate surroundings [18].
Different stages of incorporation of endovascular tropho-blast
into the vessel wall have been described by De Wolfand colleagues
[26] and similar observations have been re-ported for the rhesus
monkey [63]. Although in this species in-terstitial trophoblast is
largely absent and trophoblast invasionis mainly endovascular,
proceeding at the luminal side of thespiral arteries as far as the
decidualemyometrial junction, in-tramural trophoblast embedded into
fibrinoid also appears.Similarly, in human spiral artery explants
dissected fromnon-placental bed areas and cocultured with purified
tropho-blast which is placed into the vascular lumen,
incorporationof trophoblast into the vessel wall was observed
coincidingwith endothelial cell apoptosis [64]. In how far
endovascularand perivascular (interstitial) trophoblast represent
separatesubtypes contributing to intramural trophoblast, requires
fur-ther investigation for which the expression of specific
markerproteins may be helpful. The only exclusive endovascular
tro-phoblast marker established so far is the adhesion moleculeNCAM
[65]. A recent study indicated the presence of bothNCAM-positive
and negative intramural trophoblast in the re-modelled spiral
arterial walls, indicating a dual endovascularand interstitial
origin [66], and this supports Ramseys earlierideas which were
summarized in our historical overview[10,17].
3.4. Possible mechanisms of endovascular migration
Endovascular trophoblast migrates retrogradely within thelumina
of spiral arteries, but little is known about mechanismsexplaining
their movement against the blood stream. The factthat endovascular
invasion only occurs within arteries andnever into veins, suggests
a possible contribution of oxygencontent or haemodynamics. Although
it is now well knownthat oxygen is important for differentiation
and invasiveness[50,51], the possible role of gradients of oxygen
levels in di-rected cell migration through the vascular environment
hasbeen suggested but not proven.
The physical force of the blood stream has been consideredas a
possible trigger to induce trophoblast migration [67].Cells may
respond to flow intensities and directions by reor-ganizing their
cytoskeleton and/or motile apparatus. Exposure
nta 27 (2006) 939e958of cells to fluid streams may lead to
changes in microfilamentpolarity and alterations in membrane
recycling, both processes
-
being involved in cell motility [68]. Such changes may be
trig-gered by cell deformation and activation of a
mechanotrans-duction pathway [69]. Especially microfilament
polarizationchanges, as observed in other cell types exposed to
flow, mightbe involved in a directed e rheotactic e motile
behaviourof endovascular trophoblast. Recent work on isolated
rhesusmonkey trophoblast grown on the inner surface of
capillarytubes did show that shear stress, applied by increasing
flowin the tubes, leads to trophoblast migration. One could
argue,however, that the observed event was not really an active
mi-gration, but rather a passive displacement in the direction
ofthe fluid stream. Interestingly, a retrograde movement
againstflow in this experimental setup was observed, but only
whenthe trophoblast was cocultured with endothelial cells
[70,71].The same authors also found that shear stress induces b1
integ-rin expression in the trophoblast, indicating an involvement
ofcell adhesion molecules in flow associated migration.
The possible triggering effect of increasing blood flow
ontrophoblast migration is supported by some observationsin vivo.
In the golden hamster it was observed that intraplacen-tal maternal
arterial channels, formed in continuity with spiralarteries, appear
before the retrograde migration of endovascu-lar trophoblast into
the corresponding arteries is started [29].The appearance of the
intraplacental channels may be a resultof increased uterine blood
flow, which is known to be understeroid (estrogen) control. Indeed,
in ovariectomized pregnanthamsters treated with progesterone only,
intraplacental arterialchannels did not form in association with
all spiral arteries,and trophoblast invasion did not occur in
vessels which didnot communicate with such channels [30].
It may also be significant that in the human deep endovas-cular
migration into the myometrial spiral arteries occurs onlyafter the
alleged onset of placental circulation, reported to oc-cur around
12 weeks [41,46,47]. The decidua- or
interstitialtrophoblast-induced vascular remodelling is supposed to
causeearly vascular distension accommodating higher flow, whichmay
provide the necessary trigger for subsequent
endovasculartrophoblast migration [18]. A similar function has been
as-cribed to perivascular trophoblast in the guinea pig. It
wasshown by immunohistochemistry that this trophoblast containsthe
NOS enzyme, and it can be expected that nitric oxide hasa dilatory
action on maternal blood vessels, preceding intra-vascular
migration of trophoblast [72]. In the human, however,the presence
of NOS in extravillous trophoblast is controver-sial, both eNOS and
iNOS isoforms reported to be absent[73] and present [74,75] in
interstitial trophoblast, includingthe perivascular cells along the
blood vessels in the placentalbed. The investigations in the guinea
pig have not been re-peated by other research groups.
3.5. Decidual and myometrial invasion: the
two-wavehypothesis
Robertson et al. [76] hinted for the first time that in the
hu-man, decidual and myometrial migration within spiral
arteries
R. Pijnenborg et al. / Placis separated in time, the latter
occurring between 12 and 16weeks. In the hysterectomy collection
studied by Pijnenborget al. [18] the endovascular invasion of the
myometrial spiral ar-teries beyond the superficial myometrium
started at 14 weeks,indicating at least a 4 week difference between
their appearancein decidual spiral arteries and deeper myometrial
arteries, re-spectively (Fig. 7). The separate wave concept had
partlybeen inspired by studies on the golden hamster, where two
sep-arate endovascular migration waves occur into two
differentvascular systems, the first into the circumferential
arteries whichare associated with the yolk-sac placenta, and the
second, laterin pregnancy, into the central spiral arteries related
to the chorio-allantoic placenta [29]. This twowaves terminologywas
thenapplied to the human, with the understanding that here they
oc-cur within the same vascular system. It could never be
estab-lished whether this so-called second wave was the extensionof
invasion by endovascular cells that were lying dormantin decidual
spiral arteries, or if there was really a new wave oftrophoblast
leaving the cytotrophoblastic cell columns or theremnants of the
cytotrophoblastic shell.
The two-wave hypothesis has been criticized lately by Rob-son
and colleagues, following extensive observations in a largeseries
of early pregnancy placental bed biopsies, collected bythe newly
established punch biopsy technique under ultra-sound guidance
[77,78]. Their results supported a progressiveendovascular
migration from decidual into myometrial arteriesrather than showing
a distinct second wave. A difficulty in bi-opsy studies is of
course that the depth of invasion is difficultto evaluate because
of the random orientation of biopsy spec-imens. The original
confirmation of the two-wave idea camefrom studies on complete
hysterectomy specimens sectionedin toto allowing easy evaluation of
the depth of endovascu-lar invasion.
Lyall [79] rightly pointed out the difficulty to ascribea
physiological significance to the phenomenon and to con-ceive
cellular and molecular mechanisms controlling such a bi-phasic
invasive process. This difficulty might eventually beresolved when
the haemodynamical factor is brought into con-sideration. It could
be conceived that, as uterine blood flowprogressively increases
[80], a threshold value may be reachedaround the 12 weeks period,
allowing more substantial utero-placental flow, facilitated by
dilatation of myometrial arterieswhich have undergone interstitial
trophoblast-associatedremodelling, which is accompanied by rising
oxygen[41,46,47]. Theoretically, the crossing of this
haemodynamicalthreshold may trigger a second wave of rheotactic
migra-tion of endovascular trophoblast, possibly via activation ofa
still undefined mechanotransduction signalling pathway[69]. For a
complete understanding of the actual cellularmechanisms involved,
more insight will be needed in the bio-mechanical properties of
cells in general [81] and of tropho-blast in particular.
3.6. Intramural incorporation and the fibrinoid question
Following their migration within the arterial lumina
thetrophoblastic cells ultimately invade the vessel wall.
During
947enta 27 (2006) 939e958the last two decennia cellular
mechanisms of trophoblastinvasion have been intensively
investigated, initially focussing
-
on mechanisms of attachment and extracellular matrix break-down
[82], but this research quickly expanded to include reg-ulatory
factors such as cytokines, growth factors and oxygen[50,51,83e86].
Most studies of invasion mechanisms weremainly applied to the
interstitial invasion of stromal tissues,but it is very likely that
similar mechanisms contribute toendovascular trophoblast
incorporation into the vessel walls.Indeed intraluminal trophoblast
has been shown to containmetalloproteinases in both the human [87]
and the rhesusmonkey [53]. Such matrix-degrading enzymes may be
func-tionally important for the penetration of the
subendothelialbasement membranes and other (maternal) matrix
compo-nents. It is still unclear in how far the endothelial layer
ispenetrated by trophoblast or undergoes spontaneous (?)regression.
Although a recent report described apoptosis-induction in
endothelial cells by cocultured extravillous tro-phoblast in
explanted spiral artery segments [64], it shouldbe noted that this
trophoblast may have been of an interstitial(sub)type, not
representative for endovascular trophoblast.
A possible contributory factor to the burial of trophoblastinto
the vessel wall is the fibrinoid which is
characteristicallydeposited in physiologically changed spiral
arteries (Figs. 3and 6). In their electronmicroscopical study of
the vascularincorporation process De Wolf et al. [26] noted the
precipita-tion of fibrinoid material around the endovascular
trophoblast,
precipitation of PAS-positive fibrinoid material can be
ob-served in between intraluminal cells prior to their
intramuralincorporation [88]. Sustained release of matrix material
in thevessel wall may be responsible for the spidery shape of
in-tramural trophoblast. For a long time we assumed that fibri-noid
deposition was a hallmark of endovascular trophoblast,but Bulmer
(personal communication, reported in Ref. [89])pointed out that
also interstitial trophoblast occasionallyshows fibrinoid
deposition and thereby acquires a spideryphenotype.
In spite of extensive studies of the production of
matrixproteins by trophoblast in vivo as well as in vitro there
are,so far, very few data about the exact nature of the
fibrinoidwithin the converted spiral arteries. The matrix-type
fibri-noid of the basal plate described by Frank et al. [90,91]may
be a good candidate for the intramural fibrinoid of spiralarteries,
but their and most other published observations
ontrophoblast-associated extracellular matrix mainly relate
tointerstitial trophoblast. The fact that interstitial and
endovas-cular fibrinoids may be different, was highlighted by
immu-nolocalization studies of fibronectin in term placental
bedbiopsies, showing extensive immunopositive deposits sur-rounding
interstitial trophoblast in contrast to the absenceof staining in
the vascular fibrinoid with embedded intramuraltrophoblast [92].
Also laminin was absent from spiral artery
Fig. 7. Diagram illustrating the two wave concept of
endovascular trophoblast migration into the decidual segments of
spiral arteries in the first trimester (left) and
into the myometrial segments in the early second trimester, i.e.
from 14 weeks onwards (right). Red arrow: direction of blood flow;
black arrow: direction of
endovascular trophoblast migration.948 R. Pijnenborg et al. /
Plawhich they thought to be secretory products of this tropho-blast
[19]. Also at the light microscopical level intercellularcenta 27
(2006) 939e958fibrinoid at term, but present around interstitial
trophoblasticgiant cells (Pijnenborg and Vercruysse, unpublished
results).
-
These staining patterns contrast with reported data on
rhesusmacaque spiral arteries, which do show pericellular
lamininand also collagen IV deposits associated with
endovasculartrophoblast [93]. Also in the baboon laminin, but not
fibro-nectin, has been shown to be a component of the spiral
arteryfibrinoid, while both proteins are present in the fibrinoid
ma-trix of the cytotrophoblastic shell [94]. Further work on
spiralartery fibrinoid is clearly necessary, the more since it
alsomay highlight differences in interstitial versus
intramuraltrophoblast.
3.7. The endothelial mimicry controversy and maternalvascular
repair
The endothelial mimicry hypothesis logically followedfrom the
ill-founded idea that endovascular invasion resultsin permanent
replacement of the endothelial lining by tropho-blast. Indeed, in
such context it would not be illogical to con-sider a possible
acquisition of endothelial function togetherwith expression of
endothelium-related molecules by cells re-placing the original
endothelium. This concept was launchedfor the first time by Zhou et
al. [95,96], and especially theirreport on the alleged absence of
endothelial molecules inpre-eclamptic trophoblast drew a lot of
attention. The endothe-lial molecules investigated were PECAM-1
(CD31), VE-cad-herin and VCAM-1. As far as PECAM-1 is concerned,
itsexpression by endovascular trophoblast could not be con-firmed
in late [97] (Fig. 8) and early pregnancy [98]. LaterFACS analysis
studies, however, confirmed a high-levelexpression of VE-cadherin
in isolated trophoblast from earlypregnancies [99]. By applying
blocking antibodies in tropho-blasteendothelium coculture
experiments, the latter investiga-tors obtained evidence that
VE-cadherin, and in a lesser degree
Fig. 8. Cytokeratin (red)/CD31 (blue) double staining of a
spiral artery with
physiological change, illustrating endothelial repair (End)
after intramural in-
corporation of trophoblast (IMT). The insert is a high power
picture showing
R. Pijnenborg et al. / Plaa trophoblastic cell very close to the
lumen, covered by a very thin layer of
endothelium.PECAM-1, could play a role in the transendothelial
migrationof trophoblast, at least within this in vitro system.
In the discussion of their paper Zhou et al. [96] took
theirobservation of trophoblastic expression of endothelial
mole-cules as an indication that trophoblast started to behave asan
endothelium for the remainder of the pregnancy. Thisidea opposes
the finding that after mural incorporation of en-dovascular
trophoblast, fresh maternal endothelium overgrowsthe invaded vessel
walls (Fig. 3, stage 5). This was clearlydemonstrated by applying
double immunostaining for cytoker-atin and PECAM-1 on
third-trimester placental bed biopsies.In all cases the inner
lining of the spiral arteries was PE-CAM-1 positive but cytokeratin
negative, positive cytokeratinimmunostaining being restricted to
the mural trophoblast(Fig. 8), which refutes Zhous hypothesis
unless e applyinga reasoning ad absurdum e the endovascular
trophoblastnot only acquires endothelial markers but also loses its
tropho-blastic marker molecules including cytokeratin! Further
evi-dence of a maternal repair process after trophoblast invasionof
the spiral arteries is provided by the frequent observationof
subintimal thickening, resulting in cushions of connectivetissue
with myointimal cells interposed between the restoredendothelium
and the fibrinoid layer surrounding the mural tro-phoblast.
Especially observations on partially invaded spiralarteries
illustrate that such intimal thickenings typically over-lie streaks
of intramural fibrinoid with embedded trophoblast(Fig. 9). The
a-actin-immunopositive myointimal cells mayprobably act as
precursor cells for medial smooth muscle re-pair after
pregnancy.
3.8. The role of uterine NK cells in spiral
arteryremodelling
A characteristic feature of first trimester decidua is the
pres-ence of granulated leukocytic cells, referred to in the past
asendometrial granulocytes or large granulated lymphocytes(LGLs),
now known to be a specific uterine subpopulationof CD56
immunopositive natural killer cells. Since increasednumbers of
uterine NK cells are found in association with ex-travillous
trophoblast in the placental bed [61], a role in regu-lating
trophoblast invasion had been suspected for many years.These
morphological findings have been increasingly sup-ported by studies
of specific molecular interactions betweenuNK receptors and the
HLA-antigens on extravillous tropho-blast [100]. However, a
possible relationship between uNKcells and vascular trophoblast
invasion is less straightforward.A perivascular concentration of
uNK cells has been reported[101], but a possible involvement with
spiral artery remodel-ling e directly or indirectly via
interactions between uNKcells and invading endovascular or
perivascular (interstitial)trophoblast e has not yet been
established [102].
Turning to experimental animals there is good evidence,
es-pecially in mice, for a direct involvement of uNK cells
withvascular remodelling, as it was shown that the spiral
arterysmooth muscle layer remains intact in NK-deficient mice,
in
949centa 27 (2006) 939e958contrast to normal control animals
[103,104]. This remodel-ling occurs in the decidua as well as in
the mesometrial
-
triangle [105], formerly called the metrial gland. Because ofthe
heavy infiltration by uNK cells throughout the whole trian-gle
tissue in the mouse, this area has recently been referred toas the
mesometrial lymphoid aggregate of pregnancy [106].Differently from
the mouse, in the golden hamster [29] andthe rat [107] uNK cells
show a striking distribution as out-standing perivascular cuffs
around spiral arteries. However,the mesometrial triangle area in
these two species is also in-vaded by endovascular and interstitial
trophoblast [29,107].It is obvious that the relationship between
uNK cells and tro-phoblast invasion in these species needs
clarification. A possi-bly relevant observation was made by Ain et
al. [108], whofound a reciprocal relationship between the presence
of uterineNK cells surrounding the spiral arteries and the
perivascularinvasion by interstitial trophoblast in normal rat
pregnancy.
Two possible actions by uNK cells are emerging from theseanimal
data: (1) a direct action on vascular smooth muscle re-modelling,
which is probably related to the decidualization
human, one should realize that in our own species
trophoblastinvasion is extended deeply into the inner myometrium.
Al-though the possibility of non-trophoblast related changes
ofspiral arteries in this compartment has been considered [39],uNK
cells occur in substantial numbers only in the deciduabut not in
the inner myometrium [28], while in the aforemen-tioned rodent
species substantial infiltration of the mesome-trial triangle by
uNK cells occurs during a process clearlyrelated to decidualization
[105]. It must be kept in mind thatthe presence of uNK cells, in
human as well as in rodents, isnot limited to areas of trophoblast
invasion. Indeed, in themouse the mesometrial triangle is not
invaded by trophoblast[58]. Also the fact that deciduomata, induced
in pseudopreg-nant or unilaterally pregnant animals, are
infiltrated by highnumbers of uNK cells in the absence of
trophoblast, is a cleardemonstration of their association with the
decidualizationprocess itself. It should be added that in the human
uNK cellsalso make their appearance in the decidua vera, albeit in
lesser
Fig. 9. Partial physiological change in parallel sections
stained (A) for cytokeratin/HE, (B) with PAS to show the fibrinoid,
(C) for a-actin and (D) with acid orcein
to show the elastica. (A) Shows intramural trophoblast (IMT)
partially covered by an intimal cushion (Int). (B) Illustrates
intramural trophoblast (IMT) incorpo-
ration in fibrinoid (Fib). (C) Shows a-actin-immunopositive
vascular smooth muscle (VSM) at the noninvaded part of the vessel,
as well as a-actin-immunopositive
myointimal cells in the intima (Int) overlying the unstained
fibrinoid (Fib). In (D) the elastica (El) is obvious in the
noninvaded parts of the vessel, ending abruptly
at the beginning of the fibrinoid zone (compare with B and
C).950 R. Pijnenborg et al. / Plaprocess, and (2) a possible
controlling action on trophoblastinvasion. While trying to
extrapolate these concepts to thecenta 27 (2006) 939e958numbers
than in the placental bed [61]. Further research is ob-viously
needed to establish any firm relationship between the
-
cperivascular uterine NK cells, interstitial and/or
endovasculartrophoblast invasion and vascular remodelling in human
andlaboratory rodents.
4. Defects in spiral artery remodelling in
pregnancycomplications
As mentioned before, it was the demonstration of
impairedmaternal placental perfusion in hypertensive pregnancies
[23]that triggered the search for vasculopathies in the
placentalbed as possible causes for placental insufficiency and
fetalgrowth restriction [24]. In 1972, Brosens et al. [25]
reportedfor the first time that, in contrast to normal pregnancies,
spiralarteries with normal histological arterial features, i.e.
with-out physiological change, could be easily found in the
my-ometrial part of the placental bed of pre-eclamptic
women.According to these authors decidual spiral arteries
showednormal physiological changes, but a later study reported
theregular absence of such changes in this area as well [109].Since
in their previous study Brosens et al. had hypothesizedthat the
intramural cells of physiologically changed spiral ar-teries are
derived from invasive trophoblast [7], it was logicalto propose
that the restricted physiological change in pre-eclampsia would be
the consequence of defective trophoblastinvasion. An important
repercussion of this defect wouldthen be that noninvaded and thus
unmodified vessels retaina narrow lumen and an intact muscular
wall, which thereforemight hinder a normal blood supply to the
placenta. Moreover,some of the noninvaded vessels may develop acute
atherosis,a lesion characterized by necrosis and lipophage
infiltrationof the vessel wall [110], and it was shown that such
vesselscorrespond to infarcted areas in the placenta [111].
The initial observations on impaired vascular conversion
inpre-eclampsia have subsequently been repeatedly
confirmed[112e120]. But while originally the absence of
physiologicalchanges in myometrial spiral arteries was regarded as
a patho-gnomonic feature of pre-eclampsia, it has since then
beenrecognized that defective arterial remodelling also occurs
innon-hypertensive cases of intrauterine growth
restriction,non-proteinuric gestational hypertension and chronic
hyper-tension, and exceptionally even in an entirely normal
preg-nancy. The original black-and-white picture had therefore tobe
replaced by the concept of a spectrum of aberrations occur-ring in
different categories of pregnancy complications. Never-theless,
going back to the first reports of impaired placentalperfusion in
hypertensive pregnancies [23], the original aimof the first
generation of placental bed investigators has beenamply fulfilled.
With increasing sophistication of ultrasoundtechnologies,
correlation studies between flow measurementsand placental bed
features have provided additional evidencefor a direct link between
spiral artery histopathology and pla-cental perfusion [121e127].
Also Doppler observations onindividual spiral arteries have
confirmed this picture [49,128].
As to the detailedmechanisms of invasion defects and/or
fail-ures in vascular remodelling, all the aforementioned facts
and
R. Pijnenborg et al. / Placontroversies should be taken into
consideration. Unfortunatelyin most of the cases we can only
speculate on the causes ofplacental bed defects. Because the basic
placentation defectsnecessarily occur in early pregnancy, even if
early placentalbed samples from abortion cases were routinely
available, itwould not be possible to relate any aberrations in
invasion or ar-terial change to the (unknown) pregnancy
outcome.
4.1. Possible decidualization defects
It was suggested recently that the decidualization process,and
especially the extension of decidua-associated changesto the inner
myometrium, might be deficient in women des-tined to develop
pre-eclampsia during their pregnancy [39].Both decidua-associated
and interstitial trophoblast-associatedvascular remodelling have
been considered as essential pri-mary steps for allowing subsequent
endovascular invasion[18], and absence of the former would
therefore necessarilylead to inhibition of the latter. Such defects
should involve par-ticularly the myometrial segments of the spiral
arteries, sinceit is at this level that the major placental bed
aberrations occur.In subfertility cases with possible
decidualization defects,where pregnancy is eventually established
after IVF treatment,the incidence of pregnancy complications such
as pre-eclamp-sia would then be expected to increase and this seems
to besupported by the facts [129]. Unfortunately there are fewhard
data to support the hypothesis of impaired decidualiza-tion in
subfertility cases.
Another observation negating a possible link between re-stricted
trophoblast invasion and impaired decidualizationwas made in the
Dixon collection of complete hysterectomyspecimens. One case out of
eight post-15 weeks gestationalage showed absence of endovascular
trophoblast in myome-trial segments of spiral arteries, at a period
of time when inva-sion should have extended deep in the myometrium.
In thiscase there was no indication of a poor decidualization ofthe
spiral artery walls [18]. However, as mentioned before,a proper
definition of vascular decidualization remains prob-lematic without
the availability of suitable molecular markers.Vascular smooth
muscle cells in myometrial spiral arteriespossess progesterone
receptors, a feature shared by decidualtissue [130], but it is not
known whether there are expressionchanges in subfertility cases or
in pre-eclampsia.
4.2. Possible plugging or de-plugging defects
Plugging of arterial outlets at the basal plate is a
characteristicof early pregnancy, and dissolution of the plugs,
presumably as-sociated with the disappearance of intercellular
junctions, couldbe considered as necessary for the beginning of
invasion. Theo-retically, impaired dissociation of the plugs could
be the cause ofdefective endovascular invasion, but there is no
direct evidenceto support this view. However, Khong et al.
[109,131] made thecurious observation that a proportion of
third-trimester basalplate specimens obtained fromwomenwith
pre-eclampsia or in-trauterine growth restriction showed
intraluminal trophoblast inthe basal plate spiral artery segments.
They considered this as
951enta 27 (2006) 939e958a result of a compensatory new wave of
endovascular tropho-blast invasion, as a teleological response to
convert
-
inadequately remodelled spiral arteries [132]. Alternatively,
andprobably more realistic, such third-trimester endovascular
tro-phoblasts in the outlets of the spiral arteries may represent
rem-nants of incompletely dissolved plugs of early pregnancy.
Itwould beworthwhile to investigate such third-trimester
residualplugs for the presence of intercellular junctions. Once
againa possible relationship may be considered between the
begin-ning of endovascular migration after dissolution of the
plugsand haemodynamics. It cannot be excluded that an
inadequaterise in early uterine flow may fail to provide a timely
triggerfor de-plugging, migration and subsequent intramural
invasion.Restriction of the normal increase in blood flow must lead
topersisting low oxygen and elevated HIF-1a expression,
thuspreventing the trophoblast to differentiate to the invasive
pheno-type [51]. Then, of course, the final question to be asked is
bywhat mechanism the normal flow increase might be impairedin
pre-eclamptic women. Both systemic and local defects (inad-equate
early vascular remodelling and/or defective decidualiza-tion)
should be considered.
4.3. The extravasationeintravasation controversyrevisited
In their studies of the placental bed in pre-eclampsia,
Ger-retsen et al. [113] published striking pictures of spiral
arterieswithout physiological change surrounded by dense clusters
ofmultinucleated giant cells. Such giant cells had been consid-ered
as the result of fusion of interstitially invading tropho-blast,
and it had always been assumed that giant cells,representing the
final stage of trophoblast differentiation,show diminished invasive
activity [28]. Hence Gerretsen rea-soned that impaired
physiological change in pre-eclampticpregnancies may result from
the failure of trophoblast to reachthese vessels because of
precocious terminal differentiationinto giant cells. It is clear
that such scenario would be inline with the intravasation model of
spiral artery invasion. Al-though perivascular clustering of giant
cells has certainly beenobserved by others including ourselves
[92], it is by no meansa frequent occurrence [118]. Also Reisters
observations of in-creased apoptosis in perivascular trophoblast in
associationwith increased macrophage numbers in the placental bed
ofpre-eclamptic patients [133,134], could be fitted into the
intra-vasation model, thus explaining the failure of trophoblast
tocolonize the spiral arteries.
On the other hand, it is not yet fully clear in how
farinterstitial trophoblast invasion is indeed impaired in
pre-eclampsia. The first attempts to quantify interstitial
tropho-blast volume densities failed to show a difference
betweennormal and pre-eclamptic pregnancies [97]. Using a
moresophisticated counting system, Naicker et al. [135] did
findsignificantly lower interstitial trophoblast invasion in
pre-eclampsia. Kadyrov et al. [136] also found lower numbersof
interstitial trophoblasts in pre-eclampsia and showed thatthis was
not associated with an increased apoptosis rate. Infact the
apoptosis rate was lowered in the overall interstitial
952 R. Pijnenborg et al. / Placetrophoblast population of these
patients, while Reister hadshown that the apoptosis rate was
increased in theperivascular subpopulation of interstitial
trophoblast [133].An intriguing finding was that in anaemic women
the tropho-blast invasion occurred in higher numbers and reached
deeperlayers [136].
Whether impaired perivascular invasion of interstitial
tro-phoblast can be the whole story behind restricted
physiologicalremodelling must be questioned. The previously
discussed en-dovascular migration from the decidua, followed by
extravasa-tion, may well be the principal pathway of invasion
ofmyometrial spiral arteries. In addition, however, one
mightconsider that the initial weakening of vascular smooth
muscleby perivascular trophoblast could be impaired in women
sub-sequently developing pre-eclampsia, possibly by
macrophageattack on interstitial trophoblast or by premature giant
cell for-mation. At the moment we have not enough information on
thenormal development to decide on the relative impact of possi-ble
defects in either of the two invasive pathways in causingdefective
remodelling of myometrial spiral arteries.
4.4. Defective endothelial mimicry and exaggeratedmaternal
repair responses
After presenting evidence for the acquisition of
endothelialmarkers by endovascular trophoblast, Zhou et al. [96]
an-nounced that this endothelial mimicry was absent or occurredat a
much lower level in pre-eclampsia. The authors postulatedthat the
adhesion molecules concerned are important for form-ing vascular
connections with uterine vessels, and that down-regulation of these
molecules would be a mechanism forimpaired invasion in
pre-eclampsia. In view of the controver-sies concerning the
trophoblastic expression of endothelialmolecules, this idea e
attractive as it might be e should betreated with caution.
Furthermore, quantification of immuno-histochemical e in their case
immunofluorescent e stainingis notoriously difficult, certainly
when it comes to evaluatingstaining intensities. In order to fully
substantiate this ideaone should not rely on immunofluorescence
only.
Since a maternal repair process obviously takes place in
theinvaded spiral arteries, not only involving endothelial
restora-tion (Fig. 8) but occasionally also intimal thickening
(Fig. 9),one might wonder whether in pre-eclampsia an
exaggeratedmacrophage-mediated tissue repair response could lead
toelimination of the trophoblast which had earlier become
intra-mural. Some investigators would almost certainly be temptedto
consider the apparent narrowing of the vessel lumen, whichis
sometimes associated with intimal thickening, as patholog-ical.
However, since such situation frequently occurs in
normalpregnancies as well, there is no indication that such an
exag-gerated maternal repair in invaded (decidual!) vessels
occursmore frequently in pre-eclamptic cases.
4.5. The relevance of acute atherosis
In the search for vasculopathies in spiral arteries responsi-ble
for maternal underperfusion of the placenta, pathologists
nta 27 (2006) 939e958were particularly intrigued by the typical
lesion of acute athe-rosis, characterized by vascular necrosis,
foam cell infiltration,
-
cand perivascular leukocytic infiltrations. Although this
lesionhad already been described in decidual fragments attached
todelivered toxemic placentae [137], Robertson et al.
[110]recognized it as a real feature of the placental bed in
pre-eclamptic pregnancies which may occur in myometrial aswell as
decidual spiral arteries. Especially fascinating wasthe
morphological similarity to vascular lesions in rejectedkidney
transplants, thereby adding fuel to the then increasinginterest in
the immunological problem of pregnancy, which in-cluded the idea of
pre-eclampsia as a possible autoimmunedisease. Labarrere [138]
subsequently reviewed the histopath-ological features of acute
atherosis which might support animmunological involvement,
including the deposition of IgMand components of the complement
system and of coursethe perivascular mononuclear infiltrations.
However, althoughBrosens and Renaer [111] demonstrated a
relationship be-tween the lesion and underlying placental infarcts,
Khonget al. [139] did not find a worse outcome in pre-eclamptic
pa-tients who showed this lesion. This may relate to the fact
thatatherosis only occurs in a minor proportion of spiral
arteries[140] and therefore may not have a major impact on the
totaluteroplacental blood flow.
Since the foam cells had been identified as CD68 immuno-positive
macrophages [119], which also contain TNF-a [141],these findings
may well be linked with the recent concept thatpregnancy e and a
fortiori pre-eclampsia e represents a hy-perinflammatory state
[142]. More than 30% of the leuko-cytic cells in the decidua of
normal pregnancy aremacrophages. Since the uprising of cytokine
research, it be-came clear that macrophages at the placental site
are impor-tant cytokine producers and may be pivotal
regulatorycells for controlling trophoblast invasion [143].
Histologicalstudies have shown that macrophages form cell
extensionswhich may come in close contact with extravillous
tropho-blast [144]. Macrophages may have cytolytic effects,
presum-ably by the production of cytotoxic cytokines,
andoveractivation may possibly be related to placentation
defectsand pregnancy complications [145]. In pre-eclampsia,
highnumbers of macrophages have been seen to encircle spiral
ar-teries with few invaded trophoblasts, while the opposite oc-curs
in normal pregnancies [134]. Macrophage-derived foamcells, normally
characteristic for atherosis, have been occa-sionally described in
physiologically changed spiral arteriescontaining mural trophoblast
[146,118], but the real lesionis typically seen in spiral arteries
without physiologicalchange. Nevertheless,
cytokeratin-immunopositive remnantshave been observed occasionally
in atherotic vessel walls[118,119]. Other indications of possible
trophoblast-associ-ated vascular remodelling are no longer visible
in the necroticarteries. It is, however, a sobering fact that this
lesion is alsoreadily found in the decidua vera, a region where
trophoblastis absent [76]. It can therefore not be excluded that,
as aconsequence to the hyperinflammatory condition of
pre-eclampsia, activated macrophages may directly damagedecidual
spiral arteries in both decidua basalis and decidua
R. Pijnenborg et al. / Plavera e in serious cases also in the
inner myometrium ewhich may or may not interfere with trophoblast
invasion.4.6. Uterine NK cells in complicated pregnancies
While uterine NK cells have been postulated to play a directrole
in vascular remodelling [103,104], as well as a regulatoryrole in
trophoblast invasion [100,102], little information isavailable
about possible aberrations in complicated pregnan-cies. Croy et al.
[147] reasoned that, because of an associationbetween uterine NK
cells and vascular smooth muscle destabi-lization in mice, uterine
NK cell deficiency might equally leadto impaired early remodelling
of spiral arteries in women.However, the few available reports on
pregnancy complica-tions rather suggest an increase in uterine NK
cell numbers.Clifford et al. [148] reported an increase of uterine
NK cellsin the decidua of women with recurrent miscarriage. In
pre-eclampsia, Stallmach et al. [149] found a significant
increasein pre-eclamptic women with IUGR, but not in women
withpre-eclampsia only. Reister et al. [150], however, did find a
sig-nificant increase of CD56 cell numbers in pre-eclampticwomen,
particularly near spiral arteries. So far, the latter re-port is
the only one that relates to altered perivascular distribu-tion of
uNK cells in complicated pregnancies, but thesefindings need to be
substantiated. It is clear that the possiblerelationship between
increased uNK cell infiltration and defec-tive trophoblast invasion
or disturbed vascular remodellingneeds further investigation. The
results of such studies mayalso help to clarify the role of uNK
cells in normal pregnancy.
5. Epilogue e reconstitution of the arterial wall
afterparturition
Because of the drastic changes in spiral artery structure
dur-ing pregnancy, one might wonder how the original vessel wallis
restored after parturition. It is generally thought that
tropho-blast persists in the uterine wall for a week to 10 days
postpar-tum [151], but in order to have a complete picture of
thevascular reconstitution, a day-to-day follow-up study of
theformer placental bed site would be needed. Unfortunately,the
only materials available are curettage and hysterectomyspecimens
collected in cases of postpartum haemorrhage. Insuch material one
may observe a mixture of involutedand subinvoluted vessels, the
latter probably being thecause of the haemorrhage. While in normal
involution the for-mer uteroplacental arteries show ghost-like
hyalinized wallswith collapsed lumina which are lined by
endothelium, subin-volution shows widely dilated vessels with
thrombus forma-tion, remnants of trophoblast and absence of an
endotheliallining [151,152]. There is a hint that in subinvolution
sites ar-teries may still contain endovascular trophoblast in their
lu-mens indicating a failure of the re-endothelialization
whichshould have taken place during the third trimester of
preg-nancy [109].
Since during pregnancy the vascular smooth muscle is re-placed
by mural trophoblast and fibrinoid, reconstitution ofthe tunica
media must take place. Probable precursor cellsare the
a-actin-immunopositive myointimal cells which are
953enta 27 (2006) 939e958present in thickened intimae (Fig. 9c).
It should be notedthat there is never a complete repair of the
original arterial
-
structure. Indeed, spiral arteries of multiparous uteri show
par-tial replacement of their smooth muscle layer by
connectivetissue, together with fragmentation and duplication of
the elas-tica. Such permanent changes are thought to be related to
thehigher birth weights with increasing parity [153].
6. Conclusion
From this overview it will be clear that there are still a lotof
controversies and uncertainties about the exact pathwaysand
mechanisms of spiral artery remodelling, as well as thespecific
effects of endovascular and/or interstitial trophoblast.As a
result, plenty of placental bed stories have been toldby different
investigators, including ourselves. Althougha lot of experimental
research has already been performedon mechanisms of trophoblast
invasion, most findings relateto invasive behaviour in general, and
thus primarily to intersti-tial invasion, while observations on the
specific invasiondirected to spiral arteries are often restricted
to a shortparagraph. A major problem is the difficulty of
designingexperimental work on isolated placental bed blood
vessels.The recently developed in vitro models using explanted
spiralartery segments [35] may be helpful in the near future,
butone must realize that we are still far from in vitro modellingof
vascular function, which necessarily should
includehaemodynamics.
One firm conclusion to which everybody will agree is
thatvascular remodelling is a vital characteristic of pregnancy,
andthat trophoblast plays an important role in the complete
phys-iological change of the spiral arteries. However, vascular
re-modelling comprises a complex sequence of events, ofwhich in the
first place the earliest, decidua-associated alter-ations, should
be more precisely defined. This is vital for mak-ing a clear
distinction between decidua-induced andtrophoblast-induced
remodelling. The relationship of theseearly changes with increasing
blood flow and endovascularversus interstitial trophoblast invasion
should be clarified,and in particular the role of oxygen in
inducing invasive be-haviour specifically with regards to the blood
vessels has tobe better understood. In this respect an agreement
should beobtained on the expression of specific adhesion molecules
byendovascular trophoblast. These basic features need to betaken as
a background for understanding defects in invasivebehaviour and
failed vascular remodelling in complicatedpregnancies such as
pre-eclampsia. In the first place we wouldlike to know in how far
defective invasion in pre-eclampsia isto be regarded as an
intrinsic defect of trophoblast, or whetherextrinsic factors are
responsible for such failures. The lattermay be related to the
inflammation theory of pre-eclampsia[142], and in this light the
interference of macrophages anduterine NK cells with trophoblast
invasion and vascular re-modelling should be further analyzed.
The older generation researchers frequently referred to an-imal
studies, in order to strengthen their confidence in the re-ality of
the amazing migratory behaviour of trophoblast within
954 R. Pijnenborg et al. / Placblood vessels. When arterial
invasion was increasingly ac-cepted as a fact in the human, animal
observations tended tobe less frequently quoted by clinically
oriented researchers.Nevertheless, we believe that further use of
experimental ani-mals will be an enormous help in delineating
possible mecha-nisms of invasion and vascular remodelling that may
equallyapply to the human. Of course we should be aware of
seriousspecies diversities which doubtlessly exist in primate
[154]but also in rodent placentation. Spiral artery remodelling
inmice and rats has recently been studied more
closely[56,57,104,107,155], and there is also renewed interest
indeveloping animal models for pregnancy hypertension [156e159]. So
far no therapy exists which may cure defective tro-phoblast
invasion and vascular remodelling in pre-eclampticwomen, but if
some relevant molecules might be discoveredit would be most
suitable to test them in such animal models.At present it is
impossible to predict whether in a near futuresuch strategy may
become reality. Time e and a lot more re-search e will tell.
Acknowledgement
The authors wish to thank Tom Pijnenborg for his adviceand
practical help with the illustrations.
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