60 (2017) 134e139

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Placenta

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The cytotrophoblastic shell and complications of

* Graham J. Burton a, , Eric Jauniaux b a Centre for Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK b Department of Obstetrics and Gynaecology, University College London Hospitals and Institute for Women's Health, University College London, London, UK article info abstract

Article history: Many complications of pregnancy have their pathophysiological roots in the early stages of placen- Received 23 February 2017 tation. Impaired trophoblast invasion and deficient remodelling of the maternal spiral are a Received in revised form common feature. While malperfusion of the placenta may underpin cases of fetal growth restriction 10 June 2017 and early-onset pre-eclampsia, the mechanistic links to spontaneous , pre-term labour and Accepted 12 June 2017 premature rupture of the membranes are less obvious. Here, we speculate that formation of a well- developed cytotrophoblastic shell at the maternal-fetal interface is crucial for pregnancy success. Keywords: Initially, cells differentiate from the outer layer of the shell in contact with the fi Pre-eclampsia . Impaired development may thus contribute to reduced invasion and de cient remod- fl fi Pre-term birth elling. In addition, the extent of the shell in uences the timing and spatial con guration of onset of the maternal arterial circulation. A thin and fragmentary shell results in premature and disorganised onset, leading to spontaneous miscarriage. In less severe cases it may predispose to haemorrhage at the interface and formation of intrauterine haematomas. If pregnancy continues, these haematomas may act as a source of oxidative stress, promoting senescence and weakening of the membranes, and stimulating inflammation in the uterine wall and premature contractions. Formation of the shell is dependent on proliferation of cytotrophoblast progenitor cells during the first weeks after implanta- tion, when the developing placenta is supported by histotrophic nutrition from endometrial glands. Hence, we propose the fitness of the endometrium prior to conception, and the peri-conceptional dialogue between the endometrium and the trophoblast is critical for avoidance of later complica- tions of pregnancy. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction fibrinoid material [3]. As a result, the vessels dilate, and remodel- ling ensures a constant high volume, low velocity maternal blood The placenta is key to a successful pregnancy and the life-long flow to the placenta [4].Deficiencies in deep and health of the offspring [1]. In the human, placentation is a highly arterial remodelling have been linked to a spectrum of complica- invasive process and more complex than in most other mammalian tions of pregnancy [2,5]. Whilst it can be appreciated how some species. At the time of implantation the conceptus embeds into the complications, such as growth restriction, early-onset pre- superficial endometrium, and during the first and early second eclampsia and late spontaneous miscarriage, may arise through trimesters a sub-population of trophoblast cells, the extravillous differing degrees of malperfusion of the placenta, it is more difficult trophoblast, migrate in large numbers into the wall of the . to envisage a mechanistic link with pre-term rupture of the Under normal conditions these cells reach as far as the inner third membranes and pre-term labour. Uteroplacental ischaemia has of the , a phenomenon referred to as ‘deep placenta- been invoked in the causation of the latter, with the suggestion of tion’ [2]. The invasion is associated with remodelling of the activation of the renin-angiotensin system in the maternal spiral arteries, a process in which the smooth muscle and [6]. elastic material in the walls of the vessels is replaced by inert Here, we propose an alternative hypothesis to link the patho- physiology of this spectrum of placentally-related complications of pregnancy. Central to the hypothesis is the correct formation of the

* Corresponding author. Physiological Laboratory, Downing Street, Cambridge, cytotrophoblastic shell, the layer that represents the interface be- CB2 3EG, UK. tween the maternal and placental tissues during early pregnancy. E-mail address: [email protected] (G.J. Burton). http://dx.doi.org/10.1016/j.placenta.2017.06.007 0143-4004/© 2017 Elsevier Ltd. All rights reserved. G.J. Burton, E. Jauniaux / Placenta 60 (2017) 134e139 135

2. The cytotrophoblastic shell studies have shown that mitotic figures and immunohistochemical markers of cell division are only seen in cytotrophoblast cells either Initial growth of the placenta is prolific, and considerably in in contact with the villous basement membrane or within a few cell advance of that of the . Shortly after implantation the layers of it [11,12], leading to the concept that this represents a stem chorionic sac is covered over its entire surface by a mass of devel- cell niche [13]. Cytologically the cells appear undifferentiated, and oping villi, each consisting of a core of mesodermal cells and a their cytoplasm contains only a small amount of endoplasmic re- bilaminar trophoblastic epithelium composed of an outer layer of ticulum and Golgi bodies [14]. As the cells move away from the and an underlying layer of progenitor cyto- basement membrane they undergo differentiation involving Notch trophoblast cells. The syncytiotrophoblast is absent at the distal signalling pathways [15], and enter a post-mitotic state [16]. ends of the villi where they make contact with the , and Glycogen progressively accumulates within the cytoplasm, and instead the cytotrophoblast cells form an elongated mass of cells consequently the cells often appear conspicuously pale in histo- referred to as a cytotrophoblast cell column. At their furthest extent logical sections as the deposits are eluted during routine fixation. these columns make contact with the decidua basalis, and in doing Intermediate filaments become abundant, and numerous desmo- so spread laterally and merge with neighbours to form the cyto- somes link the cells [14]. The amount of endoplasmic reticulum trophoblastic shell. increases, and extracellular matrix material begins to be seen in the One of the most comprehensive descriptions of the shell was interstices between the cells. The columns and the shell are provided by Hamilton and Boyd [7], who had the opportunity to continuous with one another (Figs. 1 and 3), and cells within the study 37 specimens ranging from 11-12 dayse90 days post- shell retain a similar rounded morphology surrounded by matrix- (embryonic crown-rump length of 60 mm). These au- type fibrinoid [17]. More extensive deposits of fibrinoid are seen thors described the shell as being ‘thick’ at 14e18 days, ‘attenuated’ at the interface between the shell and the maternal tissues, where at 20e30 days, and ‘markedly thinned’ from the 10 mm stage, they form an irregular and commonly incomplete layer referred to 37e38 days post-fertilisation, onwards. We have been able to re- as Nitabuch's stria (Fig. 3). This marks the future plane of separation view some of the same specimens from day 26 onwards contained of the placenta at the time of delivery. within the Boyd Collection. At 26 days, the shell extends across the At present, the factors regulating cytotrophoblast cell prolifer- placental bed and continues beneath the decidua capsularis, ation are not fully understood, but two facets of the intrauterine forming an almost complete layer, 5e10 cell thick, that constitutes environment during the first trimester are thought to be important. the fetal-maternal interface around the implanted conceptus First, is the histotrophic support from the endometrial glands. The (Fig. 1). Anchoring villi attaching to the placental side of the shell by endometrial glands deliver carbohydrate and lipid-rich secretions cytotrophoblastic cell columns are numerous at this stage, and into the during early pregnancy [18], and these closely approximated together. By day 40 post-fertilisation, the secretions contain powerful mitogenic growth factors, including shell is variable in thickness, remaining several cells thick where epidermal and fibroblast growth factors [19]. Application of such cell columns are attached, but gradually reducing to a single cell growth factors to first trimester villus explants results in increased layer in the intervals between the columns (Fig. 2). Expansion of the proliferation of the cytotrophoblast population [20,21]. Indeed, in chorionic sac means that the distance between the cell columns many species there is evidence that the trophoblast is able to signal increases, and so in later specimens, the shell becomes discontin- to the glands and upregulate the expression of growth factors [22], uous, persisting only where cell columns are attached [7e9]. In the and in this way stimulate its own development. Experimental ev- intervals, fibrin is laid down at the fetal-maternal interface, idence for such a mechanism operating in the human is lacking, generating Nitabuch's stria. Later in gestation, the remnants of the although the key components appear to be in place [23]. In addi- shell and Nitabuch's stria are incorporated into the developing tion, it is well-recognised that the gland cells adopt a characteristic basal plate [10]. hypersecretory morphology during early pregnancy, the Arias- The cells of the shell are derived from the proliferative zone at Stella reaction [24]. On the placental side, it is notable that the the proximal end of the cytotrophoblast columns (Fig. 3). Many proliferative cells in the putative niche at the proximal end of the column immunoreact positively for the fibroblast growth factor receptor 2, and signalling from this receptor enhances expression of CDX2 and ELF5 [13]. These genes encode two tran- scription factors that are essential for stem cells of the trophoblast lineage. Second, a low oxygen concentration prevails within the devel- oping placenta during early pregnancy [25], and this may favour proliferation of the cytotrophoblast progenitor cells [26]. There may well be interactions between the two facets, for the levels of CDX2 and ELF5 drop sharply at the end of the first trimester [13], coinciding with the transition from histotrophic to haemotrophic nutrition and a three-fold rise in intra-placental oxygenation [25]. Some proliferation may continue in the niche at the proximal end of a column, but the implication is that the proliferative potential of the trophoblast is greatly reduced during the second and third trimesters.

Fig. 1. Photomicrograph of a 26 day post-fertilisation placenta-in-situ specimen (H710) 3. The importance of the cytotrophoblastic shell in normal illustrating the cytotrophoblastic shell (CS) forming the maternal-fetal interface. The pregnancy main illustration is taken from the area marked by the box on the low-power insert, towards the margin of the implantation site and the junction of the decidua basalis and The integrity of the shell is critical during the early stages of decidua capsularis. Note the spaces (asterisk) within the shell that communicate with the intervillous space and the maternal vasculature. CCC, cytotrophoblast cell column. pregnancy for several reasons. It provides anchorage to the extra- Stain, Masson's trichrome. Scale bar ¼ 0.5 mm. cellular matrix of the maternal endometrium [9], but it is primarily 136 G.J. Burton, E. Jauniaux / Placenta 60 (2017) 134e139

Fig. 2. Photomicrographs of a 40 day post-fertilisation placenta-in-situ specimen (H673) illustrating the variable thickness of the cytotrophoblastic shell (CS) at this stage of gestation. A) At points of attachment of cell columns (asterisks) the shell remains thick, but in intervening areas it is very thin (arrows). B) Higher power view of the central area shown in A), illustrating the gradual reduction in thickness of the shell with increasing distance from a cell column. Stain, Masson's trichrome. Scale bars; A ¼ 0.5 mm, B ¼ 100 mm.

Fig. 3. Photomicrograph of a 26 day post-fertilisation placenta-in-situ specimen (H710) illustrating how cells from a cytotrophoblast cell column (CCC) feed into the cytotrophoblastic shell (CS). Cytotrophoblast cells proliferate in a progenitor niche (asterisk) at the proximal end of a cytotrophoblast cell column, extending from an anchoring villus (AV). The columns spread laterally at their distal ends and merge with neighbours to form the shell. Note the deposition of fibrin (Nitabuch's stria) (arrowed) Fig. 4. Photomicrograph of a 26 day post-fertilisation placenta-in-situ specimen between the shell and the decidua (D). Stain, Masson's trichrome. Scale bar ¼ 50 mm. (H710) illustrating the differentiation and migration of interstitial extravillous trophoblast cells from the shell. The cytoplasm of the cells within a cytotrophoblast cell column (CCC) and the cytotrophoblastic shell often appears empty as the high glycogen content is eluted during routine fixation. Cells near the maternal surface of its functions relating to onset of the maternal arterial circulation to the shell undergo a partial epithelial-mesenchymal transition, becoming darker the placenta that are the focus of this review. Firstly, it is the source staining and spindle-shaped (black arrow), and invade into the maternal tissues (white of the extravillous trophoblast cells that are involved in the arrows) Immunostaining for cytokeratin 7 on equivalent age sections (insert) confirms remodelling of the spiral arteries. Cells towards the outer surface of the spindle-shaped morphology of many of the invading trophoblast cells. Stain, Masson's trichrome. Scale bar ¼ 100 mm. the shell undergo a partial epithelial-mesenchymal transition to form interstitial trophoblast cells [9,27,28]. This transition is asso- ciated with a marked change in their morphology, for they adopt a the shell [31]. The magnitude of this migration is sufficient to spindle-like shape with a dark-staining nucleus (Fig. 4) [8,12]. This virtually occlude the spiral arteries during the first six weeks of transition is possibly induced by the higher oxygen concentration pregnancy, restricting any flow into the intervillous space to a within the decidua with which they are in contact [25,29], but may seepage of plasma through the network of narrow intercellular also be initiated by hormones and cytokines released by the clefts [32]. The clefts gradually expand and coalesce over the next . Interstitial trophoblast cells migrate through the few weeks, until free flow of arterial blood is established around decidua and into the inner third of the myometrium where they 10e12 weeks of pregnancy [25,33]. Restriction of maternal arterial fuse to form multinucleated trophoblast giant cells [30]. Interstitial inflow is essential during early pregnancy to protect the developing trophoblast are particularly numerous surrounding the spiral ar- embryo from exposure to the oxygen in the maternal circulation, teries, and their presence appears to be essential for vascular and free radical-mediated oxidative teratogenesis [34,35]. Devel- remodelling [8,31]. Increased rates of apoptosis and reduced inva- opment of the shell assists by providing a source of endovascular siveness of these cells have both been invoked as reasons for trophoblast cells over a broad area, ensuring there is a sufficient deficient remodelling of the arteries in cases of growth restriction supply to plug any maternal vessels encountered by the expanding and pre-eclampsia [12], but it is equally possible that a reduced placenta irrespective of their precise location. This will be the case supply of cells from the shell, and ultimately from the progenitor in the central region of the implantation site where the shell is niche at the proximal end of the cytotrophoblast cell columns, thickest [2]. Towards the periphery the shell is thinner, and so the might also contribute. opportunity for plugging of the spiral arteries is less in these areas Secondly, when the advancing margin of the shell penetrating (Fig. 5A). Hence, onset of the maternal circulation is seen prefer- the decidua basalis encounters the distal portion of a spiral , entially in the periphery, and results in locally high levels of trophoblast cells migrate down the lumen of the artery as endo- oxidative stress as the villi display very limited antioxidant de- vascular trophoblast [8]. These cells retain their rounded fences at this stage of development [36]. This stress is thought to morphology and appear identical to those of the shell, although induce villus regression and formation of the smooth or free they do show immunoreactivity for CD56 that is not seen within membranes of the definitive placenta, and may be considered G.J. Burton, E. Jauniaux / Placenta 60 (2017) 134e139 137

Fig. 5. In normal (A), extravillous trophoblast cells originating from the cytotrophoblast shell invade into the mouths of the maternal spiral arteries during the first trimester, preventing full arterial inflow into the intervillous space. Formation of the shell and plugging of the arteries is least in the periphery of the developing placenta where some inflow may occur, causing villus regression and formation of the smooth membranes. In pathological pregnancies (B), the cytotrophoblast shell is poorly developed. In the most severe cases this leads to early onset of the maternal arterial circulation to the placenta and miscarriage. If the pregnancy continues, there will be deficient spiral arterial remodelling due to inadequate extravillous trophoblast invasion. There may also be bleeding at the maternal-fetal interface and formation of an intrauterine haematoma (red), which may induce senescence in the membranes and their premature rupture or an inflammatory response in the placental bed, increased uterine contractility and premature delivery. Adapted from Ref. [58]. physiological as it occurs in all ongoing pregnancies. arterial remodelling is also deficient in cases of growth restriction, Once the shell becomes fragmented from 40 days post- and even more so in those with accompanying pre-eclampsia when fertilisation (8 weeks of pregnancy) onwards, the source of extra- obstructive arterial lesions may also be present [2,39,40], but to a villous trophoblast cells must be principally from the remnants lesser extent than what is observed in early pregnancy failure. located where the distal ends of cell columns make contact with the These vascular changes likely also reflect reduced trophoblast in- decidua (Fig. 2A). This spatial rearrangement will have little impact vasion into and around the arteries, and so it might be expected on plugging of the arteries, as onset of the maternal circulation that arterial plugging was less extensive in these during begins progressively from around this time [35]. Equally, interstitial early pregnancy. Consequently, onset of the maternal circulation trophoblast will continue to flow from the cell columns and migrate may have been abnormal, both temporally and spatially. Currently, through the endometrial stroma, homing in on the spiral arteries. no data are available to support or refute this hypothesis, and future Although the cell columns shorten as gestation advances, cyto- prospective studies are required to test the concept. However, the trophoblast cells remain proliferative in the proximal progenitor fact that placentas from pregnancies complicated by growth re- niche until at least 16e20 weeks of pregnancy [12]. The number of striction often display irregular margins and excessive villous cell columns may increase during pregnancy through subdivision regression provides some circumstantial support [41]. of the early anchoring villi, possibly facilitated by the faster Besides influencing timing of the onset of the maternal circu- expansion of the developing basal plate in comparison to the lation, the extent of development of the shell may impact on the chorionic plate [10]. In addition, branching morphogenesis of the integrity of the maternal-fetal interface and the adhesion between villous trees may bring further villi into contact with the shell, the two sets of tissues [9]. The regression of around two-thirds of establishing new points of attachment [9]. the original villous mass of the early placenta creates an area of mechanical weakness in the periphery where the spiral arteries are unplugged, leading potentially to bleeding between the developing 4. Impaired development of the cytotrophoblastic shell and membranes and the decidua basalis at the end of the first trimester complications of pregnancy (Fig. 5B). This phenomenon is known clinically as threatened miscarriage, and is the most common complication of human While developmental differences in the extent of the shell are pregnancy. related to local variations in the timing of the onset of the maternal Sub-chorionic haematomas are well defined on ultrasonic ex- circulation in normal pregnancies, gross impairment of its devel- amination as crescentic hypoechogenic areas between the opment is associated with the pathology of spontaneous miscar- placental membranes and the decidua. If the haematoma expands riage. In 70% of these cases the shell is thin and fragmentary, to the basal plate of the definitive placenta it can lead to complete leading to deficient endovascular trophoblast migration and detachment of the placenta and a full miscarriage, which is incomplete plugging of the spiral arteries across the entire observed in around 10% of the cases within 48 h of the first bleeding placental bed [37,38] (Fig. 5B). Onset of the maternal circulation is episode [42,43]. In the 90% of pregnancies that continue, there is a precocious and spatially disorganised, with massive entry of 1.9e3.7 increased risk of premature rupture of the membranes and maternal blood resulting in overwhelming placental oxidative pre-term delivery [43]. The mechanistic link has not been fully stress and secondary degeneration of the villous tissue [36]. This determined, but it has been postulated that the clot of blood lying effect is independent of the trophoblastic karyotype, and so we against the membranes causes local oxidative stress [42].In must look beyond the conceptus for a cause. þ particular, the presence of free Fe2 ions may stimulate the for- Normal pregnancy and miscarriage represent opposite poles of mation of the highly aggressive hydroxyl ion through the Fenton pregnancy outcomes, but is it possible that other placentally- reaction [44]. Chronic exposure to reactive oxygen species can related complications of pregnancy are associated with interme- cause cellular senescence, and this has recently been put forward as diate degrees of development of the cytotrophoblastic shell? Spiral 138 G.J. Burton, E. Jauniaux / Placenta 60 (2017) 134e139 the final common pathway for weakening and premature rupture control of cytotrophoblast proliferation, but the unique first of the membranes in response to various stimuli [45]. In addition, trimester intrauterine environment appears to be essential. Mito- senescent cells secrete a cocktail of pro-inflammatory cytokines genic factors secreted by the glands are likely to be critical [46], and this may lead to the induction of a sterile inflammatory [22,23,41], possibly in combination with the prevailing low oxygen response within the uterus that results in pre-term delivery [47]. concentration. Hence, some cases of these syndromes may have Changes in maternal levels of placental specific proteins [48,49], their pathological roots in impaired endometrial function during and also of inflammatory cytokines [50] and markers of oxidative the peri-conceptional period and early pregnancy, a view sup- stress [51] in women presenting with a threatened ported by genetic analyses of chorionic villus samples from women support this concept. who went on to develop pre-eclampsia [56,57]. Further studies are If considered from the viewpoint of development of the cyto- required to test the hypothesis, but if proved correct then ensuring trophoblastic shell it is to be expected that the two sets of pa- optimal endometrial function prior to conception should become a thologies, namely early pregnancy failure, growth restriction and public health priority. pre-eclampsia on the one hand, and pre-term premature rupture of the membranes and pre-term delivery on the other should show Conflict of interest epidemiological associations, and also links to events during early pregnancy. This is indeed the case [43]. The authors have no conflicts of interest to declare.

5. Future directions Acknowledgements Human early pregnancy is a difficult period to research, and Presented at the 2016 Global Pregnancy Collaboration (CoLab) development of the cytotrophoblastic shell that we propose to be conference in Oxford, England. The Global Pregnancy Collaboration critical is occurring before and shortly after pregnancy is man- is part of the Pre-eclampsia-Eclampsia Monitoring, Prevention & ifested clinically. Data from other species indicate that the signal- Treatment (PRE-EMPT) initiative funded by the University of British ling dialogue between the conceptus and the endometrium is Columbia, a grantee of the Bill & Melinda Gates Foundation. essential for upregulation of the secretion of growth factors that The Boyd Collection of archival histological material is held by stimulate trophoblast proliferation, and hence likely formation of the Centre for Trophoblast Research (www.trophoblast.cam.ac.uk) the cytotrophoblastic shell [22]. Although recent data for the hu- at the University of Cambridge, and is available for viewing. man indicate the importance of the endometrial secretome for implantation [52,53], the full composition of the gland secretions and their impact during early pregnancy are not known. Uterine References flushing at this time may not be ethical, and in any case may not fl [1] G.J. Burton, A.L. Fowden, K.L. Thornburg, Placental origins of chronic disease, accurately re ect the activity of the glands within the placental bed Physiol. Rev. 96 (4) (2016) 1509e1565. where local trophoblast interactions may influence gland activity. [2] I. Brosens, R. Pijnenborg, L. Vercruysse, R. Romero, The “Great Obstetrical The derivation of endometrial that faithfully replicate the Syndromes” are associated with disorders of deep placentation, Am. J. Obstet. e fi Gynecol. 204 (3) (2011) 193 201. transcriptomic pro le of the glands and which respond to preg- [3] G.S. Whitley, J.E. 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