Variation in composition of the intervillous space lining in term placentas of mothers with pre-eclampsia

Placenta 31 (2010) 409e417

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Variation in composition of the intervillous space lining in term placentas of mothers with pre-eclampsia B. Hottor b, P. Bosio b, d, J. Waugh b, c, P.J. Diggle a, S. Byrne b, J. Ahenkorah b, C.D. Ockleford a, b, * a

School of Health and Medicine, Division of Medicine (CME), C14 Faraday Building, University of Lancaster, Bailrigg, Lancaster LA1 4YB, United Kingdom Laboratory for Developmental Cell Sciences, Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester LE1 9HN, United Kingdom c Newcastle Upon Tyne NHS Foundation Trust, Royal Victoria Infirmary, Newcastle Upon Tyne NE1 4LP, United Kingdom d Sandwell & West Birmingham NHS Trust, City Hospital, Birmingham B18 7QH, United Kingdom b

Condensation: Term placental inter-villus space lining is a mosaic of endothelial cells, trophoblast and fibrin: component area percentages alter in pre-eclampsia indicating “poor-repair” aetiology.

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 23 February 2010

Objective(s): To define composition of chorionic plate and test effects of pre-eclampsia on basal plate composition. Study Design: Retrospective cohort study where distinct area fractions were measured in: healthy term chorionic plate (CP: n ¼ 11), healthy placental basal plate (n ¼ 11), mild pre-eclamptic basal plate (n ¼ 10) and severe pre-eclamptic basal plate (n ¼ 11). Results: CP lining is partly endothelial. Mean anchoring villus (AV)/acellular (NS) basal plate area ratio decreased in pre-eclampsia (p ¼ 0.048). There was a decreasing trend with increasing disease severity. Basal plate endothelial cell proportion was not significantly lower in severe pre-eclampsia than in healthy or mild pre-eclamptics. Conclusion(s): An inverse relationship between the proportions of fibrin and anchoring villi indicates that increased basal plate fibrin deposition and reduced basal plate materno-fetal anchoring area are part of pre-eclamptic disease progression. Endothelium lining intervillous surfaces may originate from circulating maternal endothelial progenitor cells. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Fibrinoid lining Mosaic monolayer Endothelium Pre-eclampsia Anchoring villus Intervillous space Basal plate

1. Introduction 1.1. Morphology The intervillous space receives oxygenated maternal blood from spiral arterioles joining the basal plate surface. Blood in the intervillous space directly contacts chorionic villi defining the haemochorial placenta [1]. The intervillous space extends to the chorionic plate. It drains by maternal veins and extends maternal vascular space. The basal plate intervillous lining is composed of both fetal trophoblast and maternal endothelium [2e4]. Parts of the lining are fibrinoid which forms in layers within basal plate and at sites of damage to the trophoblastic epithelium [5,6]. The chorionic plate

* Corresponding author. School of Health and Medicine, Division of Medicine (CME), C14 Faraday Building, University of Lancaster, Bailrigg, Lancaster LA1 4YB, United Kingdom. Tel.: þ44 1524 594515 (Business), þ44 1572 722110 (Home), 07749230368, (Mobile); fax: þ44 1524 593747. E-mail address: [email protected] (C.D. Ockleford). 0143-4004/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.02.016

intervillous space lining is usually described as entirely trophoblastic. Trophoblast is identifiable using epithelial markers such as antibodies to specific cytokeratin intermediate filament proteins [7,8]. Maternal endothelial cells bind several different marker antibodies [4e9].

1.2. Pre-eclampsia Pre-eclampsia is pregnancy hypertension with proteinurea, oedema or both. It usually occurs after the 20th week of gestation but may develop earlier in the presence of trophoblastic disease. Apart from its well-appreciated importance as a cause of fetal and maternal mortality, eclampsia/pre-eclampsia is linked to challenges for infants born prematurely. Amongst these are cerebral palsy, epilepsy, blindness and deafness, learning disabilities, and possibly high blood pressure and diabetes later in life. It is recognised as a disease involving the placenta as its delivery is generally curative. Vessels supplying maternal blood to the placenta are inappropriately remodelled in pre-eclampsia [10]. Vascular hypertensive

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dysfunction and a generalised maternal systemic inflammatory reaction are key features of pre-eclampsia involving endothelial linings of maternal blood vessels [11,12]. Pre-eclampsia is also viewed as a clotting disturbance [13,14]. Therefore it is appropriate to investigate the intervillous space lining to establish whether it alters in the disease. 1.3. Experimental approach Using confocal laser scanning microscopy (CLSM) we have measured lining components in basal plate of healthy, mild and severe pre-eclamptic pregnancies. We aimed (1) to define changes in basal plate inter-villous space lining correlated with disease severity; (2) to establish if the alloeepieendothelial monolayer described recently [3,4] extends to chorionic plate intervillous space lining. 2. Materials and methods

2.4. Measurements The basal or chorionic plate was identified in series of uniform systematic sections sampled from the representative sites. Dual channel digital images were collected from the full length of the lining as it traversed the section. These were analysed using COMOS software by the first author. The lining was traced in each section where it appeared using the mouse operated length measurement function and the cut surfaces of the lining were identified as trophoblast or endothelium when associated with specific immunofluorescence or a non-immunofluorescent length later identified histochemically as fibrin then measured. To comprehensively survey the total area of the two plates, the width of anchoring villi was measured in the basal plate and the stem villi on the chorionic plate. Thus, there were length fractions of endothelium, trophoblast, fibrin and villus contact for each of the plates. These samples of length of the cut lining in sections are statistical stereological samples of the area fraction of these components following Delesse l1/l2 ¼ a1/a2. 2.5. Statistical analysis Analysis of variance was used to test for differences between the ratios of basal plate anchoring villus and fibrin/fibrinoid length percents between groups of healthy patients and patients with mild or severe pre-eclampsia. The tests were implemented as randomisation tests to protect against non-normality of the response variable. Paired sample t-tests were used to test for differences between length percents of endothelium and trophoblast making up basal plate lining within the groups. Extensive relevant raw data are available and can be accessed via an associated Ph.D. thesis [21].

2.1. Patient recruitment Recruitment was of primigravidae attending ante-natal hypertension clinics at Leicester Royal Infirmary who met International Society for Study of Hypertension in Pregnancy (ISSHP) diagnostic criteria after 20 weeks of gestation [15]. Exclusion criteria included cardiovascular disease (including underlying essential hypertension), diabetes, renal disease, hydatidiform mole and multiple pregnancy. For area measurement purposes, control (healthy) patients were gravidity matched to preeclamptic patients. It became impossible to gestational age match at delivery because of the clinical requirement to deliver severe cases earlier than mild cases. Samples were collected from a total of 32 placentas: 11 healthy placentae, 10 mild pre-eclamptic and 11 severe pre-eclamptic placentae. The classification mild and severe was based on the American College of Obstetricians and Gynaecologists (ACOG) criteria. Informed consent was obtained prior to delivery. Protocols for research and patient recruitment were approved by the Leicestershire Research Ethics Committee (ref.7144) and by University Hospitals of Leicester NHS Trust R&D Committee (Project UHL9161). 2.2. Tissue sampling Following delivery (<1 h), 4 equally distributed central to peripheral, radially oriented, one from each quadrant, 1  2  0.5 cm isotropic uniform systematic samples were each placed in a mould [16]. Once immersed in OCT cryo-embedding medium (Tissue-Tek, Bayer UK Ltd, Basingstoke, UK), tissue was freeze fixed in a slush of liquid hexane and dry ice. Samples were stored at 80  C. 2.3. Immunofluorescence microscopy Indirect immunofluorescence labelling was as previously described [17,18]. Sections, 7 mm thick, were exposed to dilutions of primary antibody in PBS containing 20% non-immune goat serum. Following serial washes, sections were exposed to fluorophore-conjugated antibodies in PBS containing 20% non-immune goat serum. 2.3.1. Primary antibodies Monoclonal mouse anti-pancytokeratin C2931 SigmaeAldrich inc. Missouri, USA, recognises human cytokeratins 4, 5, 6, 8, 10, 13 and 18. Concentration 1:800. Polyclonal rabbit anti-human von Willebrand factor, IgG fraction F3520 SigmaeAldrich Missouri, USA. Concentration 1:400. Monoclonal mouse anti-PECAM-1 Antibody EN4-sc-59169 Santa Cruz Biotechnology Inc 1:100 is an endothelial marker. 2.3.2. Second step antibodies Cy3-conjugated sheep anti-mouse affinipure IgG Fab2 fragment specific (Jackson Immunoresearch Laboratories, West Grove, PA, USA, 51787) Concentration 1:1000. FITC-conjugated goat anti-rabbit whole-molecule IgG (SigmaeAldrich, Missouri, USA, F6005); Concentration: 1:500. 2.3.3. Confocal Laser Scanning Microscopy Frozen-sections in a photobleach retardant mountant (Citifluor, Canterbury) were examined using a Biorad MRC 600 linked to a Zeiss Axiovert epifluorescence microscope as previously described [19,20].

3. Results 3.1. Demographic and obstetric parameters Maternal age, gestational age at delivery, sex of the babies and birth weight distributions of the study groups are summarised in Table 1a. The average age of mothers in the severe pre-eclampsia group is 22.9 years. The average ages of women in the mild preeclamptic and healthy control groups are 27 and 24.7 years, respectively. Maternal age is not significantly different amongst the study groups (F(2, 29) ¼ 1.967, p ¼ 0.158) The mean birth weight for the babies in the study group varies significantly between the patients groups (F(2, 29) ¼ 11.912, p ¼ 0.000) (Table 1b). The mean birth weight of babies born to mothers with severe pre-eclampsia is significantly lower than those born to women who developed mild pre-eclampsia or had healthy pregnancies, post hoc p ¼ 0.000 and 0.001 for mild preeclamptic and healthy controls, respectively. Seven of the 11 babies born to mothers who had pregnancies complicated by severe preeclampsia were females and 4 were males (Table 1a). In the mild pre-eclamptic group, 6 of the 10 babies were females and the other 4 were male babies (Table 1a). Mothers with healthy uncomplicated pregnancies had five female and six male babies (Table 1a). There were no significant differences amongst the groups with regards to the gender of the babies (p ¼ 0.686). The mean gestational age at delivery was significantly different amongst the groups (ANOVA p ¼ 0.001). Pregnancies complicated by severe preeclampsia were, as expected, terminated at an earlier gestational age (mean gestational age at delivery of 34.8 weeks) than normal pregnancies (p ¼ 0.001) and mild pre-eclamptic pregnancies (p ¼ 0.003) (Table 1b). 3.2. Immunofluorescence CLSM Fetal and maternal endothelial cells were specifically labelled with the anti-von Willebrand factor or EN4 antibody, whereas trophoblast strongly labelled with the anti-pancytokeratin antibody (Fig. 1a and c). On using dual channel detection there was negligible cross-labelling between the endothelial and trophoblast marker antibodies. In experiments where both primary antibodies were omitted and replaced with 20% fetal calf serum diluted with TBS-T or when isotype control antibodies were used no

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Table 1a Summary of the demographic and obstetric characteristics of the patient groups.

Maternal age (years) Gestational age at delivery (weeks) Birth weight (g) Female babies Male babies

Control patients (n ¼ 11)

Mild pre-eclampsia (n ¼ 10)

Severe pre-eclampsia (n ¼ 11)

24.7 (19e35) 38.7 (31e41) 3057.1 (1980e4138) 5(45.45) 6(54.54)

27.0 38.5 3200.7 6 4

22.9 (17e30) 34.8 (31e39) 2004.3 (1020e3628) 7(70) 4(30)

(17e31) (37e40) (2636e4110) (60) (40)

Values are expressed as mean (range) for maternal age, gestational age and birth weight and number (percentage) for sex of the baby. n ¼ number of patients.

fluorescence above background resulted. Labelling was similar for chorionic plate specimens. Patterns of anti-von Willebrand antibody and EN4 antibody (Figs. 1c,d and 2) binding were indistinguishable. Tandem red and green immunofluorescent strips formed a monolayer lining the intervillous space (Figs. 2e3). Their length fractions reflect the area fractions of endothelium and trophoblast. 4. Qualitative morphology 4.1. Basal plate lining cells Endothelial and trophoblast segments immunofluoresce with distinct colour over the vast majority of the basal plate lining. Different coloured regions often abut end-to-end and the colour change is abrupt (Fig. 3a and b). Small lengths do appear to dual label showing as an orange colour particularly at junctions where red meets green (Figs 2 and 3c). Given that the thickness of the frozen section is such that there can be overlap between tissues we interpret these as pixels in the image containing immunofluorescence from both trophoblast and endothelium superimposed in the section where different parts of the mosaic meet. Endothelial cells are found throughout the intervillous space basal plate lining. This monolayer was interrupted by gaps unreactive with either antibody (Fig. 2). In Martius yellow, Brilliant Crystal Scarlet and Soluble (aniline) Blue (MSB) preparations these gaps were identified as fibrin(oid) (Fig. 4a and b). Also interrupting the layer are anchoring villi (Figs. 4b and 5a and b). The alloeepieendothelial mosaic was present over the entire basal plate. There were no obvious qualitative differences of the labelling in healthy and pre-eclamptic placentas. Pre-eclamptic placentae have fewer anchoring villi interrupting the lining. The maternal surface of the basal plate was immunoreactive with either trophoblast or endothelium markers but predominantly endothelium (Figs. 1c, 3a,b). The morphological characteristics of the trophoblast and endothelium differ in the sense that the trophoblastic elements are of overall greater height than the endothelium (Figs. 1c,d and 3b) although orientation of the sheets to the section plane does change locally and the apparent thickness of the sheets may appear to differ, so minimum thicknesses are of greater value for interpreting the structure. In a number of sections, it appears that the incidence of extravillous trophoblast cells that are strongly cytokeratin Table 1b Test statistic result of the between-group analysis of variance. ANOVA

Maternal age (years) Gestational age at delivery (weeks) Birth weight (g) Sex of the baby

Sum of squares

df

Mean square

87.784 104.557

2 2

43.892 52.278

9 182 698.684 0.202

2 2

4 591 349.342 0.101

F

Sig. 1.967 9.576

0.158 0.001

11.912 0.382

0.000 0.686

The means of gestational age at delivery and the birth weight are significantly different between the groups (P ¼ 0.001 for GAD and p ¼ 0.000 for birth weight). Means of maternal ages are not significantly different between the groups.

positive varies substantially in the basal plate deep to the lining layer (Figs.1c,d and 3b) and also in the chorionic plate immediately above the lining layer (Fig. 3ced). 4.2. Chorionic plate lining cells Chorionic plate intervillous space was lined with epithelial, endothelial or non-immunofluorescent properties (Figs. 2e4a and b). These features appeared similar to those in the basal plate. The mosaic architecture of the lining is therefore detectable in the most distant parts of the intervillous space from the feeding and draining vessels. 5. Quantitative morphometry 5.1. Comparisons of basal plate lining proportions Basal plate lining components in healthy placentae had mean lengths of endothelium (62.85%; SEM 4.89) and trophoblast (27.64%; SEM 4.54), whereas fibrin and anchoring villi formed 5.93% SEM 1.28 and 3.58%; SEM 0.62. Mean percentage lengths of endothelium and trophoblast for mild pre-eclampsia placentae were 65.56% and 23.59% (Table 2). Fibrin and anchoring villi made up 8.75% and 2.10% of the lining of mild pre-eclampsia placentae (Table 2). In severe preeclampsia placentas, mean values for endothelium, trophoblast, fibrin and anchoring villi were 51.24%, 35.42%, 11.12% and 2.21%, respectively (Table 2). Multiple comparisons between various lining components of basal plate in the groups revealed that in healthy placentas and those from mild pre-eclampsias, there was a larger proportion of endothelium than trophoblast, also of fibrin/fibrinoid than anchoring villi. Within the three pre-eclamptic placental groups, there were no significant differences between the mean length proportions of endothelium and trophoblast (healthy p ¼ 0.003; moderate p ¼ 0.0005; severe p ¼ 0.143). The mean length percent of fibrin/fibrinoid and anchoring villi did not differ significantly across the three patient groups (post hoc, p ¼ 1. 00). 5.2. Comparisons of chorionic plate lining proportions In healthy placentae fibrinoid occupied 33.01% (SD ¼ 13.98474, n ¼ 16): trophoblast 34.18% (SD ¼ 9.58754, n ¼ 16) and endothelium 32.79% (SD ¼ 17.51755, n ¼ 16) of the lining, respectively. The fact that approximately one-third of the chorionic plate lining is endothelial is evidence that endothelial cells form a significant component of the structure. 5.2.1. Comparisons between groups of patients Calculating basal plate lining area percent occupied by anchoring villi divided by the area percent occupied by fibrin/ fibrinoid yielded an interesting feature of the data. This is illustrated in Fig. 5a where the ratio decreases with increased severity of disease across the three patient groups. The significance of this trend was tested in a three group ANOVA giving p ¼ 0.048. Other tests using a subtractive approach also revealed a significant trend

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Fig. 1. (a) Confocal laser scanning microscopic images of healthy placenta dual labelled with anti-pancytokeratin (red: trophoblast marker) and anti-von Willebrand factor (green: endothelial marker). It shows the pancytokeratin labelling of trophoblastic epithelium (c) and von Willebrand factor labelling the endothelium (v) of fetal capillaries in the core of the villi. The pattern of the labelling is mutually exclusive and distinctive. Scale bar 50 mm. (b) As in 1a but the tissue is from a pre-eclamptic placenta. Scale bar 200 mm. (c) This healthy human placental tissue sample contains a segment of basal plate (bp) and inter-villus space (ivs). The inter-villus space lining (li) is reactive with endothelial marker (green) as is the fetal capillary endothelium (*). All the inter-villus space lining in this image is endothelial. Extravillous trophoblast cells (evt) are seen in the basal plate. (Red channel ¼ anti-pancytokeratin; green ¼ anti-von Willebrand factor; cvt ¼ chorionic villus trophoblast). Gaps (ns) in the layer are negative for the endothelial and trophoblast markers. Scale bar 250 mm. (d) Anti- EN4 labelled tissue gives identical specific labelling patterns of the endothelium. Scale bar 200 mm.

(data not presented) when the term NS-AV was used to compare patient groups. The scatter plot of AV against NS (Fig. 5b) reveals that the ratiometric approach is better as best fit lines for data from each of the three patient groups demonstrate a progressive angular change rotating around their intersection. Severe pre-eclampsia placentas exhibited a tendency to reduced length proportion of endothelial cells (51.24%) and increased length proportion of pancytokeratin positive trophoblast cells compared to mild pre-eclampsia (mean of 65.56%) and healthy (62.85%) that

was not statistically significant. Comparison of the mean length percent of endothelium, trophoblast and anchoring villi between patients groups revealed there were no significant differences. Furthermore, there were no significant differences between the groups when endothelium: trophoblast ratios were compared. Placentas from pre-eclamptic patients with associated fetal growth restriction in pregnancy exhibited a trend towards a reduced length percent of endothelium and an increased proportion of the trophoblastic cell layer [21].

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migrated over a basal lamina from openings of utero-placental vessels as endothelial cells are never found interrupting villous syncytiotrophoblast. These cells may have replaced trophoblast that at an earlier stage of development lined the lacunar systems from which the intervillous space develops, were destroyed, replaced by fibrin in the short term then replaced by cells of endothelial lineage. 6.1.2. Extra villous trophoblast Data shown here support the views that the distribution of extravillous trophoblast cells is irregular and that they are brightly anti-cytokeratin immunofluorescent [7,8]. The reason for their irregular distribution is presently unclear but is of interest with respect to defining their function. Also the question arises whether there are associations with adjacent lining components such as endothelial sheets or with anchoring villi. Such possibilities merit further study. 6.1.3. Chorionic plate intervillous space lining The lining of chorionic plate intervillous space is similar to the basal plate [4]. Therefore, the lining-mosaic extends beyond the basal plate. Chorionic plate lining was previously described as syncytiotrophoblast and fibrin only [24e27]. The endothelial area (33.02%) is substantial, widespread, and inconsistent with the description of this layer in textbooks. 6.1.4. Alloeepieendothelial mosaic The proportion of the IVS occupied by endothelial cells reported in this and earlier studies is evidence that the “all-trophoblast” description given in most current textbooks and the opposing and largely disregarded “all-endothelial” concept both require revision [27]. In this paper, we redefine the extent of the mosaic to include the chorionic plate lining and provide evidence that it is extensive reaching all parts of the placenta. 6.2. Pathological findings in pre-eclampsia

Fig. 2. Anti-von Willebrand factor labelled indirect immunofluorescence confocal laser scanning microscope image colours the chorionic plate lining green where it is composed of maternal endothelium (e). At the stem villus (sv) the lining is red indicating the sleeve of trophoblast that is immunoreactive with anti-pan-cytokeratin. There is an orange colour at a region of overlap and an area (ns) that is not immunofluorescent. Scale bar 250 mm.

6. Discussion 6.1. Morphological findings in the healthy placenta 6.1.1. Basal plate-IVS lining Our area% measurements of endothelium (62.9%) and trophoblast (27.6%) of placentae were similar to published figures (60.8% and 18.9%, respectively) for healthy placentae [4]. Others too reported predominance of endothelium (over trophoblast with different percentages (46.5%: 27.75%) [4]. Variations in patient groups, experimental techniques or measurement classification probably underlie the differences. Theoretically, the IVS lining endothelial cells might have originated by migration from the intima of vessels that supply/drain intervillous space [4]; arisen from blood-borne endothelial progenitor cells [5]; or be transformed vascular trophoblast [22]. The latter theory can be excluded as it is incompatible with evidence from in situ hybridisation and amelogenin allelotype profiling supporting our view that endothelial cells lining basal plate are maternal [3,23]. Endothelial cells are a significant component of chorionic plate intervillous space lining. They are found remote from basal plate and are more likely to have settled from circulating blood than to have

6.2.1. Pre-eclampsia and the cellular-IVS lining In our study, pre-eclampsia had no significant effect on length percent of endothelium and trophoblast lining basal plate intervillous space surface (mean length of 65.56% for endothelium and 23.59% for trophoblast in mild pre-eclampsia and 62.85% of endothelium and 27.64% of trophoblast in controls). This contraindicates the exaggeration of the length proportion of endothelium in preeclampsia or reduction of endothelium/trophoblast ratio associated with the disease reported in previous studies. The difference in these findings could however be explained by differences in patient mix, sample size, sample preparation, method of measurement and/or disease severity [28,4]. The basal plate intervillous space lining composition does appear to vary as the extent of pregnancy aberration becomes extreme. Pre-eclampsia may increase endothelial proportion when the placental circulation is not extremely compromised and uteroplacental perfusion is adequate. Cells, either from the ends of the utero-placental vessels and/or from endothelial progenitor's cells in the circulation, may enter the intervillous space to replace damaged trophoblast cells. When placental circulation is severely compromised, with reduced intervillous space perfusion and placental oxidative stress, large segments of the syncytiotrophoblast lining the intervillous space of the basal plate may be damaged and will not be replaced adequately by endothelial cells, so the fibrin component may be expected to expand. 6.2.2. Pre-eclampsia and non-cellular effects 6.2.2.1. Fibrin. Pre-eclamptic placentas exhibit increased fibrin covering basal plate intervillous space and mean length percent of anchoring villi linking the basal plate is reduced in pre-eclampsia.

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Fig. 3. (a and b) Confocal laser scanning microscopy of pre-eclamptic placental basal plate showing an anchoring villus (av) and the intervillus space border lining (li). The latter is positively immunoreactive with anti-von Willebrand factor (green). Fetal capillary endothelium (*) is similarly positive. The endothelial marker positive cells lining the intervillous border form a thin layer characteristic of endothelium and also cover an extensive length of the abscission layer (al). Scale bar 250 mm. (c) This section of chorionic plate is a dual channel confocal laser scanning microscopy image of a dual-labelled specimen with the mosaic architecture of the chorionic plate lining cells apparent. The chorionic plate nature is defined by the overlying amniotic epithelium (ae). Sections of the chorionic plate lining are labelled green with specificity for endothelium (e) cytokeratin (c) and bear stem villi (sv). Chorionic villi are covered with anti-cytokeratin labelled (red) trophoblast. Scale bar 100 mm. (d) An immunofluorescence confocal laser scanning image of the chorionic plate of a healthy pregnancy placenta showing anti-endothelial antibody (green) outlining the chorionic plate intervillous border lining cells (e). Some of the chorionic plate cells in this image are positive for anti-pancytokeratin antibody. These are chorionic plate extravillous trophoblast cells (evt). Villous trophoblast labels less intensely with anti-pancytokeratin antibody than extravillous trophoblast as in the basal plate [8]. Scale bar 100 mm.

The mean length percent in control placentae (5.93%) is close to 5.1% given in an independent study [3]. This fibrin-type fibrinoid forms on the superficial layer of the basal plate [29,30]. It derives largely from clotted maternal blood in the intervillous space [31]. The reduction in attachment area of anchoring villi observed may be relevant to “shallow” implantation models of pre-eclampsia [10] as the reduced intravascular penetration of the uterus by

trophoblast in pre-eclampsia serves to limit the area of fetomaternal contact in implantation. The reduction in anchoring villus (the source of extra-villus trophoblast) cross-sectional area may represent another zone where the feto-maternal contact area is reduced. Data show a trend caused by an increase in fibrin/fibrinoid covering the basal plate-intervillous space and a decrease in

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Fig. 4. (a) Upper panel showing a selectively stained histological section where red indicates fibrin using the triple stain Martius Yellow, Brilliant Crystal Scarlet and Soluble Blue (MSB). This portion of the chorionic plate is identifiable by the amniotic epithelium (ae) above a probable chorionic vessel indicated by chorionic blood (cbv) within the chorionic plate (cp). The inter-villus space lining layer has endothelial properties (e), trophoblast (t) and patches of fibrin (f) just below the red staining fibrin (oid) stria that runs from left to right obliquely. Chorionic villi (cv) are present in the intervillous space. Scale bar 100 mm. (b). Lower panel showing placental basal plate (MSB stained). Chorionic villi (cv) fibrin (f) in the villus tree and anchoring villi (av) are all visible. The image reveals a thick band of fibrin (f) beneath the cellular layer lining the basal plate intervillous surface. This fibrin layer (f) is Rohr's fibrinoid it replaces the cellular layer at places where the cellular layer is absent; bp ¼ basal plate; ivs ¼ intervillus space; e ¼ endothelial cell; av ¼ anchoring villous. Scale bar 100 mm.

anchoring villi attachment area in pre-eclamptic placentae compared to healthy placentae. The differences in the means of NS and AV between patient groups are not independently significant. However by combining the two and calculating the ratio of length percent of fibrin/fibrinoid (NS) and length percent of anchoring villi (AV), we have overcome lack of power in the individual raw data. The means of NS/AV data are also significantly different between the three groups. There was an increased deposition of fibrin on the basal plate-intervillous space as well as a decrease in the area of anchoring villi that make contact with the basal plate in pre-eclampsia. Our statistically significant findings are consistent with the increased placental fibrin deposition seen on the chorionic villus tree in hypertensive pregnancy disease [31]. Fibrin because of its low overall coverage is not likely to be an important contributer to antigen masking processes rather it is as a sign of the extent of tissue pathology. The cause of this damage is still unknown but probably related to genetic (innate), immunological [32,49], biochemical, developmental, or physiological factors.

Fig. 5. (a) A graph that shows the ratios anchoring villus/non-immunofluorescent areas in placentae from three groups of patients. Group 1 (black discs) are measurements from healthy pregnancy placentas. Group 2 (red discs) are from mild preeclamptic patients. Group 3 (blue discs) are measurements from severe pre-eclamptic patients. (b) Scatter diagram of the three patient groups (healthy, mild pre-eclamptic and severe pre-eclamptic). It plots fibrinoid (NS length percent) against anchoring villus (AV length percent) with least squares lines of best fit for each patient group (healthy, black line and discs; mild pre-eclamptic, red line and discs; severe preeclamptic, blue line and discs). These lines intersect and pass close to the origin not through it. The increasing slope with severity suggests a ratiometric relationship rather than a summative or subtractive one.

6.3. Repair of the IVS lining? Endothelial cells usually undertake haemostasis, regulate vascular tone, mediate inflammation and angiogenesis. As probable replacements for trophoblast, we suggest that they are also involved in chorion repair. Pre-adherent placental endothelial cells may thus form a source of stem cells for use in treatments for vascular lesions. Activation of coagulation and fibrinolysis is

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Table 2 Descriptive statistics showing the length percent of the basal plate intervillous space lining composed of endothelium, trophoblast, fibrinoid and anchoring villi in the three groups of healthy, mild pre-eclamptic and severe pre-eclamptic pregnancies. Length % of endothelium Patient group Mean SEM Median Minimum Maximum Patient group Mean SEM Median Minimum Maximum Patient group Mean SEM Median Minimum Maximum

Length % of trophoblast

¼ healthy controls (n ¼ 11) 62.85 27.64 4.89 4.54 63.49 28.71 30.68 7.98 86.80 59.04 ¼ mild pre-eclamptic (n ¼ 10) 65.56 23.59 4.71 3.70 67.91 23.95 48.01 7.07 90.53 41.71 ¼ severe pre-eclamptic (n ¼ 11) 51.24 35.42 7.63 5.86 60.94 34.84 12.25 7.96 79.06 63.21

Length % of fibrin/fibrinoid

Length % of anchoring villi

5.93 1.28 4.32 2.12 16.43

3.58 0.63 3.69 0.00 6.16

8.75 1.43 9.14 1.26 15.14

2.10 0.59 1.28 0.00 5.04

11.12 2.07 9.54 0.85 22.10

2.21 0.54 1.83 0.00 5.93

increased with gestational age [33]. Coagulation predominates towards parturition controlling haemorrhage [34e38]. Intervillous space lining fibrin is probably deposited at places of trophoblastic loss as with perivillous fibrin [39,40] where it initiates re-epithelialization at denudation sites by differentiation of cytotrophoblasts [41,42]. Beneath basal plate lining syncytiotrophoblast there are no epithelial cytotrophoblast cells, so damaged syncytiotrophoblast may be replaced by endothelial cells there faux-de-mieux. Increased deposition of fibrin within the basal plate, particularly on the intervillous space lining will lead to an increased area of fibrin barrier, impairment of anchoring villus development and remodelling of spiral arteries by extravillous trophoblast cells emanating from anchoring villi. Resulting poor placentation may be fundamental to progression to systemic pre-eclampsia [43e47]. Massive perivillous fibrin deposition in placentas of diabetic women causes poor fetal outcomes and is associated with miscarriage and intra uterine growth retardation in failed antigcoagulant treatment of antiphophospholipid antibody syndrome [48e50]. Fibrin was thought to act as an immunological barrier inhibiting antibody binding to tumour antigens but this is not a widely held view now [46]. Rather the greater fibrin deposition may be a reaction to immuno-toxic aspects of pre-eclampsia. In pregnancy fibrinolytic activity reduces and blood fibrin increases. In pre-eclampsia, return of these factors to normal levels post-partum is delayed [51]. Women who develop pre-eclampsia during pregnancy release less plasminogen activator and their thromboxane: prostacyclin ratios favour platelet aggregation [52]. Incomplete transformation of spiral arterioles results in stasis and also promotes coagulation [53e57]. These features of pre-eclampsia all favour deposition of placental fibrin. Pre-eclampsia is also associated with hypoxia and oxidative stress which damage trophoblast cells [44,58e60]. Based on this model, the length percent of the intervillous space lining covered by fibrin is expected to increase in pre-eclampsia. Confirming this role of placental fibrin may justify use of antiplatelet drugs to prevent pre-eclampsia and limit its severity. Aspirin is effective in the first trimester for treatment of high-risk patients [61e64]. 7. Future research The stringent criteria for recruiting pure primigravidae preeclamptic patients into the study group limited the statistical power of this study.

To impair early placental formation and contribute to preeclampsia, some fibrin must form early in gestation. There are apparently no studies of fibrin deposition in first trimester placenta in women who later develop pre-eclampsia and the present study is restricted to term placentae. Any future studies showing an increase in the fibrin component in early pre-eclamptic pregnancy would support its role suggested here. Earlier work by Redline et al. [65] showed that increased intervillous fibrin was prone to diagnostic uncertainty yet was a key pathological feature that correlated with underperfusion and hence IUGR. Fibrin is widespread in its distribution and therefore less prone to sampling errors than other histopathological correlates when few sections are scored. Limiting our ability to assess fibrin deposition from microscopy of samples taken at a single point in time (term) is its turnover and re-epithelialisation. Since it appears from the current and related studies that trophoblast re-epithelialisation is confined to the villous tree and endothelial repair is the fibrin replacement mechanism used in the basal plate lining then a combined assessment may be of value. It may be that assessment of villous fibrin volume fraction provides a helpful insight into fibrin incidence at the time of sampling. Endothelium/trophoblast basal plate area fraction measurement may provide a better cumulative measure of fibrin deposition as fibrin on this model is replaced by endothelium. This idea should be tested to see whether it is useful in practice. Basal plate area fractions of fibrin and anchoring villi have not previously been investigated as possible histopathological signs of other fetal and placental growth restriction conditions and should now be. Acknowledgment We thank staff of the pregnancy hypertension unit at Leicester Royal Infirmary for clinical coordination. References [1] Ockleford CD, Wakely J, Badley RA. Morphogenesis of human placental chorionic villi: cytoskeletal, syncytioskeletal and extracellular matrix proteins. Proc Roy Soc Lond Ser B 1981;212:305e16. [2] Byrne S, Cheent A, Dimond J, Fisher G, Ockleford CD. Immunocytochemical localisation of caveolin-1 in human term extra-embryonic membranes using confocal laser scanning microscopy. J Anat 1998;193:312e3. [3] Richani K, Romero R, Soto E, Nien JK, Cushenberry E, Kim YM, et al. Genetic origin and proportion of basal plate surface-lining cells in normal and abnormal pregnancies. Hum Pathol 2007;38:269e75. [4] Smith R, Ockleford CD, Byrne S, Bosio P, Sanders R. Healthy and pre-eclamptic placental basal plate lining cells: quantitative comparisons based on confocal laser scanning microscopy. Microsc Res Tech 2004;64:54e62. [5] De Boer K, Ten Cate JW, Sturk A, Borm JJJ, Treffers PE. Enhanced thrombin generation in normal and hypertensive pregnancy. Am J Obstet Gynecol 1989;160:95e100. [6] Lang I, Hartmann M, Blaschitz A, Dohr G, Kaufmann P. Differential lectin binding to the fibrinoid of human full-term placenta: correlation with a fibrin antibody and the PAF-Halmi method. Acta Anatom 1994;150:170e7. [7] Ockleford CD, Smith RK, Byrne S, Sanders R, Bosio P. A confocal laser scanning microscope study of cytokeratin immunofluorescence differences between villous and extravillous trophoblast: cytokeratin downregulation in preeclampsia. Microsc Res Tech 2004;64(1):43e53. [8] Ahenkorah J, Hottor B, Byrne S, Bosio P, Ockleford CD. Immunofluorescence confocal laser scanning microscopy and immuno-electron microscopic identification of keratins in human maternofetal interaction zone. J Cell Mol Med 2009;13:735e48. [9] Byrne S, Ahenkorah John, Hottor Bismarck, Lockwood C, Ockleford CD. Immuno-electron microscopic localisation of caveolin 1 in human placenta. Immunobiology 2007;212:39e46. [10] Brosens JJ, Pijnenborg R, Brosens IA. The myometrial junctional zone spiral arteries in normal and abnormal pregnancies. Am J Obstet Gynecol 2002;187:1416e23. [11] Roberts J. Endothelial dysfunction in preeclampsia. Semin Reprod Endocrinol 1998;16:5e15. [12] Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: An endothelial cell disorder. Am J Obstet Gynecol 1989;161: 1200e4.

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