Hum. Reprod. Advance Access originally published online on June 1, 2007
Human Reproduction 2007 22(8):2254-2260; doi:10.1093/humrep/dem143
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The architecture of first trimester chorionic villous vascularization: a confocal laser scanning microscopical study
1 Department of Obstetrics and Gynaecology, Academic Medical Centre, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands 2 Department of Anatomy and Embryology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands 3 Department of Pathology, Spaarne Ziekenhuis Hoofddorp, PO Box 770, 2130 AT Hoofddorp, The Netherlands 4 Department of Obstetrics and Gynaecology, Spaarne Ziekenhuis Hoofddorp, PO Box 770, 2130 AT Hoofddorp, The Netherlands
5 Correspondence address. Tel: +31 20 566 6429, Fax: +31 20 696 3489. E-mail: b.a.lisman{at}amc.uva.nl
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Abstract |
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BACKGROUND: The aim of this study was to investigate normal chorionic villous vascularization using CD31 immunofluorescence and confocal laser scanning microscopy (CLSM) to elucidate the spatial arrangement in terms of connections between vessels and cords and their branching patterns compared to deficient chorionic villous vascularization in complicated pregnancies.
METHODS: A descriptive morphologic study using CLSM after CD31 immunofluorescence staining of placental biopsies from normal pregnancies (n = 20), complete hydatidiform molar pregnancies (CHMs; n = 3) and empty sacs (n = 3), with a well documented gestational age (GA).
RESULTS: In this three-dimensional study, first trimester chorionic villi were occupied by a complex network of mainly cords with redundant connections as early as 5+5 weeks GA. With increasing GA cords transform into vessels. From about 9 weeks GA onwards, vascular development is characterized by the presence of two large vessels located centrally and surrounded by and connected to a capillary network. In first trimester CHM and empty sacs, we observed a primitive network of mainly cords.
CONCLUSIONS: This first visualization of the spatio-temporal patterns of blood vessel formation in placental villi is characterized by the development of the vasculosyncytial membrane from a complex network of cords and can be regarded as the placental development before it becomes functional at the end of organogenesis.
Key words: first trimester pregnancy/CD31/vasculogenesis/chorion villus/confocal laser scanning microscopy
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Introduction |
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Detailed knowledge of the spatial arrangement of angiogenetic cords and luminized blood vessels is important for a complete understanding of placental development and function. Only limited data are available on early chorionic villous vascularization in human, as recently reviewed by Benirschke et al. (2006). Electron microscopic analysis of human and macaque placental material (Dempsey, 1972
; King, 1987; Demir et al., 1989), revealed that vasculogenesis (i.e. de novo formation of blood vessels) is first observed in exactly dated placentae of the macaque at 4+5 weeks gestational age (GA). Because of ethical and technical considerations, it is not possible to study vasculogenesis in exactly dated pregnancies in human before 4+5 weeks GA. The limited data on human placenta (Dempsey, 1972; Demir et al., 1989) suggest that vasculogenesis is comparable to the macaque, although the onset is slightly later at 5 weeks GA. Vasculogenesis is the process of haemangioblastic cells from the mesenchyme forming cords or aggregates of cords. Formation into vessels with a lumen takes place by dilatation of intercellular clefts.
Valuable detailed information on placental vascularization became available in numerous anatomical studies using scanning electron microscopy on corrosion vessel casts, sometimes performed in combination with three-dimensional (3D) reconstruction. This work concerns, however, only human placentae at term (Arts NFT, 1961; Habashi et al., 1983; Kaufmann et al., 1985; Leiser et al., 1985). First trimester chorionic villous vascularization in normal pregnancies (GA: 5–12 weeks) was studied more recently by te Velde et al. (1997), based on morphometric analysis using CD34 immunohistochemically stained sections. It was concluded that the development of the chorionic villous vascular system is characterized by maturation of luminized vessels from primitive angiogenetic cords and margination into peripherally located vessels forming the vasculosyncytial membrane. This membrane is essential for an optimal exchange of nutrients and gasses between mother and fetus starting in the second trimester of pregnancy. The spatial arrangement between vessels with a lumen and cords as well as the arrangement between cords, however, could not be elucidated using a 2D technique.
Confocal laser scanning microscopy (CLSM) proved to be useful for 3D visualization of human placental tissue (Jirkovska et al., 1998, 2002; Kubnov et al., 2004). This technique allows perfectly registered stacks of thin serial optical sections to be obtained from which computer 3D reconstructions can be made and eliminates the need to solve the problem of alignment of images of successive sections. CLSM provides an opportunity to examine in 3D the location and branching pattern of villous vessels and cords in first trimester chorionic villous vascularization, in normal and complicated pregnancies.
The purpose of this study was, therefore, to investigate normal first trimester chorionic villous vascularization after CD31 immunofluorescence staining using CLSM, comparing the results with 2D microscopical sections of the same villi. In this analysis, we aimed to elucidate connections between vessels and cords and their branching patterns in normal pregnancies as well as in complete hydatidiform moles (CHMs) and pregnancies with an empty sac (3D).
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Case selection
Twenty placentae of uncomplicated singleton pregnancies were collected and studied after legal abortion, terminated by dilatation and curettage (D&C). The age of the 20 patients undergoing legal abortion ranged between 18 and 37 years (mean: 26 years). Three CHM and three empty sac pregnancies were collected after D&C. Ultrasound and clinical data were used to calculate the exact GA, which ranged between 5+5 and 12+3 weeks. The GA of the CHM ranged between 9+1 and 11+4 weeks and empty sac pregnancies ranged between 8+2 and 11+1 weeks. It was not possible to obtain normal human placental tissue before 4+5 weeks GA, because the Dutch law prescribes a 5-day period to reconsider the request for legal abortion, starting the day when the first request for legal abortion is made.
Placental tissue was fixed directly after collection in 4% (w/v) paraformaldehyde dissolved in PBS (10 mM H2NaPO4/HNa2PO4, 150 mM NaCl, pH 7.6). After fixation for 4 h, the samples were stored in 70% ethanol at 4C till further histological processing. The endothelial marker CD31 was used to study vascularization in first trimester chorionic villi using CLSM. A representative sample of the free (terminal) villi of the chorion frondosum closest to the decidua basalis of uncomplicated singleton pregnancies was drawn for every week of gestation during the first trimester. Contours of vessel walls were distinct from contours of hemangiogenic cords: an angiogenetic cord is visualized by a dense thin line and a luminized vessel, as well as being thicker, is also less dense. Local connections between trophoblast, vessels with a lumen and cords in the mesenchymal and immature intermediate chorionic villi could be revealed from the images captured by CLSM. The study design was approved by the Institutional Medical Ethics Committee. All samples were included in the study after informed consent of the patients.
Immunofluorescent staining of placentae, CLSM
Placentae were stained whole mount as previously described by van den Hoff et al. (1999). In short, a sample of the placenta—chorion frondosum closest to the decidua basalis—was taken and hydrated in a graded series of ethanol-PBS (75%, 50% and 25%) and permeabilized by incubation in PBST (0.25% Triton x 100 in PBS) for 30 min. To reduce background staining, the villi were incubated in PBS-A (1% BSA in PBS) with 5% Goat serum. The endothelial cells were immunofluorescently stained using the CD31 monoclonal antibody (DAKO, Glostrup, Denmark) and Goat-anti-Mouse Alexa-568 (Molecular Probes), as secondary antibody. Following extensive washing in PBS-A, the villi were mounted in PBS with 50% (v/v) glycerol. Fluorescence was visualized using CLSM (Bio-Rad MRC 1024). Auto-fluorescence of the tissue was used to visualize the contours of the villi. All images are shown as brightest point projections.
Morphometric analysis, CLSM
The specimens were examined by CLSM, Bio-Rad MRC 1024 using the planapochromat objective with a magnification of 10. Digitized images (512 x 512 pixels) of serial optical sections (15–20 µm apart) of individual mesenchymal or immature intermediate villi were captured and analysed using the brightest point projection, for demonstration of the spatial arrangement of vessels and cords inside chorionic villi. In order to demonstrate the connections between vessels and cords, manifestations of placental vasculogenesis and angiogenesis, respectively, villi of different developmental stages were studied. The luminal diameter of the vessels was measured.
Immunofluorescent staining of sections
After fixation the placental tissue was dehydrated in a graded ethanol series and embedded in paraplast (Klinipath, Duiven, The Netherlands). About 7 µm thick sections were prepared and mounted onto aminoalkylsilane-coated slides. To reduce background staining, the sections were incubated in PBS-A with 5% Goat serum. The endothelial cells were immunofluorescently stained using the CD31 monoclonal antibody (DAKO, Glostrup, Denmark) and Goat-anti-Mouse Alexa-568 (Molecular Probes), as secondary antibody. Following extensive washing, the sections were mounted in Vectashield H-1000 (Brunschwig chemie, The Netherlands) and fluorescence was visualized using the CLSM.
Morphometric analysis of sections
To compare the vascularization of first trimester chorionic villi visualized by CLSM using the brightest point projection with those of classical sections of the same placental tissue, we used CLSM, Bio-Rad MRC 1024 with a magnification of 20. The presence of CD31 positive hematopoietic cords and vessels with a lumen were described as well as their localization in complete chorionic villi.
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Results |
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Between 5 and 6 weeks GA, a complex network of cords and vessels with redundant connections in chorionic villi is seen. This network comprises mainly cords, already connected together. All vessels and cords are connected to each other without any interruptions. The chorionic villus is completely dominated by a network of vascular elements. Vessels and cords are located centrally as well as peripherally and as a consequence contact the overlying trophoblastic layer (Fig. 1A and B). The luminal diameter of the vessels ranges between 10 and 15 µm (Table 1).
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Between 7 and 8 weeks GA, the chorionic villi are dominated by a capillary network consisting of vessels and cords. The capillary network contains more vessels than cords. At the tip of the chorionic villus, regular small branched off (mesenchymal) chorionic villi are observed containing a conglomeration of CD31 positive cells (Fig. 2). The luminal diameter of the vessels, ranging between 10 and 26 µm, has increased compared with the earlier stage (Table 1).
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Between 9 and 10 weeks GA, the chorionic villi are characterized by the presence of two large vessels located centrally and surrounded by and connected to a capillary network at the periphery of the villus. The capillary network contains mainly vessels with a lumen that are in tight contact with the overlying trophoblastic layer (Fig. 3A and B). From this GA onwards, we observed villous projections containing blindly ending capillary sprouts arising from the underlying capillary network. The luminal diameter of the two centrally located vessels varies between 60 and 75 µm, whereas the vessels of the capillary network range between 26 and 34 µm (Table 1).
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Between 11 and 12 weeks GA, the immature intermediate villi are characterized by the presence of two large vessels surrounded by a capillary network. Within the network, cords are infrequently present. Blindly ending capillary sprouts branching off the capillary network are present (Fig. 4). The centrally located large vessels range between 70 and 90 µm in diameter and are wider than the vessels between 9 and 10 weeks GA. However, the diameter of the vessels of the capillary network is similar to the previous stage (Table 1).
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The 3D representations of the whole mount stained chorion villi were compared with sections from the same specimen. In the sections, the endothelial cells were visualized using CD31. Between 5 and 6 weeks GA, primarily cordsperipherally as well as centrally locatedare observed together with a minimal amount of vessels. Between 7 and 8 weeks GA, mainly peripherally located cords are seen together with an increased amount of vessels, centrally as well as peripherally located. Between 9 and 10 weeks GA, a decrease in amount of peripherally located cords is seen in favour of an increase in peripherally located vessels, whereas in some villi, large calibre centrally located vessels are present. A representative example is shown in Fig. 5. In this section, two large calibre vessels are present in the centre of the villus and at the periphery small vessels and cords are present, confirming the observations of the whole mount staining. Between 11 and 12 weeks GA, primarily vessels are seen that are mainly located peripherally with large calibre centrally located vessels in some villi together with a minimal amount of cords, located peripherally as well as centrally.
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The results of images captured by CLSM of a CHM and an empty sac pregnancy are depicted in Figs 6 and 7. CHM of 10 weeks GA shows a primitive vascular network consisting of cords with conglomerations of CD31 positive cells. The empty sac pregnancy of 8+1 weeks GA is characterized by a primitive network of cords, primarily centrally located.
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Discussion |
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Until recently, the common methodological approach of 3D placental studies consisted mainly of the application of corrosion casts observed in a scanning electron microscope, injection of contrast medium and observation using a classical microscope or manual 3D reconstructions of paraffin sections (Arts NFT, 1961
; Bøe, 1969; Dempsey, 1972; Habashi et al., 1983; Kaufmann et al., 1985; Leiser et al., 1985; King, 1987; Demir et al., 1989).
Although these methods provided valuable information on the micro vascular architecture and morphometry of the placenta, these techniques have limitations. The most important disadvantages of these methods are the incomplete filling of the vessels and the impossibility to fill cords. They are therefore not suited to study vessels and cords, neither the connections between them nor their relation with the trophoblastic layer. Recently, CLSM has been used to study the spatial arrangement of the capillary bed in the placenta (Jirkovska et al., 1998, 2002; Kubinova et al., 2004; Resta et al., 2006) at term and in first trimester of anaemic women. In this paper, we present the results of, to our knowledge, the first 3D reconstruction of the development of chorionic villous vascularization in exactly dated normal first trimester pregnancies compared to deficient first trimester chorionic villous vascularization in complicated pregnancies using CLSM. CLSM of CD31 immunostained chorionic villi provides the possibility of focussing through the villus, visualizing the intact vascular network of vessels and cords. This technique does not have to solve the problem of alignment of images of successive sections, nor does it shows the limitations of the injecting method, i.e. incomplete filling, and per definition being unable to prove the presence of cords with no lumen.
CD31 immunohistochemistry, an endothelial marker, was used to study early chorionic villous vascularization. As described in earlier studies (Demir et al., 1989, 2004; Charnock-Jones et al., 2004; Tertemiz et al., 2005), hemangioblastic cell cords and formation of a primitive lumen can be determined using CD31 immunohistochemistry.
In our study, we observed in first trimester chorionic villi, a complex network of cords and vessels with redundant connections as early as 5+5 weeks GA. In the literature, lumen formation out of hemangioblastic cords during vasculogenesis is described between 5+2 and 6+0 weeks GA (Demir et al., 1989; Zygmunt et al., 2003) The process of vasculogenesis results in an extensive network in which all vessels and cords are connected with each other. Benirschke et al. (2006) observed fetal capillary segments formed by vasculogenesis until 6+4 weeks GA and did not report a 'fetoplacental capillary network' until 7–8 weeks GA. This discrepancy with our results might possibly be explained by the limitations of the technique used by Benirschke et al. (2006).
In our material, a conglomeration of CD31 positive staining was frequently observed in small mesenchymal villi branching off at the top of the intermediate immature villus until 9 weeks GA, a finding which we have interpreted as the initiation of vasculogenesis. Benirschke et al. (2006) states that de novo formation of capillaries (vasculogenesis) usually can be found at the tips of newly sprouting mesenchymal villi, as seen in our material, but only until 6+4 weeks GA. According to this group, from this date onwards, vasculogenesis is an exception and further expansion of the villous vascular system mainly occurs by angiogenesis (i.e. the formation of new blood vessels from pre-existing ones). Our results, however, show the presence of vasculogenesis until at least 9 weeks GA, being consistent with the results of our sections and the study of te Velde et al. (1997) who described the process of maturation (i.e. development of angiogenetic cords into luminized vessels) and marginalisation (i.e. development of centrally localized into peripherally localized vessels) in CD34 stained sections of first trimester normal pregnancies. The limitation of the latter, however, was the fact that no information could be obtained concerning the spatial arrangement of these cords and vessels. Therefore, the present study is complementary to the results of te Velde et al. (1997).
From 8+4 weeks GA onwards, we observed the presence of two large calibre vessels located centrally, which are surrounded by and in complete contact with a capillary network adjacent with the overlying trophoblastic layer. These large calibre vessels most probably develop centrally in the villus within the vascular network. This was also observed by Bøe (1969) in his study on the vascularization of young placentae by injecting Indian Ink into the umbilical artery. However, it does not become clear from his publication, at which GA he first observed this paravascular network, other than it concerned 'young placentae'. Starting at 9 weeks GA, we observed a villous projection containing blindly ending capillary sprouts arising from the underlying capillary network, suggesting a branching form of angiogenesis. From this point onwards, expansion of the villous vascular system occurs predominantly by angiogenesis with vasculogenesis playing only a minor role.
From the results in the present study, we conclude that the network of cords is formed by vasculogenesis and that transformation into a capillary network which develops into two centrally located vessels surrounded by a capillary network, most probably occurs on the one hand by elongation of the network of cords and on the other by formation of intercellular clefts forming vessels with a lumen, followed by angiogenesis. Little information is available regarding the exact mechanisms on how placental vasculogenesis and angiogenesis occur, how angiogenic precursor cells differentiate and which signals are inducing differentiation of these cells (Hanahan, 1997; Risau and Lemmon, 1998; Demir et al., 2004). According to Tertemiz et al. (2005), studying placental vasculogenesis and angiogenesis using CD31 immunohistochemistry, CD31 terminal dUTP nick-end labelling (TUNEL) and electron microscopy apoptosis (in the centre of the capillary lumen) as a novel approach for lumen formation during vasculogenesis, and angiogenesis is required for a normal and vigorous vessel development. How this apoptosis cascade is triggered could not be concluded from their study.
Bøe described a paravascular network as a system of anastomosing vessels of capillary size which constitutes a system of arterio-venous communications between the afferent arteries and the efferent veins of the terminal branches of the villi, acting as a buffer system for the regulation of the hemodynamics in the villi. Benirschke et al. (2006) however stated that one vein and one artery surrounded by a capillary network are characteristic for immature intermediate villi in the first trimester of pregnancy. This was confirmed in our study. Because the blood pressure during the first trimester of pregnancy is low (Pijnenborg et al., 2006), efficient arterio-venous shunting as suggested by Bøe (1969) seems unlikely. In our opinion, the paravascular network, being in contact with the trophoblastic layer, is used for an efficient exchange of nutrients and gases at the end of the first trimester. The centrally located vessels on the other hand most likely transport nutrients and gases towards the fetus.
The images of normal first trimester chorionic villous vascularization captured by CLSM were compared to those of CHM and empty sac pregnancies. Although the latter two showed vasculogenesis in a primitive form, according to the GA, one would expect an extensive network of cords and vessels at this stage combined with signs of angiogenesis. In earlier publications studying first trimester chorionic villous vascularization using classical histological sections of CHM and empty sac pregnancies compared to normal first trimester pregnancies, we concluded that vascularization is diminished in empty sac pregnancies and CHM (Lisman et al., 2004, 2005). The results of the present study support the conclusions of our earlier studies and by visualizing the spatial arrangement of the vascularization, we conclude that initiation of vasculogenesis is seen in these types of pregnancies but further development of vasculogenesis and angiogenesis does not occur. Although we are aware of the fact the number of analysed CHMs and empty sac pregnancies is limited, the observations underscore that blood vessel formation in the placenta starts by the formation of cords by vasculogenesis. During subsequent development, the cords mature into a primitive plexus of blood vessels that further expands by angiogenesis.
In conclusion, the morphological and morphometric evaluation of first trimester chorionic villous vascularization is feasible using CLSM technology. Our data concerning the spatial arrangement of first trimester chorionic villous vessels and cords using CLSM technology are consistent and complementary with the results obtained by classical microscopy. The spatio-temporal patterns of blood vessel formation in placental villi, visualized for the first time, are characterized by the development of the vasculosyncytial membrane from a complex network of cords and can be regarded as the placental development before it becomes functional at the end of organogenesis.
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Submitted on February 1, 2007; resubmitted on April 22, 2007; accepted on May 1, 2007.
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