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Reproduction (2004) 128 107-116
DOI: 10.1530/rep.1.00208
Copyright © 2004 Society for Reproduction and Fertility
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RESEARCH

Angiogenesis and microvascular development in the marmoset (Callithrix jacchus) endometrium during early pregnancy

Amanda J Rowe, Christine Wulff1 and Hamish M Fraser

Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh Chancellor’s Building, 49 Little France Crescent, Edinburgh, EH16 4SB, UK and 1 Department of Obstetrics and Gynaecology, University of Ulm, Prittwitzstraße 43, 89075 Ulm, Germany

Correspondence should be addressed to H M Fraser; Email: h.fraser{at}hrsu.mrc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The aim of the study was to describe and quantify the changes in the maternal vasculature and angiogenesis during early pregnancy in the marmoset endometrium using bromodeoxyuridine (BrdU) to identify proliferating cells, CD31 to label endothelial cells and dual staining to identify proliferating endothelial cells. Non-pregnant animals from mid- and late secretory stages were studied and compared with pregnant animals at weeks 2, 3 and 4 of pregnancy. Qualitative and morphometric analyses of angiogenesis and vascular area were performed. The results show that pregnancy is associated with increasing angiogenesis in the upper zone of the endometrium, becoming significantly increased at 3 weeks. This is associated with an increase in the vessel area and diameter in this zone. These results provide the platform from which to design studies in which specific angiogenic factors can be targeted in vivo during early pregnancy in order to determine their role in regulating these vascular changes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Angiogenesis, the formation of new blood vessels from pre-existing capillaries, is of importance in supporting the reconstruction of endometrium after menstruation and in providing a vascularised, receptive endometrium for implantation and placentation (Torry et al. 1996, Ahmed & Perkins 2000). In early pregnancy, complications may arise from defects in angiogenesis associated with luteal insufficiency, intra-uterine growth retardation and pre-eclampsia (Ahmed & Perkins 2000, Maynard et al. 2003). With recent advances in the ability to manipulate angiogenesis in vivo it may be possible to identify the defects in angiogenesis at a cellular and molecular level which in turn should lead to the design of more effective therapeutic approaches (Fraser & Lunn 2001, Maynard et al. 2003, Wulff et al. 2003). Direct study of the human implantation site is neither ethical nor practical. Hence, in order that the potential to manipulate angiogenesis be realised, it is important that relevant animal models be identified in which the changes in angiogenesis and the microvasculature during early pregnancy may be quantified.

The marmoset is widely used in reproduction research and has the advantage of an extremely high rate of reproductive efficiency, 90% of ovulated follicles giving rise to healthy offspring (Nubbemeyer et al. 1997). Implantation in the marmoset occurs on day 12 after ovulation and previous studies have described the morphological and ultra-structural changes involved (Enders & Lopata 1999, Enders 2000, Niklaus et al. 2001). We have recently described the changes in gene expression of vascular endothelial growth factor (VEGF) and angiopoietin families and their receptors during early pregnancy in this species (Rowe et al. 2002, 2003). We have also shown how changes in angiogenesis in the ovary of the marmoset can be monitored using bromodeoxyuridine (BrdU) to identify proliferating cells, CD31 to label endothelial cells and dual staining to identify proliferating endothelial cells (Fraser et al. 2000). In the current study, we employed this approach to describe the changes in angiogenesis, cell proliferation and the microvasculature during early pregnancy in the marmoset endometrium. Animals from the mid- and late secretory stages of the cycle were compared with pregnant animals at weeks 2 (the time of implantation), 3 and 4 of pregnancy. Qualitative studies were conducted and morphometric analyses of angiogenesis performed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Tissue collection
Experiments were carried out under the Animals (Scientific Procedures) Act (1986), and were approved by the local Ethical Review Committee. Marmosets were mated and stage of pregnancy determined by measuring plasma progesterone concentrations and examining serial sections of the uterus for the presence of a trophoblast as described previously (Rowe et al. 2003). Uteri were collected 1 h after administration of 50 mg BrdU per kilogram body weight – the BrdU was made up in 1 ml saline and injected i.v. at 2 weeks (n = 4), 3 weeks (n = 6) or 4 weeks (n = 6) post-ovulation; the procedure was as described in previous studies in which the changes in expression of angiogenic factors associated with early pregnancy in the corpus luteum (Rowe et al. 2002) and uterus (Rowe et al. 2003) were reported for the same animals. Non-pregnant animals were studied at the mid- and late secretory phase (n = 5 per group), 2 and 3 weeks post-ovulation. Uteri were weighed and fixed in 4% paraformaldehyde.

Cellular changes were studied by: (1) examining sections stained with haematoxylin and eosin; (2) Quantifying the number of mitotic cells stained for BrdU; (3) dual labelling to record the incidence of co-localisation of BrdU and CD31.

Haematoxylin and eosin staining and immunocytochemistry
Tissue sections (5 µm) were cut onto Super-frost plus slides (Sigma) dewaxed in xylene, rehydrated in descending concentrations of ethanol and washed in water. For morphological evaluation, sections were stained with haematoxylin and eosin, dehydrated and mounted in pertex. For immunocytochemistry, antigen retrieval was performed in a Tefal Clypso pressure cooker (Tefal, Essex, UK) in 0.01 M citrate buffer (pH 6), for 6 min at high pressure setting 2. Slides were then left in hot buffer for 20 min and washed in Tris-buffered saline (TBS: 0.05 mol/l Tris, pH 7.4; 9 g/l NaCl). To reduce non-specific binding, sections were blocked in normal rabbit serum (NRS) (diluted 1:5 in TBS/bovine serum albumin) for 30 min.

To visualise BrdU-stained cells, mouse monoclonal anti-BrdU (Boehringer Mannheim) (diluted 1:30 in NRS block) was applied overnight at 4 °C. Slides were washed 3 times in TBS before the secondary antibody, rabbit anti-mouse (Dako, Glostrup, Denmark) (diluted 1:60 in NRS block), was added for 30 min at room temperature. After three TBS washes, slides were incubated with mouse APAAP (alkaline phosphatase-anti-alkaline phosphatase; 1:100 in NRS block at room temperature) for 40 min, washed with TBS and transferred to nitroblue tetrazolium (NBT; Sigma) buffer for 10 min at room temperature then stained with NBT. The reaction was stopped in tap water, sections were counterstained with haematoxylin before being dehydrated in graded alcohols and mounted in pertex.

To visualise endothelial cells, the first antibody CD31 (Dako) was diluted 1:20 in NRS, added to slides and incubated overnight at 4 °C. This was then detected the following day by addition of a rabbit anti-mouse secondary antibody (1:60 in NRS), followed by mouse APAAP (diluted 1:100 in NRS). TBS washes were performed between each antibody. Visualisation was performed using NBT as described for BrdU.

For detection of proliferating endothelial cells, dual staining was obtained by immuncytochemistry with CD31 and BrdU. For CD31 detection the protocol was followed as described above but visualisation was performed with Fast Red (Sigma). Sections were then washed with TBS before the second primary antibody, sheep antibody to BrdU (Fitzgerald, Concord, MA, USA) was added, diluted 1:500 in NRS, and incubated overnight at 4 °C. After post-incubation washes with TBS, a biotinylated rabbit anti-sheep secondary antibody (Vector, Peterborough, UK) was added, followed by avidin–biotin conjugated alkaline phosphatase (ABC-AP; Dako). After incubation with the ABC-AP complex, slides were transferred to NBT buffer for 10 min before staining with NBT. Reactions were stopped in tap water, then slides were counterstained in ascending concentrations of alcohol, cleared in xylene and mounted in pertex.

Volume fraction measurements
Volume fraction analyses were performed in three directions, from luminal epithelium to myometrium, each perpendicular to the luminal epithelium. The first, at the fundic end of the uterus, the second and third on the right- and left-hand side of the cross-section respectively (see Fig. 1Go). In each direction, every field of view was analysed, ensuring no overlap occurred between each. This gave rise to between 30 and 40 grids per animal. The test grid, superimposed on the sections, consisted of 588 points. The number of test points falling on glands (including lumen), proliferating glandular epithelium, uterine lumen, stroma, proliferating stromal cells, myometrium, luminal epithelium, proliferating luminal epithelial cells, endothelial cells and proliferating endothelial cells (characterised by co-localised BrdU and CD31 immunostaining) were counted. The volume fraction occupied by each component was then calculated by expressing the number of points hitting that component as a percentage of the total number of test points applied. If, for example, the volume fraction of proliferating endothelial cells was required, it was expressed as volume fraction of proliferating endothelial cells as a percentage of volume fraction of total endothelial cells.



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  		Figure 1 Schematic illustrating zones of the marmoset uterus and grid placement. The grid consists of 588 points (21 x 28). This grid size was chosen as it allows differentiation of individual cells with no cell being counted by more than one point. At each point on the grid, the cell type was recorded and expressed as a volume fraction of the total 588 points. The number of proliferating cells (black-stained nuclei) were expressed as a volume fraction of the total volume fraction of that cell type. For example, the number of points overlaying proliferating endothelial cells was divided by the total volume fraction of endothelial cells (black-stained nuclei, red cytoplasm) to gain a volume fraction of proliferating endothelial cells. Volume fraction analyses were performed in three directions, from luminal epithelium to myometrium, each perpendicular to the luminal epithelium. The first, at the fundic end of the uterus (1), and the second and third on the right- and left-hand side of the cross-section respectively (2 and 3). In each direction, every field of view was analysed at x 40 objective magnification, ensuring no overlap occurred. This gave rise to between 30 and 40 grids per uterus. Scale bar, 50 µm.

 
The endothelial cell area (i.e. CD31-positive cells) was measured at x 400 magnification in four sections of each uterus. The field was placed into the upper zone so that no embryonic tissue which is also positive for CD31 was measured. The captured grey-scale image was thresholded and converted to a binary image. The CD31-positive area was then calculated per unit area of the upper zone and expressed as a mean value of the number of sections analysed. Diameter of microvessels beneath the luminal epithelium was measured in the same sections by taking the mean of three measurements of diameter for each blood vessel.

Results were analysed statistically by ANOVA multiple comparison followed by a Bonferroni post hoc test. Differences were considered to be significant at P < 0.05 or less.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Changes in uterine morphology
Uterine weights in the mid- and late secretory phase were 341 ± 26 and 327 ± 15 mg, and at 2, 3 and 4 weeks of pregnancy they were 377 ± 26, 354 ± 4 and 462 ± 34 mg respectively. Uteri at pregnancy week 4 were significantly heavier (P < 0.05) than both groups of non-pregnant controls.

In the late luteal phase of the non-pregnant cycle the endometrium contains abundant glands. By the appearance of the shape of the glands, the endometrium may be divided into three zones (Fig. 2Go). (1) The luminal zone is a small strip containing the luminal epithelium with accumulation of larger blood vessels underneath. (2) The upper zone contains cross-sections of glands which are almost round in shape; in between the glands, condensed stroma and a few vessels are found. (3) The lower zone contains more elongated longitudinal glands and densely packed stroma.



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  		Figure 2 Haematoxylin and eosin sections of endometrium of the non-pregnant late luteal phase (a) and of pregnancy (b). The endometrium can be divided into three zones: the luminal zone (1), the upper zone (2) and the lower zone (3). During the late luteal phase (a), the luminal zone (1) contains luminal epithelium (LE) with large blood vessels beneath the surface (arrows) and stroma (S). The upper zone (2) and the lower zone (3) consist of numerous glands (Gl) and stroma (S). The glands in the upper zone are round in shape, while glands in the lower zone are more elongated. During pregnancy (b), morphological changes occur within these zones. In the luminal zone (1) the trophoblast (T) becomes attached to the luminal epithelium. The vasculature beneath the luminal epithelium increases in diameter. The upper zone (2) is characterised by decidualising stroma (D) which leads to a decrease in the number of glands (Gl) in that region. Glands of the lower zone (3) have changed their appearance, being convoluted and containing enlarged lumina. Scale bar, 100 µm.

 
At day 14 of the non-pregnant cycle, no luminal epithelial proliferation (Fig. 3aGo) or glandular epithelial proliferation was present in the upper zone (Fig. 3bGo), although proliferation was found in the stroma (Fig. 3bGo). While stroma proliferation was absent in the lower zone (Fig. 3cGo) glandular proliferation was observed in this zone (Fig. 3cGo). By day 21 of the luteal phase, again there was no luminal epithelial proliferation visible in the luminal zone (Fig. 3dGo) and glandular proliferation was absent in the upper (Fig. 3eGo) and lower (Fig. 3fGo) zones. Stromal proliferation remained in the upper zone.



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  		Figure 3 Immunocytochemical staining for BrdU in the endometrium of day 14 (a–c) and day 21 (d–f) of the luteal phase (LE, luminal epithelium; S, stroma; Gl, glands). The arrows depict the vasculature beneath the luminal epithelium. At day 14, proliferation (dark-stained nuclei) is found in the stroma, but not in the luminal epithelium and glands of the luminal zone (a). In the upper zone (b), proliferation is found in the stroma but not in the glands, while in the lower zone (c) proliferating cells are localised exclusively to the glands. At day 21 of the cycle (d–f) proliferation is detected in the stroma of the luminal zone (d) but is less in the stroma of the upper zone (e) and is virtually absent in the glands of the lower zone (f). Note the increase in vasculature beneath the luminal epithelium at day 21 as compared with day 14. Scale bar, 100 µm.

 
After conception, morphological changes occurred throughout the whole thickness of the endometrium (Fig. 2bGo). In the luminal zone, the vessels appear to enlarge (Fig. 2bGo, inset 1). The trophoblast is superficially attached to the luminal epithelium. The glands of the upper zone disappear due to the process of stroma decidualisation after implantation has taken place (Fig. 2bGo, inset 2). Decidualisation is found around the entire upper zone of the endometrium and progresses to the lower zone with ongoing pregnancy until the whole upper zone is decidualised. The glands in the lower zone enlarge showing a more convoluted shape with a larger lumen (Fig. 2bGo, inset 3).

In the following, the description of the results focuses on the luminal and upper zone since the most obvious changes during pregnancy regarding angiogenesis occur herein. By week 2 of pregnancy (Fig. 4aGo) maternal vessels, particularly those immediately beneath the implantation site, appear larger than those in the non-pregnant, lateluteal-phase endometrium. Decidualisation begins to take place in the stroma directly under the luminal vasculature. In the glandular epithelium of the upper zone, proliferation was virtually absent while stroma proliferation was apparent, particularly immediately surrounding the glands of this region (Fig. 4bGo). By week 3 of pregnancy, the vasculature has expanded containing a higher number of luminal vessels in the further decidualised upper zone (Fig. 4cGo). Stroma proliferation is intense especially in decidual cells (Fig. 4dGo). By week 4 of pregnancy (Fig. 4eGo) stroma decidualisation of the upper zone is complete. Large vessels rest beneath the luminal epithelium around the entire uterine lumen but also pass through the whole width of the decidua. Penetration of the trophoblast beneath the luminal epithelium is becoming more evident (Fig. 4eGo). Proliferation appears to be still high but is more evident in the lower parts of the decidua at the border to the lower zone (Fig. 4fGo).



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  		Figure 4 Comparison of the morphology (haematoxylin- and eosin-stained sections: a, c and e) and cell proliferation (BrdU-stained sections: b, d and f) of the endometrium at two (a, b), three (c, d) and four weeks (e, f) of pregnancy (T, trophoblast; D, decidua; Gl, glands; arrows mark the vasculature of the decidua). Note the increase in the vasculature (arrows) and the enlargement of the decidua from 2 weeks to 4 weeks of pregnancy. The depth of invasion of the trophoblast is indicated by the black line. Proliferation increases in the decidua from 2 weeks to 4 weeks of pregnancy. Proliferation in the decidua at 4 weeks of pregnancy is mainly localised at the border to the glands of the lower zone. Scale bar, 100 µm.

 
Endothelial proliferation
To identify proliferating endothelial cells, dual staining for CD31 and BrdU was carried out. By gross inspection, changes in endothelial cells were most evident in the luminal and upper zone of the endometrium and typical examples are shown in Fig. 5Go. In the late-luteal-phase endometrium (Fig. 5aGo), dual-stained endothelial cells were found in the vasculature beneath the luminal epithelium and in the stroma. There appears to be a slight increase in endothelial cell proliferation especially around the glands at week 2 of pregnancy (Fig. 5bGo). A further increase in endothelial proliferation but also in proliferation of decidual cells is observed during week 3 of pregnancy (Fig. 5cGo), while in the vessels beneath the luminal epithelium, endothelial cell proliferation has ceased. By week 4 of pregnancy (Fig. 5dGo) no further increase of endothelial proliferation is evident whereas numerous decidua cells are proliferating.



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  		Figure 5 Dual staining for CD31 (red-stained cytoplasm) and BrdU (dark-stained nuclei) (arrows) to identify endothelial proliferation in the luminal and upper zone in late luteal controls (a), and at 2 (b), 3 (c) and 4 weeks (d) of pregnancy (LE, luminal epithelium; Gl, glands; D, decidua). Note the increase in endothelial proliferation from late luteal controls to three weeks of pregnancy. Scale bar, 100 µm.

 
These observations are in accordance with volume fraction measurements of endothelial proliferation as shown in Fig. 6Go. By comparing the mean volume fraction of proliferating endothelial cells as a percentage of total endothelial cells (Fig. 6aGo) between lateluteal-phase endometrium and week 2 of pregnancy there was a tendency for increasing endothelial cell proliferation (although not significant). When compared with week 3 of pregnancy endothelial proliferation has significantly increased (P < 0.05). No further increase was observed in week 4 of pregnancy. When comparing endothelial cell proliferation with total cell proliferation (Fig. 6bGo), there was a significant decrease in the mean volume fraction of proliferating endothelial cells from week 2 to week 4 of pregnancy which is consistent with the observation of increasing decidual cell proliferation at that time. No difference in endothelial proliferation was found in the lower zone between the stages.



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  		Figure 6 Quantification of endothelial proliferation in the upper zone of the endometrium of the late luteal phase (non-pregnant), and at 2 (P 2), 3 (P 3) and 4 weeks (P 4) of pregnancy. (a) Mean volume fraction of proliferating endothelial cells as a percentage of total endothelial cells. (b) Mean volume fraction of proliferating endothelial cells as a percentage of total proliferating cells. Different letters denote significant differences.

 
Vascularisation of the luminal and upper zones
To visualize and quantify changes in the vasculature of the luminal and upper zone, CD31-stained sections were examined (Fig. 7Go). In lateluteal-phase endometrium (Fig. 7aGo) large vessels beneath the luminal epithelium and a vascular network of small vessels in the upper zone are observed. At 2 weeks of pregnancy (Fig. 7bGo) the vessels beneath the luminal epithelium are markedly increased in size and the vascular network in the upper zone has enlarged. A further expansion of the vasculature in the upper decidualising zone is evident by week 3 of pregnancy (Fig. 7cGo). By 4 weeks of pregnancy the vasculature of the decidua has been established, characterised by large vessels beneath the luminal epithelium and by a fine network of capillaries and luminal vessels in between.



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  		Figure 7 CD31 staining of the vasculature in the luminal and upper zone in the endometrium of late luteal phase (a), and at 2 (b), 3 (c) and 4 weeks (d) of pregnancy (B, blastocyst; D, decidua; the black bar denotes the thickness of the upper zone). Note the increase in vascularisation of the upper zone with ongoing pregnancy and the enlargement of the decidua. Note also, CD31 staining of the blastocyst (b). Scale bar, 100 µm.

 
To quantify the observed changes in the vasculature, the vascular diameter of the vessels under the luminal epithelium and the area of CD31 staining were measured using image analyses. The results are shown in Fig. 8Go. For the area of CD31 staining, there was a significant increase(P < 0.05) at week 2 of pregnancy and a further significant increase at week 3 compared with non-pregnant controls. The visible increase in vascular diameter of the vessels beneath the luminal surface was confirmed by a significant increase of the mean diameter from non-pregnant endometrium to week 2 of pregnancy (Fig. 8bGo). No further increase in the vessel diameter was detectable from week 2 to weeks 3 or 4 of pregnancy.



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  		Figure 8 Quantification of the endothelial area by CD31 staining (a) and vascular diameter of vessels beneath the luminal epithelium (b) in the upper zone of late luteal endometrium (non-pregnant), and at 2 (P 2), 3 (P 3) and 4 weeks (P 4) of pregnancy. Different letters denote significant differences.

 
Cellular compartment analysis
Volume fraction of glands in non-pregnant animals was 64 ± 3% at 2 weeks and 65 ± 5% at 3 weeks. Volume fraction of glands decreased as pregnancy progressed being 51 ± 11, 38 ± 9 and 11 ± 7% at 2, 3 and 4 weeks respectively, the 4-week group being significantly lower (P < 0.05) compared with all other stages.

Glandular epithelial proliferation
Proliferation in the upper zone glands was virtually absent in non-pregnant and 2-week pregnant animals (Fig. 3Go). At weeks 3 and 4 of pregnancy, proliferation increased to 1.8 ± 0.7 and 1.74 ± 0.06% respectively, being significantly higher than in all other groups. This proliferation was localised to the glands bordering the decidua. Proliferation in the lower zone was present only in the non-pregnant group at week 2 post-ovulation; at 0.8 ± 0.24%, this was significantly higher (P < 0.05) than all other groups, in which proliferation of glands in this area was virtually absent.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
This investigation has identified and quantified the changes in endometrial angiogenesis and maturation of the micro-vascular system during early pregnancy for the first time in the marmoset. It was found that the main changes take place in the upper and luminal zones of the endometrium. Specifically, pregnancy is associated with increasing angiogenesis in the upper zone of the endometrium, becoming significantly increased at 3 weeks. This is also associated with an increase in the vessel area. In the luminal zone, vascular diameter significantly increased during pregnancy. These results provide the platform from which to design studies in which specific angiogenic factors can be targeted in vivo during early pregnancy in order to determine their role in regulating these vascular changes.

After implantation of the blastocyst, the trophoblast remains avascular for a period of time which varies between species (Enders 1993, 2000). It is therefore essential that the mechanisms required to meet the growing metabolic demands of the embryo be in place throughout the early implantation stages. Nutrients and oxygen from the maternal circulation will be supplied to the trophoblast by diffusion, and so a well-developed maternal vascular system is likely to be critical. Increased angiogenesis at this time may be an important mechanism involved in ensuring that an adequate vasculature is available to support the conceptus. Accordingly, we found that angiogenesis was consistently more intense in the upper zone than the lower zone with a significant increase from the late luteal phase to week 3 of pregnancy. The vascular area subsequently increased during early pregnancy. In the luminal zone, the vascular diameter of the maternal vessels beneath the luminal surface increases at as early as 2 weeks of pregnancy as compared with lateluteal-phase controls. It appears that these luminal vessels are the first source from which the very early trophoblast receives nutrients and oxygen, hence their rapid development. With the growing demands of the trophoblast, angiogenesis subsequently increases from 2 weeks to 3 weeks of pregnancy leading to the development and expansion of the decidual vasculature until decidualisation is complete at 4 weeks of pregnancy. It is this decidual vasculature which is the source for maternal blood that fills the placental labyrinth after trophoblast invasion during later stages of pregnancy (Wulff et al. 2002). Similar observations have been made in other species. In the ewe, increased luminal diameter and decreased endometrial thickness, increased abundance of large microvessels and development of a subepithelial capillary plexus were described on days 24 and 30 after mating (Reynolds & Redmer 1992). Thus, early pregnancy is a time of major remodelling in the uterus and this includes development and expansion of the maternal blood vessels.

The endothelial cell proliferation observed is likely to be mediated by angiogenic factors, such as the VEGF and angiopoietin families and their receptors (Ahmed & Perkins 2000). In the specimens studied here, we previously reported increased expression of VEGF and angio-poietin-1 (Ang-1) mRNA in stroma of the upper zone particularly adjacent to the implantation site, during the peri-implantation period. Expression of mRNA for the receptors was detected in endothelial cells immediately surrounding the glands in the upper zone nearest to the luminal epithelium, highest expression being adjacent to the implantation site (Rowe et al. 2003). VEGF is also a potent permeability factor which is likely to have an important role in facilitating the diffusion process through the maternal vessels. The co-expression of VEGF with Ang-1 may enhance angiogenesis and subsequently lead to vessel stability.

Implantation is followed by proliferation and differentiation of stromal cells forming the decidua. This serves to displace the glandular tissue further away from the lumen. Accordingly, volume fraction of the glands decreased. The current study also revealed an increase in proliferation at the junction with the decidua as pregnancy progresses from the time of implantation. Associated with this increased proliferation, the decidual plate is expanding as it is the docking side to the trophoblast.

Understanding of angiogenesis at the time of implantation and in early pregnancy is of fundamental importance. Early miscarriage within the first few weeks after conception is a common phenomenon in women. Genetic and congenital abnormalities account for some early pregnancy loss, but it is also possible that failure of the conceptus and endometrium to establish an effective implantation may be involved; angiogenesis is likely to be a key component in this process (Smith et al. 1987, Reynolds & Redmer 2001). Studies are beginning in rodents to address the role of VEGF in early pregnancy and to show that inhibition of VEGF inhibits implantation by blocking oestrogen-induced oedema (Rabbani & Rogers 2001, Rockwell et al. 2002). Marmosets have a very high fertility rate compared with most other primates (Nubbemeyer et al. 1997), and as such have a practical advantage as a model for early pregnancy where the role of angiogenic events occurring therein can be addressed. Now that we have described and quantified the vascular changes in the endometrium of the pregnant marmoset, future studies should help to elucidate whether an inadequately developed endometrial vasculature results from specific inhibition of angiogenic factors.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Helen Wilson for expert assistance, I Swanston, F Pitt and G Johnstone for assays, K D Morris and staff for animal care and collection of tissue and Dr S Riley for discussions. We are indebted to Professor S K Smith and Dr S J Charnock-Jones, University of Cambridge, for guidance on the volume fraction measurements.


   Footnotes
 
Received 19 February 2004
First decision 21 April 2004
Accepted 6 May 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Rowe AJ, Wulff C & Fraser HM 2003 Localistion of mRNA for vascular endothelial growth factor (VEGF), angiopoietins and their receptors during the peri-implantation period and early pregnancy in marmosets (Callithrix jacchus). Reproduction 126 227–238.[Abstract]

Smith CA, Moore HD & Hearn JP 1987 The ultrastructure of early implantation in the marmoset monkey (Callithrix jacchus). Anatomy and Embryology (Berlin) 175 399–410.[CrossRef]

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H. M. Fraser, H. Wilson, A. Silvestri, K. D. Morris, and S. J. Wiegand
The Role of Vascular Endothelial Growth Factor and Estradiol in the Regulation of Endometrial Angiogenesis and Cell Proliferation in the Marmoset
Endocrinology, September 1, 2008; 149(9): 4413 - 4420.
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A. Silvestri and H. M Fraser
Oestrogen and progesterone receptors in the marmoset endometrium: changes during the ovulatory cycle, early pregnancy and after inhibition of vascular endothelial growth factor, GnRH or ovariectomy
Reproduction, August 1, 2007; 134(2): 341 - 353.
[Abstract] [Full Text] [PDF]

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