Molecular Human Reproduction, Vol. 6, No. 3, 276-282,
March 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
VEGF, its receptors and the Tie receptors in recurrent miscarriage
1 Department of Obstetrics and Gynecology, Helsinki University Central Hospital, PL 140, 00029 HYKS, 2 Department of Pathology, Haartman Institute, University of Helsinki, 00014 Helsinki, and 3 Family Federation of Finland, Helsinki, Finland
Abstract
The aetiology of recurrent miscarriage (at least three consecutive miscarriages) usually remains unsolved. The vascular endothelial growth factor (VEGF) family of proteins, together with their receptors and the Tie (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) receptors, are crucial for embryonic development. Therefore, we used immunohistochemistry to analyse the expression of VEGF, the VEGF receptors (VEGFR)-1, -2, and -3, and the Tie-1 and Tie-2 receptors in placental and decidual tissue of women with a history of recurrent miscarriage and missed abortion (MA; n = 12) or blighted ovum (BO; n = 6), and from normal early terminated pregnancies (n = 12). Compared with controls, the MA and BO groups showed: (i) diminished placental trophoblastic VEGF immunoreactivity; (ii) weaker VEGFR-1 and -2 immunoreactivity in decidual vascular endothelium; (iii) reduced placental trophoblastic Tie-1 receptor immunoreactivity; and (iv) reduced decidual vascular endothelial Tie-1 and -2 receptor immunoreactivity. The absence of VEGFR-3 immunoreactivity in decidual vascular endothelium was also noted in all study groups. Interestingly, placental villi from the BO group presented blood vessel-like structures negative for von Willebrand factor, but positive for VEGF, VEGFR-1, -2, -3, Tie-1 and Tie-2 receptor. We conclude that the expression of these antigens may be altered in recurrent miscarriages.
recurrent miscarriage/Tie receptors/VEGF/VEGF receptors
Introduction
Of all women in early pregnancy, ~12–15% miscarry (Stirrat, 1990
). The occurrence of recurrent miscarriages, defined as three or more consecutive miscarriages, has been estimated to be from 0.3 to 0.8% of all diagnosed pregnancies (Houwert-de-Jong et al., 1989; Stirrat, 1990).
Recurrent miscarriages have a multifactorial aetiology (Li, 1998). Parental chromosomal rearrangements have been observed in ~9% of recurrent miscarriages (Houwert-de-Jong et al., 1989), and up to 20% of women experiencing recurrent miscarriages have uterine defects such as myomas, endometrial polyps or intrauterine adhesions at hysteroscopy (Houwert-de-Jong et al., 1989; Tulppala et al., 1993). Also, a disturbance in the maternal immunological response, e.g. the formation of antiphospholipid antibodies (Rai et al., 1995), as well as increased sharing of major histocompatibility complexes between couples (Thomas et al., 1985), have been proposed as aetiological factors for recurrent pregnancy loss. Yet it seems that the aetiology mostly remains open. It is not known, for example, whether abnormal changes in the balance of the utero–placental vascular development and growth would underlie recurrent miscarriages, even though adequate and appropriate vasculo- and angiogenesis are requirements for the successful continuation of pregnancy.
The vascular endothelial growth factor (VEGF) family of proteins constitutes of VEGF, VEGF-B, VEGF-C, VEGF-D and placenta growth factor, and their receptors VEGFR-1/Flt-1, VEGFR-2/KDR and VEGFR-3/Flt-4 (Eriksson and Alitalo, 1999; Ferrara, 1999; Persico et al., 1999). They play an essential role in fetal angiogenic development, as mice lacking the expression of VEGF or any of the three receptors die in utero due to inadequate vascular development (Dumont et al., 1998; Ferrara, 1999). They are also expressed in the placenta (Kaipainen et al., 1995; Clark et al., 1996; Vuorela et al., 1997), as is the Tie-1 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) receptor (Vuorela et al., 1997). Similarly, based on animal studies, lack of Tie-1, or its close relative Tie-2/tek (tunica interna endothelial cell kinase) expression results in vascular maldevelopment and intrauterine death (Sato et al., 1995).
The present study was designed to find out whether the expression of VEGF, VEGFR-1, -2 or -3 or the Tie-1 or Tie-2 receptors in the placenta or decidua is altered in recurrent miscarriage.
Materials and methods
Patients
All study subjects volunteered for the study and were recruited following informed consent and approval by the local ethics committee. A total of 18 women with a history of recurrent first trimester pregnancy loss (three or more consecutive miscarriages) participated. They had been investigated previously in terms of possible causes of miscarriage, but ended up with unexplained aetiology (Tulppala et al., 1993). Their present pregnancies were followed regularly at 1–2 week intervals by transvaginal ultrasound from 6–7 weeks gestation until `missed abortion' (MA, n = 12, fetal death) or blighted ovum (BO, n = 6, only a gestational sac present) was diagnosed. None of these patients had experienced any vaginal bleeding before diagnosis and subsequent evacuation. Women undergoing surgical termination of normally progressing pregnancies served as controls. Clinical data of the study subjects are given in Table I.
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Tissue samples
Tissue samples including placental, decidual, and endometrial structures were collected by curettage. Samples were immediately fixed in 10% neutral buffered formalin at 4°C overnight, dehydrated and embedded in paraffin. Tissue sections (5 µm) were cut onto slides coated with 3-aminopropyltriethoxy-silane (Sigma Chemical, Poole, UK).
Immunohistochemistry
Tissue sections were dewaxed, rehydrated, and incubated in 0.5% pepsin (Merck, Darmstadt, Germany) in phosphate-buffered saline (PBS; pH 7.4), and endogenous peroxidase activity was then quenched in 0.6% H2O2 (Perhydrol®; Merck) in methanol for 10 min at room temperature. The sections were blocked with normal swine serum for 30 min and reacted with polyclonal rabbit antibodies against VEGF (2 µg/ml), VEGFR-1 (2 µg/ml), VEGFR-3 (2 µg/ml), Tie-1 (2.5 µg/ml), or Tie-2 (2.5 µg/ml); or blocked with normal rabbit serum for 30 min at room temperature and reacted with monoclonal mouse antibodies against VEGFR-2 (2 µg/ml). Primary antibodies, all purchased from Santa Cruz Biotechnology (Santa Ana, CA, USA), were incubated at 4°C overnight. The sections were then incubated with biotinylated swine-anti-rabbit or rabbit-anti-mouse antibody (Dako A/S, Glostrup, Denmark), when appropriate, for 1 h at room temperature, followed by incubation with avidin–biotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) for 1 h more at room temperature. Between each step, excluding that between blocking serum and primary antibody, sections were washed three times for 5 min each in PBS.
Protein localization was visualized by aminoethylcarbazole (Sigma Chemical Co, St Louis, MO, USA). Sections were counterstained with Mayer's haemalum (Merck) and Aquamount Improved (BDH Laboratory Supplies, Poole, UK) was used as mounting medium.
Polyclonal rabbit antibodies against von Willebrand factor (5 µg/ml, Dako A/S) were used to identify blood vascular endothelial cells, monoclocal mouse antibodies against vimentin (0.65 µg/ml, Dako A/S) to identify cells of mesenchymal origin, and monoclonal mouse antibodies against a cytokeratin cocktail (cytokeratins 8, 14, 15, 16, 18 and 19) to identify trophoblasts (1:100, MAK®, Zymed Laboratories Inc, San Francisco, CA, USA). Non-immune immunoglobulin G (IgG) served as a negative control, and term placenta and haemangioblastoma tissues were used as positive controls (Clark et al., 1996; Hatva et al., 1996; Vuorela et al., 1997).
Tissue sections were analysed for protein localization and staining intensity was scored from negative (–) to faint (+), medium (++) and strong (+++) staining. All sections were analysed by two independent observers blinded to the origin of the sections.
Results
The staining intensity of von Willebrand factor, vimentin and the cytokeratin cocktail MAK® was equal in all study groups, indicating no general protein degradation and thus allowing comparisons of the staining intensities of the antigens under study.
Placental villi
In all study groups, faint VEGF immunoreactivity was observed in the villous stroma and blood vascular endothelium. In the MA and BO groups VEGF immunoreactivity was absent in the cyto- and syncytiotrophoblasts, whereas faint staining was observed in tissues from the healthy controls (Table II, Figure 1).
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Faint or no staining for VEGFR-1, -2 and -3 was observed in the stroma of the MA and BO groups, whereas in the controls the staining intensity ranged from faint to medium. In the vascular endothelium the staining intensity for VEGFR-1 and -2 was equal between the MA and control groups. That of VEGFR-3, however, was absent in the MA group, while faint staining was seen in the controls. The trophoblastic staining for VEGFR-1 was similar in all study groups. In all study groups, VEGFR-2 showed faint to medium immunoreactivity in the syncytiotrophoblast, whereas medium to strong staining was seen in the cytotrophoblast. VEGFR-3 immunoreactivity was absent in both the cyto- and syncytiotrophoblast, except for faint staining seen in the cytotrophoblasts of the MA group (Table II
, Figure 1).
The MA and BO groups showed faint to medium trophoblastic staining for Tie-1 and Tie-2 receptors, while in the control tissues medium to strong staining was observed. In the stromal cells, only faint Tie-1 receptor staining was seen in the MA and BO groups, whereas control tissues showed medium staining. Median stromal cell Tie-2 receptor staining in the BO group was opposed to faint staining in the other groups (Table II, Figure 2).
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Decidua
Decidual stromal cells showed faint VEGF and medium VEGFR-3 immunoreactivity in all study groups. Faint stromal VEGFR-1 staining in the MA and BO groups, as well as faint VEGFR-2 staining in the BO group, were opposed to medium staining in the control group. In all study groups, the vascular endothelium was negative for VEGFR-3, and in the MA and BO groups also for VEGF and VEGFR-1 (Table II
, Figure 1).
In the decidual stroma of the MA group Tie-1 and Tie-2 receptors showed faint immunoreactivity, but in the BO group medium immunoreactivity was shown (Table II, Figure 3). In the MA and BO groups the decidual vascular endothelium stayed negative for Tie-1 and Tie-2 receptors, whereas control tissues showed faint immunoreactivity (Table II, Figure 4).
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Endometrial glands
In the hypersecretory endometrial glands no VEGF staining was observed in the BO and MA groups, whereas control tissues were faintly positive (Table II
, Figure 1). The staining intensities of VEGFR-1 or Tie-1 or Tie-2 receptors did not differ between the study groups (Table II, Figures 1 and 3). Faint and medium VEGFR-2 staining in the MA and BO groups respectively, was opposed to strong staining in the control group. Faint VEGFR-3 immunoreactivity was observed in the BO group, whereas the other groups stayed negative (Table II, Figure 1).
Invading trophoblasts
In one sample of the control group we managed to see a massive invasion by trophoblasts into the decidual stroma. The identity of these cells was confirmed by positive staining for cytokeratin and negative staining for vimentin. This trophoblast population was strongly positive for Tie-1 and Tie-2 receptors but negative for VEGF, VEGFR-1, -2 and -3 (Figure 5).
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Blood vessel-like structures in placental villi
Interestingly, in placental villi of samples from the BO group, we observed blood vessel-like structures which were positive for all VEGF, VEGFR-1, -2, -3 and Tie-1 and Tie-2 receptors but, however, negative for von Willebrand factor (Table II
, Figure 6).
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Discussion
Apart from being a potent vascular endothelial mitogen, VEGF also maintains newly formed capillaries, induces vascular permeability and macrophage chemotaxis (Ferrara, 1999). It may thus give a novel aspect to the aetiology of recurrent miscarriages. In tissues of uncomplicated pregnancy, we observed VEGF immunoreactivity in the placental trophoblasts as well as in the decidua, thus confirming previous findings (Clark et al., 1996). However, the cyto- and syncytiotrophoblasts of women with MA or BO were negative for VEGF, giving support to the hypothesis of the role of VEGF in the pathology of miscarriage. Unfortunately we were unable to confirm the result by Western or Northern blotting due to small tissue volumes, but in view of animal data our results are not surprising (Ferrara, 1999).
In the placenta, vasculogenesis, i.e. the de-novo synthesis of new blood vessels, accounts for the majority of new vessel formation during the first trimester, and is initially observed around day 19 post-coitum (Demir et al., 1989). VEGFR-1 and VEGFR-2 have largely been regarded as specific for vascular endothelial cells, and are crucial for embryonic vascular development (Ferrara, 1999). Their mRNA signal has been detected in human placenta studied at 9 weeks gestation (Clark et al., 1996). In women with MA, no difference in the VEGFR-1 or -2 immunoreactivity in the placental vascular endothelium was observed, compared with the controls. However, in the decidual vascular endothelium, the MA group showed weaker immunoreactivity. It may thus be speculated that a possible association between recurrent miscarriages and altered expression of VEGFR-1 and -2 in the vascular endothelium would rather relate to the maternal decidua than to the placenta.
Interestingly, in the two trophoblast layers lining the placental villi, VEGFR-2 immunoreactivity was constantly stronger in the cytotrophoblasts underlying the syncytiotrophoblasts. Such a staining pattern has earlier been shown for VEGF and VEGFR-1 (Clark et al., 1996), whereas for these two antigens we observed equally strong staining in both types of trophoblasts. The reason for this controversy is not known but might, at least in the case of VEGF, be due to the different antibodies used. Altogether, these results may implicate a role for VEGF and its receptors in the function of trophoblasts, cells that secrete a wide range of proteins and hormones, and, therefore, have an important endocrinological and nutritional role (Ross and Reith, 1985; Talamentes and Ogren, 1988).
VEGF-C is a potent cytokine for the lymphatic vasculature, but it is also capable of inducing vascular endothelial cell growth (Eriksson and Alitalo, 1999). At the time of this study, antibodies against VEGF-C were not available for immunohistochemistry, but we showed placental expression of its receptor VEGFR-3 in stromal and vascular endothelial cells in tissues of both women with recurrent miscarriages as well as controls. Surprisingly, the endothelium of decidual blood vessels stayed negative for VEGFR-3 in all study groups.
Both Tie-1 and Tie-2 receptors were earlier considered blood vascular endothelial specific markers. Their expression is not, however, limited to the vascular endothelium, as Tie-1 is also expressed in fetal liver megakaryocytes and in the trophoblast cells of healthy term placentae, and Tie-2 in bone marrow haematopoietic stem cells (Vuorela et al., 1997; Partanen and Dumont, 1999). The importance of Tie-1 and Tie-2 in embryonic vascular development has been shown in mice (Partanen and Dumont, 1999). They might also play a role in recurrent miscarriages in humans as, compared to healthy controls, their expression was diminished in the placental villous trophoblasts and in the decidual vascular endothelium of affected women. Interestingly, Tie-1 and Tie-2 were also expressed in a trophoblast population invading the maternal decidua, whereas VEGF or VEGFR-1, -2 or -3 were not. Tie-1 and Tie-2 might thus also play a role in implantation, where the trophoblast invasion is a crucial event. The roles of the recently found Tie-2 agonist and antagonist ligands, angiopoietin 1 and angiopoietin 2 (Partanen and Dumont, 1999), also have to be evaluated in recurrent miscarriages in future.
In placental villi from the group of women with blighted ovum we observed lumens that morphologically resembled blood vessels usually seen in placental villi. Interestingly, however, these lumens were negative for von Willebrand factor, but positive for VEGF, the three VEGF-family receptors, and the two Tie receptors. One might speculate that in a pregnancy with blighted ovum the placental villous blood vessels have not matured enough to express von Willebrand factor. It is known, for example, that during embryogenesis in mice the expression of VEGF receptors, followed by the expression of Tie receptors, both precede that of von Willebrand factor, which is considered to be a marker of mature endothelium (Partanen and Dumont, 1999).
Taken together, our results imply that recurrent miscarriage might be associated with alterations in the expression of VEGF, VEGF receptors -1, -2 and -3, and Tie-1 and -2 receptors. Further research, most likely by animal models, is needed to find out the exact roles of growth factors and their receptors in recurrent miscarriages.
Acknowledgments
Anu Harju and Pia Lehtinen are thanked for skilful technical assistance, and Antti Huittinen for expert desktop publishing. Financial support was provided by the Clinical Research Institute, Helsinki University Central Hospital, the Finnish Foundation of Obstetric and Gynecologic Research, and by the Finnish-Norwegian Foundation for Medical Research.
Notes
4 To whom correspondence should be addressed 
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Submitted on September 27, 1999; accepted on December 16, 1999.
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