Molecular Human Reproduction, Vol. 5, No. 11, 1059-1065,
November 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of implantation |
Interleukin-10 receptors are expressed by basement membrane anchored, 
6 integrin+ cytotrophoblast cells in early human placenta*
1 Department of Obstetrics and Gynaecology, WHO Collaborative Centre for Research in Human Reproduction, Albert Szent-Györgyi Medical University, Szeged, H-6725, and 2 Department of Dermatology, Albert Szent-Györgyi Medical University, Szeged, H-6720, Hungary
Abstract
Cytotrophoblast cells produce interleukin (IL)-10 and express IL-10 receptor mRNA in culture. Furthermore, IL-10 dramatically reduces the synthesis of matrix metalloproteinase (MMP)-9 and the invasivity of cytotrophoblast cells in vitro, suggesting that an autocrine regulatory role in vivo is also possible. To test this hypothesis we investigated the expression of IL-10 receptor protein by first trimester cytotrophoblasts both in vitro and in situ, using flow cytometry and immunohistochemistry. Flow cytometric analyses demonstrated that 75–80% of cytotrophoblasts are able to bind labelled IL-10, suggesting that these cells possess IL-10 receptors in vitro. Serial sections of early human placentae stained for either 
5 and 6 integrin subunits, or for IL-10 receptors respectively, revealed that placental cytotrophoblasts possess cell surface IL-10 receptors not only in vitro, but also in vivo. IL-10 receptors were present mainly on 6 integrin expressing villous cytotrophoblast cells and on 6-positive cells of invasive cell columns located nearest the villous stroma. Differentiated trophoblasts (i.e. 5-positive cells and villous syncytiotrophoblasts) showed no reactivity. This differential expression of IL-10 receptors suggests that IL-10 might suppress the invasivity of undifferentiated cytotrophoblast cells, in vivo, preserving their non-invasive state in an autocrine manner. The possible involvement in cytotrophoblast proliferation and/or differentiation is also discussed.
cytotrophoblast/IL-10 receptor/integrins/placenta
Introduction
The human placenta is an important site of both the production and action of various cytokines and growth factors. Cytokines and growth factors play an important role in regulating implantation and placental development (Robertson et al., 1994
; Chard, 1995; Jokhi et al., 1997). Although numerous studies have been published on the biological role of cytokines in the regulation of critical reproductive events, such as trophoblast proliferation, differentiation and function, the complex autocrine, paracrine and juxtacrine regulatory mechanisms involving these molecules are still far from being completely understood. Among the various cytokines and growth factors the recently described pleiotropic cytokine interleukin-10 (IL-10) has been proposed to be a factor that might protect the semiallogeneic fetus from maternal allorecognition and rejection by driving the maternal (both local and systemic) immune reaction toward a T helper (Th)2-type immune response (Lin et al., 1993; Cadet et al., 1995).
IL-10 was described initially as a cytokine synthesis inhibitory factor (CSIF) that shifts the body's immune reaction away from an inflammatory response (Fiorentino et al., 1989; Howard et al., 1992; Moore et al., 1993; Mosmann, 1994). Although its serum concentrations were found to be stable throughout the menstrual cycle, once the pregnancy is established the circulating values of IL-10 rise significantly (Maskill et al., 1997). IL-10 inhibits proinflammatory cytokine production including IL-1ß, IL-6, IL-8, tumour necrosis factor (TNF)- and interferon (IFN)-
(Wang et al., 1994; Takeshita et al., 1996) therefore prevents the development of Th1-type immune reactions deleterious for both the establishment and maintenance of pregnancy (Wegmann et al., 1993; Ragupathy, 1997). Several studies have demonstrated that IL-10 can prevent naturally occurring fetal wastage in a murine model of immunologically-mediated spontaneous early pregnancy loss (Chaouat et al., 1995). Earlier studies (Szekeres-Bartho et al., 1996a, 1996b, 1997) stressed the role of IL-10 as a possible mediator of the immunological pregnancy-protective effect of progesterone.
In human pregnancies, the protective role of IL-10 has been demonstrated by several findings concerning the disregulation of IL-10 production in some reproductive pathologies, including recurrent spontaneous abortion (Hill et al., 1995; Marzi et al., 1996), intrauterine growth restriction (Heybourne et al., 1994) and intrauterine infection-associated preterm labour (Greig et al., 1995). Roth et al. were the first to describe that highly purified human cytotrophoblasts secrete physiological amounts of IL-10 in vitro, independently of gestational age (Roth et al., 1996). Furthermore, trophoblast-derived IL-10 was able to suppress the IFN- production of alloreactive lymphocytes in a mixed lymphocyte reaction, indicating that IL-10 may contribute to the placental immune protection of the semiallogeneic fetus in a paracrine manner. Recently, these authors (Roth et al., 1999) have shown that cytotrophoblast cells express IL-10 receptor mRNA in vitro suggesting that, as well as its role as a regulator of materno–fetal relationship, this cytokine might also possess autocrine regulatory properties. Furthermore, in their experimental conditions, IL-10 significantly decreased the matrix metalloproteinase (MMP)-9 expression of cytotrophoblast cells in culture both at the protein and mRNA level, suggesting that IL-10 is an autocrine inhibitor of cytotrophoblast MMP-9 activity and invasiveness. However, they did not provide any direct evidence for the expression of IL-10 receptor at the protein level by cytotrophoblast cells in vitro. Data concerning the placental localization of IL-10 receptor expressing cytotrophoblasts in vivo, that could specify more precisely the possible autocrine regulatory role of this cytokine are also missing. Therefore, in the present study we aimed to elucidate the placental localization of IL-10 receptor expression in vivo, to define the subset of placental cytotrophoblasts constituting the predominant target for the autocrine regulatory influence exerted by IL-10 during the first trimester of pregnancy.
Materials and methods
Isolation of first trimester human cytotrophoblast cells
Cytotrophoblast cells were isolated from first trimester placenta as previously described (Bischof et al., 1991). Trophoblastic tissue was obtained from legal terminations of pregnancies performed in accordance with the Hungarian Abortion Law at 6–12 weeks gestation. Villous tissue was dissected manually, rinsed and minced in Hanks' balanced salt solution (HBSS) containing 200 IU/ml penicillin and 200 mg/ml streptomycin (both from Sigma, Budapest, Hungary). The minced tissue was then incubated at 37°C four times for 20 minutes in Hanks' balanced salt solution (pH 7.4) containing 0.25% trypsin (Sigma), 50 U/mol deoxyribonuclease I (Sigma), 4.2 mmol/l magnesium sulphate (Sigma), 25 mmol/l HEPES (Sigma), and antibiotics (200 IU/ml penicillin and 200 mg/ml streptomycin). The supernatant containing the dissociated mixed placental cells was collected, and the trypsin activity was neutralized by addition of 10% fetal calf serum (FCS; Gibco, Life Technologies, Vienna, Austria). The neutralized supernatant was centrifuged at 800 g for 10 min and the resulting cell pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 25 mmol/l HEPES, 200 IU/ml penicillin, and 200 mg/ml streptomycin. The cell suspension obtained was placed immediately into an incubator and was maintained at 37°C until the end of the entire dissociation procedure. Concomitantly, the remaining villous tissue was subjected to another 20 min trypsinization step. At the end of the dissociation procedure the remaining villous fragments were discarded. The four fractions of cell suspensions were pooled, filtered over a 100 µm nylon mesh to remove remaining villous fragments, centrifuged at 800 g for 10 min, and resuspended in 2–3 ml of the same medium without FCS. This cell suspension was layered over a 5–70% preformed discontinuous Percoll gradient, according to a previously described method (Kliman et al., 1986). The fraction containing the cytotrophoblast cells (densities 1.048–1.062 g/ml) was washed and resuspended in DMEM. These Percoll-enriched cells were further used for flow cytometric analyses. The viability estimated by Trypan Blue exclusion was consistently >95%.
Sorting experiments using fluorescence as the method of detection and an anticytokeratin antibody specific for cytokeratins 5, 6, 8, 17 and probably also 19 (Clone MNF116; Dako A/S, Glostrup, Denmark), which within the placental villi stains only trophoblasts (Aboagye-Mathiesen et al., 1996), showed that the purity of Percoll isolated cells was ~75%.
Analysis of IL-10 receptor expression on first-trimester cytotrophoblasts
The expression of IL-10 receptor was determined for Percoll gradient-purified cytotrophoblasts using an IL-10 Fluorokine Kit (R & D Systems GmbH, Wiesbaden, Germany) according to the manufacturer's protocol. Briefly, the freshly isolated cells were incubated overnight at 37°C in serum-free culture medium, under conditions that did not allow them to attach (i.e. in solution, on a rocking platform or on an agarose-coated culture dish). Next day the cells were washed (in order to remove any residual growth factors present in the culture medium) and separated into two groups to which the biotin-conjugated IL-10 or the biotin-labelled control protein (soybean trypsin inhibitor) were added. After 1 h incubation at room temperature avidin–fluorescein isothiocyanate (FITC) was added, followed by an additional 1 h incubation, washing and resuspension in a specific buffer designed by the manufacturer to stabilize specific staining and minimize background. Analysis was conducted on a FACStarPLUS (Becton Dickinson, Erembodegem-Aalst, Belgium) flow cytometer with a 488 nm argon-ion laser excitation device. The cells were first analysed for forward and side scatter to gate out debris, and to select the cytotrophoblasts based on their relatively large size and primitive cytoplasmic structure.
Immunohistochemistry
The anti-IL-10 receptor monoclonal antibody, specific for the extracellular, ligand binding domain of the human cell surface IL-10 receptor, was obtained from R & D Systems. The mouse monoclonal antibodies to 5 (CD 49e, clone SAM1) and 6 (CD 49f, clone 4F10) integrin subunits were obtained from Immunotech (Coulter, Marseille Cedex, France) and Serotec (Kidlington, Oxford, UK) respectively. Negative control slides were stained with mouse immunoglobulin G (IgG) class-matched irrelevant antibodies, obtained from Dako. All antibodies and negative control reagents were diluted to a working concentration of 1 µg/ml in Tris-buffered saline (TBS), containing 0.1% Triton X-100 and 0.5% bovine serum albumin (both from Sigma).
Samples of first trimester placental tissue (6–12 weeks gestation, n = 12) were taken from routine vaginal terminations of pregnancies. Tissue fragments were immediately frozen in Cryomatrix (Shandon, Cheshire, UK). Six µm frozen sections were thawed, air-dried, fixed in acetone for 10 min at 4°C, and rehydrated in TBS, containing 0.1% Triton X-100. Slides were then incubated for 30 min at 4°C with human IgG (0.5 mg/ml, heat aggregated at 65°C for 20 min) (Jackson ImmunoResearch Laboratories Inc, West Baltimore Pike, USA), in order to block cell surface Fc receptors. Excess liquid was blotted without washing, and the sections stained for IL-10 receptors were incubated overnight at 4°C. Slides stained for 6 and 5 integrin subunits were incubated for 1 h at room temperature. The antigen-antibody reaction was revealed by using a StreptABComplex Duet (mouse and rabbit) Reagent Set (Dako). Briefly, the slides were incubated for 1 h at room temperature with the biotin-labelled second antibody (goat anti-mouse/rabbit Ig). After washing, avidin–horseradish peroxidase (HRP) was added and the incubation was performed in similar conditions as described for the second antibody. The peroxidase was developed with 3-amino-9ethyl-carbazol (AEC; Sigma). Finally the sections were washed in tap water, counterstained with haematoxylin, rewashed and mounted in Glycergel® aqeous mounting medium (Dako).
Results
Flow cytometric analysis of IL-10 receptor expression in vitro on first trimester cytotrophoblast cells
Binding experiments performed on Percoll-purified first trimester cytotrophoblasts using biotinylated IL-10 and avidin-FITC have demonstrated that >75–80% of the cells were able to bind the labelled cytokine, as indicated by the FACS histogram (Figure 1b). Cells were also stained with a monoclonal antibody against cytokeratins (clone MNF116). Of the cells, 75–80% showed positive staining (Figure 1d) relative to the isotype control (Figure 1c), indicating that the cytokeratin positive population (i.e. cytotrophoblasts) and IL-10 receptor expressing cells might be the same. Non-specific binding was ruled out by the negative control experiments, where the biotinylated IL-10 was substituted by biotin-labelled soybean trypsin inhibitor (Figure 1a). This protein is unable to bind specifically to any known cell surface receptor, therefore the fluorescence of these cells was identical to that of the background. For the specific binding, it was essential to maintain the cells in serum-free medium overnight under conditions that did not allow them to attach (i.e. in solution, on a rocking platform or on an agarose-coated culture dish). Freshly isolated cells, as well as cultured cells, treated with trypsin to remove them from the culture plates, did not bind labelled IL-10 at all (data not shown). This finding indicated that IL-10 receptors expressed by early human cytotrophoblast cells were trypsin sensitive.
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Immunohistochemical detection of IL-10 receptors in human first trimester placental tissues
Reactivity to IL-10 receptor specific monoclonal antibody was detected in all (n = 12) first trimester preparations. Figure 2
shows the result of a representative experiment. The most intense staining was observed in trophoblast cells forming the internal layer of villi (i.e. villous cytotrophoblast), whereas the external syncytiotrophoblastic layer was negative (Figure 2A). The cytotrophoblast cells which expand from the tip of anchoring villi shortly after implantation and form cytotrophoblast cell columns also reacted with the IL-10 receptor specific monoclonal antibody (Figure 2B). The most intense reactivity was observed in the proximal cells of these columns, located nearest the villous stroma. In contrast, cells located more distally stained only weakly, if at all. In all experiments, reactivity to IL-10 receptor specific antibody was localized to the cell membrane with no reactivity in the cytoplasm of the cells. Moreover, cytotrophoblast cells resting on the villous basement membrane (villous cytotrophoblasts and the first row of cytotrophoblast cell columns) stained intensely along the apical and lateral microdomains of their cellular membranes. This staining pattern suggested that cytotrophoblast cells expressed cell surface IL-10 receptors in a polarized manner. Cells of the villous stroma were always negative, showing that in the first trimester of pregnancy these cells did not express IL-10 receptors.
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Expression of
5 and 6 integrin subunits in human first trimester placentaIn order to specify more precisely the subsets of IL-10 receptor bearing cytotrophoblast cells in vivo, in parallel we have studied the pattern of integrin expression in our first trimester placental tissue preparations. Villous cytotrophoblast cells resting on villous basal lamina expressed
6 integrin subunit clustered along the basement membrane (Figure 2E
). The proximal cells of cytotrophoblast cell columns resting on the basement membrane of the villous tip have preserved this pattern of expression. In contrast, more distal but still closely packed cytotrophoblasts of cell columns expressed 6 integrin subunit in an unclustered way. Concomitantly, a gradual reduction of staining intensity towards the distal, deeply invasive cells was observed. Villous syncytiotrophoblasts and deeply invasive cells of cytotrophoblast cell columns were always negative. When serial sections stained for 5 integrin subunit were examined and compared with those stained for 6 integrin, a reciprocal staining pattern was observed: 5 integrin expression reached its maximum in distal cells and gradually decreased towards the proximal part of cell columns (Figure 2F). In villous cytotrophoblast, no staining for the 5 integrin subunit was seen. Discussion
In human placenta at least two major morphologically and functionally distinct cytotrophoblast populations can be identified. Villous cytotrophoblasts are polarized immotile cells anchored to the basement membrane of chorionic villi. They can differentiate by fusion to form the overlying syncytium which is in direct contact with maternal blood, mediating nutrient and gas exchange for the developing fetus, and representing the major endocrine component of human placenta. Extravillous, invasive cytotrophoblast cells rise from cytotrophoblasts that rest on the basement membrane surrounding the tip of anchoring villi. These cells differentiate by leaving their basement membrane and form non-polarized cellular aggregates (i.e. cytotrophoblast cell columns). They give rise to the invasive cytotrophoblast population, which invades the endometrium, its arterial system thus connects the developing fetus to the maternal circulation. No relevant differences were reported between basement membrane-anchored cells giving rise to the extravillous trophoblast and those underlying the villous syncytial trophoblast, therefore it is very likely that both kinds of cells represent a uniform cell population, which can equally differentiate in either of two major pathways already mentioned (Kaufmann and Castellucci, 1997).
Previous immunohistochemical (Damsky et al., 1992; Aplin, 1993; Bischof et al., 1993) and functional (Burrows et al., 1993) studies have shown that cytotrophoblast cells modulate their integrin repertoire during invasion of the endometrium. These authors have demonstrated that villous cytotrophoblast cells, resting on a laminin-rich basement membrane, express the integrin 6ß4 (a laminin receptor) in a clustered manner towards the basement membrane. When leaving their basement membrane at the tip of anchoring villi to form cell columns, they continue to express the integrin 6ß4, but in an unclustered way. Cytotrophoblasts located deeper in the placental bed have lost their capacity to express the integrin 6ß4, and instead express 5ß1 integrin, the major fibronectin receptor. Therefore, as trophoblast cells are gradually transformed from polarized villous epithelial layer into non-polarized extravillous population, a down-regulation of 6ß4 integrin expression with a reciprocal up-regulation of 5ß1 integrin occurs. Our immunohistological findings regarding the placental localization of 6 and 5 subunit expressing cytotrophoblast subpopulations fit these reports perfectly. This gradual switch from the basal lamina receptor 6ß4 to interstitial receptors such as 5ß1 was regarded as a mechanism by which invasive cytotrophoblast cells adapt to their successive environments (Bischof et al., 1996, 1997). These authors have shown that this integrin switch is paralleled by significant changes in the invasive behaviour of cytotrophoblasts (Bischof et al., 1995). If villous syncytiotrophoblast and deeply invasive extravillous cytotrophoblast cells are considered terminally differentiated forms of the same 6 integrin-positive cytotrophoblast stem cell, the above-mentioned integrin switch might equally be an expression of cellular differentiation.
The presence of IL-10 receptor mRNA in human placental cytotrophoblast cells has been demonstrated recently (Roth et al., 1999). However they have not offered any direct evidence, either for the presence of IL-10 receptor protein on these cells in vitro, or for placental localization of IL-10 receptor expressing cytotrophoblasts, in vivo. Our flow cytometric analyses clearly show the ability of cytotrophoblast cells to bind biotin-labelled IL-10. However, freshly isolated cells which had been exposed recently to the proteolytic activity of trypsin during the dissociation of placental villi, as well as cultured cells treated with trypsin to remove them from the culture plates, failed to bind IL-10. Binding was only seen when the cells were incubated overnight either in solution, on agarose or on a rocking platform in order to prevent their attachment to the culture dish. Throughout culture the use of FCS which might promote both attachment and differentiation was avoided. Under these conditions, cytotrophoblasts remained floating in the medium, but at least some of them formed small aggregates. Based on these observations we cannot exclude the possibility that for the recovery of IL-10 receptor, cytotrophoblasts have to make contact with each other. According to our best knowledge, these experiments provide the first clear-cut evidence for IL-10 receptor expression by cytotrophoblast cells in vitro.
Our immunohistochemical studies demonstrate that IL-10 receptor protein is expressed by early human cytotrophoblasts not only in vitro, but in vivo as well. The most intense staining for IL-10 receptors occurred on the polarized villous cytotrophoblast cells, and on the very proximal cells, resting on the basal lamina of the villous tip of cytotrophoblast cell columns. The remaining invasive cells stained much more weakly if at all, and villous syncytiotrophoblast did not stain. Our results also show that positive staining for IL-10 receptors and that for 6 integrin subunits largely overlap. In contrast, 5 integrin expressing cells appear to be devoid of receptors for IL-10. Therefore recent findings (Roth et al., 1999) could be interpreted as a mechanism by which basement membrane-anchored 6 integrin expressing cytotrophoblast cells, including villous cytotrophoblasts, suppress their own potentially invasive behaviour. It has not been clearly elucidated whether villous cytotrophoblast cells produce gelatinases or possess gelatinase coding mRNA in vivo, but logically they have no reason to be invasive. The up-regulation of MMP-9 production by cytotrophoblasts has been reported to occur after a 12 h culture period, at a time when IL-10 production by these cells ceases (Roth et al., 1996; Roth and Fisher, 1999). Since IL-10 is a potent inhibitor of trophoblastic MMP-9 synthesis in vitro, one could speculate that the gelatinase-secreting activity of cytotrophoblast cells starts later, when the cells escape from the suppressive effect of endogenous IL-10. Recent findings (Roth and Fisher, 1999) provide the first clear-cut evidence for the molecular mechanism which might be responsible for this sequence of events in vitro, whereas our results identify the target cell population for this IL-10-mediated autoregulatory mechanism in vivo.
It is interesting to note that the expression pattern of IL-10 receptor in early human placenta mirrors the proliferation marker Ki-67 expression reported previously (Mühlhauser et al., 1993). Therefore it is tempting to speculate that IL-10 might act as an autocrine factor in regulating placental growth either by promoting or by inhibiting cellular proliferation within the human trophoblast lineages. Reports providing evidence for either the proliferation-promoting or the antiproliferative effect of this cytokine in other cell types support this hypothesis (Ho et al., 1994; Michel et al., 1997; Yue et al., 1997; Seppanen et al., 1998; Wang et al., 1999). Taken together these studies suggest that the IL-10-dependent regulation of cellular proliferation might be cell-type-specific. However, signals involved by the IL-10/IL-10 receptor system in the regulation of cell-cycle progression that might give a comprehensive explanation for these presumably cell-type-specific differences are poorly understood. Therefore it is rather difficult to predict exactly how IL-10 would affect trophoblast proliferation.
The profound down-regulation of the IL-10 receptor as soon as 5 integrin expression is turned on might suggest that IL-10 could also affect the production of the integrin family of adhesion molecules which, in turn, impacts both cytotrophoblast proliferation and invasiveness. Another possibility is that IL-10 might play a role in cytotrophoblast differentiation. However, the possible involvement of IL-10 in these processes would have interesting implications from the perspective of the pregnancy-specific disorder pre-eclampsia, in which enhanced trophoblast proliferation, decreased invasiveness and the inadequate regulation of adhesion molecules have been reported (Zhou et al., 1993, 1997; Lim et al., 1997).
Near term and during parturition the effects of IL-10 are likely to be profoundly modulated by the pro-inflammatory mediators released in increasing concentrations by gestational tissues. A recent report (Denison et al., 1998) demonstrates that the basal production of monocyte chemotactic peptide (MCP)-1, IL-8 and Regulated on Activation and Normally T-cells Expressed and presumably Secreted (RANTES) by explants of fetal membranes, decidua and placenta at term greatly exceeds that of IL-10. Furthermore, prostaglandin E2, which might rise near term has been able to further augment this basal placental production of MCP-1 and IL-8 thus promoting a local inflammatory reaction and favouring parturition. Prostaglandin E2 has also been shown to stimulate release of IL-10 by perfused placental cotyledons thereby probably enhancing local immunosuppression and protecting the fetal allograft during the `high-risk' inflammatory process of parturition. Although the pattern of IL-10 receptor expression by various gestational tissues in the late pregnancy is not yet elucidated it seems logical to suppose that, as in keratinocytes, the overproduced IL-8 would suppress the expression of IL-10 receptors by placental cytotrophoblasts making them unresponsive to IL-10 (Michel et al., 1997). One of the multiple consequences this unresponsiveness might have is the possible elevation of MMP-9 production, which might contribute to the remodelling of the uterus and placenta in parallel with the accelerated growth of the fetus in the second half of pregnancy. A role favouring the separation of the placenta from the uterine wall after delivery is also possible.
Therefore IL-10 appears to serve important functions throughout pregnancy as well as during parturition. Beside its role in placental immune protection of the fetal allograft and in the regulation of cytotrophoblast invasiveness, IL-10 might be involved in basic reproductive events, such as placental growth, cytotrophoblast differentiation and tissue remodelling. However, further evidence is needed to support these hypotheses.
The overall conclusion of our immunohistochemical and cytological studies is that first trimester cytotrophoblast cells express IL-10 receptors both in vivo and in vitro. IL-10 receptor expression is localized mainly to the 6 integrin subunit-expressing cytotrophoblast cells resting on the basal lamina of both floating and anchoring villi, whereas differentiated trophoblasts (i.e. villous syncytiotrophoblast and 5 integrin expressing invasive cells) seem to be devoid of receptors. Therefore, we propose that one major autocrine role of IL-10 in vivo could be to suppress the invasive potential of basement membrane-anchored cytotrophoblast cells and to preserve their non-invasive state. Another interesting possibility is that in early pregnancy IL-10 might be an autocrine regulator of cytotrophoblast proliferation and/or differentiation.
Acknowledgments
We thank Dr Anna Kenderessy-Szabó and Dr Imre Földesi for their helpful advice. We also thank Dr Imre Ocsovszky for carrying out the flow cytometric analyses. We are greatly indebted to Dr Edit Olasz for her guidance in early phases of cytotrophoblast isolation. We also acknowledge the co-operation of Dr Attila Pál and the medical and nursing stuff at the Department of Pathological Pregnancy in providing abortion material. Finally we express our thanks to Ms Edit Gordos and Mrs Katalin Hudák for technical assistance. B.J.SzoISOdia''ny is a recipient of a Ph.D. fellowship from the Hungarian Ministry of Education. This work was supported by grants from the National Scientific Research Found (OTKA Nr. T017031) and from the Hungarian Ministry of Welfare (Nr. K25 ETT II.)
Notes
* Part of the results described here were presented at the XI Congress of Perinatal Medicine, XIX Alpe Adria Meeting held on 3–4 October 1997, Alsópáhok-Hévíz, Hungary 
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, WHO Collaborative Centre for Research in Human Reproduction, Albert Szent-Györgyi Medical University, H-6725 Szeged, Semmelweis u. 1. Hungary
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Submitted on April 26, 1999; accepted on August 17, 1999.
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