Leukocyte populations, hormone receptors and apoptosis in eutopic and ectopic first trimester human pregnancies

Lore Marx1, Petra Arck1,2,3, Michaela Kapp1, Christian Kieslich2 and Johannes Dietl1

1 Department of OB/GYN, Julius-Maximilians University Würzburg and 2 Charité, Campus Virchow Klinikum, Humboldt University Berlin, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The implantation of trophoblast cells at extrauterine sites still results in decidualization. The objective of the present study was to compare decidualization at eutopic and ectopic implantation sites. Tissues from women undergoing elective termination of uterine pregnancy and from women with ectopic pregnancy were used to detect the presence of cells important for the maintenance of pregnancy, such as BCL-2+, CD56+, CD3+, CD8+ and CD68+ cells, and the presence of oestrogen (ER) and progesterone receptors (PR) by immunohistochemistry. In-situ detection of fragmented DNA was performed to identify apoptotic cells. The percentage of CD3+ cells among all immunocompetent cells in the tubal epithelium was 46.6% (39.9% of CD3+ were also CD8+); the other 53.4% were CD68+ cells. CD56+ cells were undetectable in ectopic decidua at the feto-maternal interface in ectopic tissue. In uterine decidua, we found 29.9% CD3+ cells (2.2% of CD3+ were CD8+), 51.6% CD56+ cells and 18.5% CD68+ cells. The ratio of BCL-2+ to CD3+ cells in ectopic pregnancy was 0.41. In uterine pregnancy, the ratio of BCL-2 to CD3 was 0.44 and 0.39 for CD56. Tissues from both ectopic and uterine pregnancies were positive for PR. Fewer apoptotic cell bodies were present in ectopic pregnancy. The use of tissue obtained from ectopic pregnancy may become an excellent model to identify the mechanism of trophoblast invasion in eutopic pregnancies.

Key words: apoptosis/ectopic pregnancy/leukocytes/progesterone receptor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In human reproduction, the fertilized oocyte develops into an embryo which normally attaches to the uterine tissue from which it obtains nutrition sufficient to develop to maturity (Bell, 1985Go). Trophoblast cells, which form the outer layer of the preimplanting blastocyst, attach to the uterine epithelium, invade or destroy the epithelial barrier, and ultimately establish direct contact with maternal blood and maternal uterine lining stromal cells (Enders, 1991Go). The latter rapidly differentiate into large glycogen-filled cells called decidual cells (Bell, 1985Go). Enders (1991) has proposed that decidualization represents a key element in creating a relationship that allows a successful pregnancy to occur.

In ectopic pregnancies, the fertilized ovum implants into the Fallopian tube (Breen, 1970Go). Immunohistochemical characterizations have suggested that ectopic pregnancy represent abnormal implantation of a morphologically normal blastocyst, giving rise to immunologically normal and hormonally active trophoblast cells (Vassiliadou and Bulmer, 1998Go). On the other hand, decidualization cannot occur normally in the epithelium of the Fallopian tube (Earl et al., 1987Go; Maruyama et al., 1992Go; Stewart-Akers et al., 1997Go). Therefore, this pathological situation provides an excellent model in helping us to understand immunological reactions at the eutopic feto-maternal interface during early gestation.

Decidualized endometrium is more complex than just a collection of enlarged glycogen-filled stromal cells. There are immunocompetent cells in direct contact with trophoblast cells expressing paternal antigens (Clark, 1991Go; King et al., 1997Go). The decidua contains intraepithelial lymphocytes, and in the stroma, T cells, B cells, CD56+ natural killer (NK) cells, macrophages and mast cells are present (Bulmer et al., 1988Go; King and Loke, 1991Go; Hunt 1994Go; Clark 1995Go; Marx et al., 1999Go). There are changes in the composition of decidual tissue as pregnancy progresses, differences between species, and differences depending on the nature of the stimulus initiating decidualization, like that given in ectopic pregnancies (Pace et al., 1991Go; Chernyshov et al., 1993Go; Clark, 1995Go).

Programmed cell death by apoptosis has been accepted as a mechanism for maintaining tolerance in the immune system. Expression of BCL-2 and Fas ligand (FasL) by human trophoblast cells has been proposed as a mechanism providing protection against the lytic action of decidual immune cells (Hunt et al., 1997Go; Lea et al., 1997aGo; McLaren et al., 1997Go; Jones et al., 1998Go; Quenby et al., 1998Go). However, very little is known about the protection mechanism of decidual cells for FasL-mediated apoptosis. Therefore, it was an aim of the present study to investigate decidual cells for the presence of BCL-2. The proto-oncogene product BCL-2 is associated with the inhibition of apoptosis (Nunez et al., 1994Go). BCL-2 immunopositive cells have been identified in non-pregnant endometrial stroma and glandular epithelium (Koh et al., 1995Go; Rodger et al., 1995Go; Tabibzadeh et al., 1995Go). Lea et al. (1997a) have shown that during first trimester healthy pregnancies, a majority of stromal BCL-2+ cells were CD56+ large granular lymphocytes. We performed double staining immunohistochemistry for various decidual and tubal leukocytes and examined their expression of BCL-2. In addition, we examined the tissue for apoptotic cell bodies by in-situ detection of fragmented DNA. The following experiments were designed to investigate if the distribution of immunocompetent decidual cells, hormone receptor expression and apoptosis show similarities between intrauterine decidua, reflecting normal invasion of trophoblast, and ectopic decidua. This may help us to improve our understanding of the mechanism underlying trophoblast invasion in normal or failing pregnancies.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
All formalin-fixed, paraffin-embedded tissues were retrieved from archive files of the Department of OB/GYN, University of Würzburg, Germany. Patient data are summarized in Table IGo. The study protocol was approved by the ethics committee of the University of Würzburg. First trimester intrauterine decidual biopsies were obtained from healthy female patients undergoing elective termination of normally progressing pregnancies (n = 10). Tubal implantation sites from patients who had an ectopic pregnancy were obtained from therapeutic terminations by salpingectomy (n = 10). All tissue was chosen after histological assessment of sections stained with haematoxylin and eosin, and we exclusively used fresh tissue samples; any histological signs of tissue damage such as severe acute inflammation, necrosis, old blood clots or infections were excluded from the present study. Intrauterine tissue from women with ectopic pregnancies was not available for the present study, since the evacuation of the uterus in the case of ectopic pregnancy does not have therapeutic consequence for the patient and is therefore not a standard procedure in Germany.


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Table I. Demographic details of biopsies
 
Biopsies
All tissues were routinely fixed in 6% neutral buffered formalin and embedded in paraffin. For each patient we examined two different sections of tissue. Sections were cut 2 µm and stained with a monoclonal antibody against pancytokeratin (Immunotec, Hamburg, Germany) to confirm the presence or absence of invasive trophoblast and proliferating endometrial glands. On this basis, the intrauterine decidua was classified as decidua basalis (invasive trophoblast or proliferating glands present) or decidua parietalis (no trophoblast or proliferating glands). Cytokeratin staining of salpingectomy samples revealed either tubal implantation sites or tubal mucosa away from the implantation site. Published data have shown that there is no difference in the number or distribution of immunocompetent cells with regard to the absence or presence of trophoblast. (Vassiliadou and Bulmer, 1998Go). Therefore we pooled all our ectopic pregnancy samples in one group. Consecutive slides were stained with monoclonal antibody against CD56 (Novo Castra, Hamburg, Germany), CD3, CD8, CD68, BCL-2 (all from Dako, Hamburg, Germany) and progesterone receptor (PR) and oestrogen receptor (ER) (Immunotec) and double stained for CD56, CD3 and BCL-2 respectively. To confirm that we were dealing with two separate populations, double staining for CD56 and CD3 was also performed.

Immunolocalization of single staining for cytokeratin (CK), CD56, CD3, CD8, CD68, BCL-2, ER, PR
Tissue sections were dewaxed in xylene and rehydrated through a descending ethanol series. Non-specific endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in methanol for 30 min at room temperature. All stainings required microwave boiling in citrate buffer pH 6.0 for 15 min. Tissue sections were then washed with Tris-buffered saline (TBS; pH 7.4) for 2x5 min and exposed to a non-immune, serum-free protein block (Dako). Monoclonal mouse anti-human cytokeratin antibody was applied to the different sections at a dilution of 1 in 100 for 60 min at room temperature; on different sections anti-CD3 was used at 1:75, anti-CD8 at 1:25, anti-CD68 at 1:75, anti-BCL-2 at 1:40, and anti-PR and anti-ER at 1:50. This was followed by biotinylated multilink anti-mouse antibody for 15 min at room temperature, and an avidin–biotin–peroxidase detection system (LSAB-Kit; Dako) for a further 15 min at room temperature. Finally, the sections were developed with diaminobenzidine (DAB; Sigma, Munich, Germany) for 5 min to generate a brown-coloured product and lightly counterstained with haemalaim (Roth, Munich, Germany) prior to mounting. Negative controls were performed by replacing the primary antibody with a serum-free protein block (Dako) at the same concentration as the primary antibody.

Immunolocalization of double staining for BCL-2, CD56, CD3
Staining with mouse monoclonal antibody to CD56 or CD3 was performed as described earlier in this paper and followed by multi-link antibody. The same avidin–biotin detection kit as in the single staining was used, and developed with DAB to generate a brown-coloured product. Mouse monoclonal antibody to BCL-2 was incubated overnight at 4°C at a dilution of 1:40, followed by biotinylated multi-link antibody. Then the avidin–biotin peroxidase system was applied and developed with Vector VIP substrate kit (Linaris, Bettingen, Germany), which generated a purple-coloured product. For the CD3/CD56 double staining the same protocol was used except that anti-CD56 was used instead of anti-BCL-2. Negative controls were carried out by replacing either BCL-2 antibody or CD56 and CD3 respectively by normal mouse IgG, or by replacing both primary antibodies with normal mouse IgG.

In-situ cell death detection
The in-situ detection of fragmented DNA was performed on one slide of all samples used. We used a commercially available kit (TUNEL test kit; Boehringer Mannheim, Germany) and performed the assay according to the guidelines of the distributor with the following modification: after step 8, we washed in PBS/Triton 100 twice for 5 min at 60° C and incubated in PBS plus 10% fetal calf serum for 15 min at room temperature, instead of washing in PBS, as recommended by the distributor.

Evaluation
The evaluation of immunohistochemistry, double immunostaining and detection of apoptotic cell bodies was performed by two independent observers using a light microscope with scaled eye pieces. The number of positive cells/mm2 tissue was counted at a magnification of x25.

Statistics
Mean scores and SD of all samples were calculated for absolute and relative cell numbers within the same groups. Statistical analysis was performed on relative cell numbers using Student's t-test, and significance was set at P <= 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As shown in Table IIGo, in normal pregnancy decidua we observed 157.3 ± 42.7 CD3+ T cells/mm2 in tissue from uterine pregnancy and 95.1 ± 11.9 in tissue from ectopic pregnancy. Among the total number of CD3+ T cells in uterine pregnancy, 3.5 ± 2.4 cells/mm2 were also positive for CD8, which has been determined by making the simplifying assumption that CD8 is expressed only by CD3+ cells. Although Chernyshov et al. (1993) demonstrated by flow cytometry that the majority of decidual CD8+ lymphocytes expressed CD56, we could not confirm these observations by double staining immunohistochemistry for CD3 and CD56. In addition, double immunohistochemical labelling, as published by Lin et al. (1991), where the main focus was to demonstrate the co-expression of perforin and CD56 cells, also revealed that CD56+ cells express the T-cell surface antigens CD2 and CD3 (which contradicts our results), but not CD4 or CD8 (which contradicts Chenychow et al.). The CD8+ cell numbers we observed seem surprisingly low, compared to published data on endometrial or decidual T lymphocytes, where usually ~10% CD8+ cells among all immunocompetent cells have been described (Gulan et al., 1997Go). One explanation might be that quantitative data on cell distribution have been assessed by flow cytometry, where blood contamination generates disturbances of the actual decidual cell proportion. In the slides of ectopic tissue stained for CD8 in the same experiment, we found much higher numbers of CD8+ cells, therefore we believe in the validity of our results. In the case of ectopic pregnancy, we observed 37.9 ± 11.6 CD8+ T cells/mm2 among the total number of CD3+ cells. CD56+ cells were only present in uterine pregnancy at 276.2 ± 20.3 cells/mm2; like other authors we observed a complete lack of CD56+ cells in tissue from ectopic pregnancy (Vassiliadou and Bulmer, 1998Go). Further, we counted 98.6 ± 46.2 CD68+ cells/mm2 tissue from uterine pregnancy and 109.8 ± 42.1 CD68+ cells/mm2 in ectopic pregnancy. The cells were evenly distributed among the stroma and the basal glands of the epithelium; however, islands of all-stained cells could be observed in the myosalpinx and were not included in the quantitative evaluation at the microscope. There was no difference between the tubal implantation site and the tubal mucosa away from the implantation site, as already described by Bulmer et al. (1990).


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Table II. Absolute cell numbers/mm2 tissue
 
In order to obtain comparable results between uterine pregnancy and ectopic pregnancy, we calculated the percentages of each cell population by using the sum of CD3, CD56 and CD68 cell counts, as depicted by Figure 1Go. In tissue from patients with salpingectomy due to ectopic pregnancy, the percentage of CD3+ T cells was 46.4, and 39.8% of the CD3+ cells were positive for CD8. In addition to the lack of CD56+ cells, we calculated 53.6% CD68+ cells. In uterine pregnancy, we observed 29.5% CD3+ T cells (of which 2.2 % were CD8+ T cells), 51.9% CD56+ cells and 18.5% CD68+ cells, this distribution is already well established for uterine pregnancy. The differences betwen ectopic pregnancy and uterine pregnancy of all cell populations were statistically significant.



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Figure 1. Percentage of immuncompetent cells in uterine pregnancy (UP) decidua and ectopic pregnancy (EP) tubal epithelium. One star (*) shows statistically significant increase of each investigated cell type between UP and EP, P <= 0.05. Two stars (**) show a statistically significant reduction of percentages between UP and EP. Striped bars within the CD3+ T cells reflect the percentage of CD8+ T cells among the total cells.

 
The ratio of BCL-2+ cells to CD3 or CD56 respectively is shown in Figure 2,Go the corresponding immunohistochemistry is depicted in Figure 3AGo–D. In uterine pregnancy the ratio of BCL-2+/CD3+ cells was 0.44 and 0.39 for BCL-2 to CD56. We compared these results to ectopic pregnancy tissue and observed a ratio of 0.41 for BCL-2+/CD3+ cells. Due to the lack of CD56+ cell in ectopic pregnancy, the BCL-2 ratio for this population could not be calculated. We also observed BCL-2 expression on syncytiotrophoblast, suggesting protection from apoptosis, as described by the group of G.Vince (Quenby et al., 1998Go). However, since our main focus was to determine differences between ectopic pregnancy and uterine pregnancy, the majority of our tissue samples contained decidua or tubal epithelium; syncytiotrophoblast was only occasionally present and the observations regarding its immunohistochemistry were therefore not included in the present study.



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Figure 2. Ratio of BCL-2+ cells to CD3+ or CD56+ cells respectively in uterine pregnancy (UP) decidua or ectopic pregnancy (EP) tubal epithelium.

 


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Figure 3. Immunohistochemical analysis of human eutopic (A, C, E, G) and ectopic (B, D, F, H) decidua tissue. (A) CD3/BCL-2 double staining on eutopic pregancy decidua; open arrow heads depict BCL-2 single positive cells, solid arrow heads show CD3 single positive cells and half-filled arrow heads point to BCL-2/CD3 double positive cells. (B) Corresponding staining as in A on ectopic tubal epithelium. (C) CD56/BCL-2 double staining on eutopic pregancy decidua, open arrow heads depict BCL-2 single positive cells, solid arrow heads show CD56 single positive cells and half-filled arrow heads point to BCL-2/CD56 double positive cells. (D) Corresponding staining as in A on ectopic tubal epithelium. No CD56+ cells were present. (E) Progesterone receptor (PR) positive cells (brown staining) in eutopic pregnancy decidua and (F) ectopic pregnancy tubal epithelium. (G) Oestrogen receptor (ER) positive cells (brown staining) in eutopic pregnancy decidua and (H) ectopic tubal epithelium. Scale bars: AD = 30 µm; EH = 50 µm.

 
We obtained conflicting data analysing the TUNEL assay, as summarized in Table IIIGo. We only observed pieces of fragmented DNA in two out of 10 samples from patients with ectopic pregnancy; here we counted 6.3 ± 4.1 positive signals/mm2 tissue. In four out of 10 samples from patients with uterine pregnancy, we observed apoptotic cell bodies with 94.8 ± 51.1 signals/mm2 tissue. The presence or absence of fragmented DNA was random and could not be related to either the age of gestation or number of cell populations investigated. Due to the small number of positive samples we did not perform statistics on the TUNEL assay results.


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Table III. Detection of fragmented DNA (TUNEL test)
 
From all the results published on leukocyte distribution in ectopic pregnancy and uterine pregnancy, one might conclude that hormones are playing a major role in regulating decidualization in uterine pregnancy and fail to initiate decidualization and inhibit the presence of CD56 cells in ectopic pregnancy tissue. We therefore performed immunohistochemistry to detect the presence or absence of PR and ER in our pool of ectopic pregnancy and uterine pregnancy tissue. Interestingly, we observed the presence of PR in tissue from both ectopic pregnancy and uterine pregnancy, as shown in Figure 3E,FGo. PR positive cells were evenly distributed in the stroma of eutopic decidua, as well as in the stroma of ectopic pregnancy. Interestingly, the tubal epithelium was highly positive for PR. Staining for ER led to very faint immunohistochemistry signals (Figure 3G,HGo).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study we observed a shift of leukocyte distribution in favour of CD3+, CD8+ T cells and macrophages in the epithelium of tubal pregnancies. A complete lack of CD56+ NK cells characterized the tubal epithelium, whereas CD56+ NK cells are the most abundant population in uterine pregnancy. The first report on immunocompetent cells in ectopic pregnancy tissue and uterine pregnancy decidua was published by Bulmer and Earl in 1987. They observed leukocyte infiltration at the ectopic implantation site in cases of early tubal pregnancy and characterized these cells as HLA-DR positive macrophages and mature T lymphocytes. Interestingly, it has been shown that no differences were detected in the number or proportion of eutopic decidual leukocytes in women who had a normal pregnancy compared with those who had an ectopic pregnancy (Bulmer and Earl, 1987Go). The evacuation of the uterus in the case of an ectopic pregnancy is not routine procedure in Germany, therefore we do not have additional data on this topic for patients with an ectopic pregnancy.

In ectopic tubal pregnancy, fetal trophoblast cells show an identical reaction pattern with regard to their MHC antigen expression in comparison to that seen in intrauterine pregnancy, suggesting that ectopic implantation is not related to an inherent immunological abnormality of fetal trophoblast (Earl et al., 1985Go). Therefore we suggest that the increase in CD3 and CD68 positive cells is a sign of inflammation due to the mechanical irritation of the Fallopian tube through the implanted and growing embryo. However, the increased proportion of CD8+ cells among the total number of T cells is striking. CD8+ T cells are thought to be pregnancy-protective in humans, as well as in mice (Arck et al., 1996Go; Szekeres-Bartho and Wegmann, 1996Go). Recent data have shown that peripheral CD8+ lymphocytes with PR of women during early pregnancy also bear the g{delta} T cell receptor. These cells produce progesterone-induced blocking factor (PIBF) which contributes to the success of gestation via cytokine-mediated inhibition of NK activity (Szekeres-Bartho et al., 1997Go; Barakonyi et al., 1998Go).

It has been suggested that decidual CD56+ NK cells may control key events in trophoblast migration and placentation (King and Loke, 1991Go). However, CD56+ NK cells are absent from the tubal implantation site, as demonstrated by the present study and others (Maruyama et al., 1992Go; Vassiliadou and Bulmer, 1998Go) These results suggest that sufficient placentation and invasion may also occur in the absence of CD56+ cells, since in ectopic pregnancy we observe a lack of CD56+ cells but progressing pregnancy until the mechanical rupture of the Fallopian tube.

The immunomodulation of CD56+ cells is most likely not induced by the local presence of trophoblast due to the lack of CD56+ cells in the tubal epithelium in the case of ectopic pregnancy. The data published by others and presented in this study allow us to postulate that CD56+ cells are relatively inactive in the endometrium. A stimulus, other than invading trophoblast cells, might induce immunomodulation. This certainly requires receptor(s) which are present in the intrauterine decidua and absent in the tubal epithelium. It is quite possible that hormones may induce stimulation of CD56+ immunocompetent cells in the decidua. It has been shown that the number of stromal leukocytes in human endometrium varies during the menstrual cycle and, in particular, CD56+ cell numbers increase in the late secretory phase (Jones et al., 1995Go). The stimulus responsible for these cyclic changes is unknown, but it is likely that steroid hormones such as oestrogen and particularly progesterone play a role. The investigation of steroid hormone receptors on leukocytes in non-pregnant and pregnant human endometrium demonstrated that, despite the prominence of CD56+ endometrial granulated lymphocytes in late secretory phase endometrium and early pregnancy decidua, there was no expression of either PR or ER by these cells or other endometrial leukocyte populations. Rather than acting directly, steroid hormones are more likely to influence endometrial leukocyte populations indirectly via products of endometrial stromal or epithelial cells that express steroid hormone receptors (Stewart et al., 1998Go).

Progesterone is secreted by trophoblast cells; the production can be doubled in vitro in the presence of pure HCG (Yagel et al., 1989Go). Assuming that both ectopic and eutopic trophoblast produces progesterone, and keeping in mind that CD56+ NK cells do not express PR, we evaluated the distribution of PR in ectopic pregnancy and uterine pregnancy tissues and observed a high density of PR positive cells in both types of tissue. ER and PR expression in Fallopian tubes had been investigated by Land and Arends (1992) using ectopic pregnancy tissue; no PR expression was seen in glandular epithelium or stroma of the Fallopian tube and it was postulated that the absence of PR is correlated to poor decidualization. We believe that the improved availability of more specific antibodies to PR led to the results we observed with regard to PR expression in ectopic pregnancy and uterine pregnancy.

Cell death by apoptosis is regarded as an important feature in reproductive biology. Recent reports have suggested that BCL-2, a proto-oncogene responsible for the suppression of apoptosis, is expressed in the endometrium and may be involved in the regulation of menstruation (Koh et al., 1995Go). On the other hand, the presence of BCL-2 on decidual CD56+ cells has recently been described, resulting in the hypothesis that the CD56+ population has been selected for survival during early pregnancy (Lea et al., 1997aGo). In the present study it was therefore our aim to identify the phenotype of the remaining BCL-2+ cells in the decidua. Our data suggest that BCL-2 expression among the decidual leukocyte population is not restricted to CD56+ cells since we observed comparable percentages of BCL-2/CD3 double positive cells. The co-expression of BCL-2 with CD56 or CD3 may be a reflection of a normal cell turnover. If BCL-2 expression would only be detectable on `selected cells', further studies are needed to identify the mechanism of selection for the various BCL-2+ cells in the human decidua.

Only very few data are currently available on apoptosis in reproduction (Hunt et al., 1997Go). We observed apoptotic cell bodies in the stroma of eutopic early pregnancy decidua. The presence of fragmented DNA as a sign of apoptosis might be attributed to the presence of tumour necrosis factor (TNF)-{alpha} in the decidua (Tabibzadeh et al., 1995Go). Induction of apoptosis in decidual epithelium may also be related to the amount of TNF-{alpha} present at the feto-maternal interface (Lea et al., 1997bGo). In ectopic pregnancy we observed fewer apoptotic cell bodies, which supports recently published data (Kokawa et al., 1998Go). These apoptotic differences between ectopic pregnancy and uterine pregnancy may be related to the increase in CD8+ cells producing Th2 type cytokines in ectopic pregnancy.

The results discussed in the present paper suggest that reproductive biologists should refrain from the hypothesis that CD56+ confers privilege on foreign embryonic tissue. Normal placentation appears to be possible in the absence of these cells, as demonstrated by the use of tissue from ectopic pregnancies lacking CD56+ cells. In addition, the expression of BCL-2, which had previously been thought to be present only on `selected' CD56 cells, was also detectable on CD3 T cells. Rather, the results presented in this paper allow us to conclude that CD8+ cells may be of particular importance during early pregnancy whereby further experiments are neccessary to identify their Th1/Th2 and TcR {alpha}ß or {gamma}{delta} phenotypes. In addition, the use of tissue obtained from ectopic pregnancies has been demonstrated to be an excellent model to identify the mechanism of trophoblast invasion in normal pregnancies.


    Acknowledgments
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft.


    Notes
 
3 To whom correspondence should be addressed at: Charité, Campus Virchow Klinikum, Medizinische Klinik mit Psychosomatik/Biomedizinisches Forschungszentrum, Augustenburger Platz 1, 13353 Berlin, Germany Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on September 2, 1998; accepted on December 11, 1998.