1 Department of Gynaecology and Obstetrics, University of Vienna, Waehringer-Guertel 1820, 2 Department of Medical Statistics, University of Vienna and 3 Bone and Biomaterials Research Laboratory, Institute for Histology and Embryology, University of Vienna, Schwarzspanierstrasse 17, 1090 Vienna, Austria
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Abstract |
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Key words: Bcl-2/ectopic pregnancy/immunohistochemistry/tubal rupture
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Introduction |
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In the regulation of apoptosis the bcl-2 protein favours cell survival and thus inhibits apoptosis, while in contrast, other members of the bcl-2 family such as bax and bcl-xs appear to be proapototic. During the apoptosis cascade, the proapoptotic proteins of the bcl-2 family regulate execution caspase activation and thus initiate irreversible progression of apoptosis, a process known as a `point of no return' (Huppertz et al., 1999). Bcl-2 is a 25 kDa protein, which is localized at the inner mitochondrial membrane, the endoplasmic reticulum, the nuclear envelope and the plasma membrane, and is often topographically restricted to long-lived or proliferating cell zones (Hockenbery et al., 1990
). While bcl-2 acts by protecting the cell against apoptosis, high bcl-2 expression extends cell survival and proliferation (Wyllie, 1994
; Marzioni et al., 1998
).
Bcl-2 has already been immunolocalized in the syncytiotrophoblast of the chorionic villi in human placenta from the first to the third trimester; undisturbed regulation of apoptosis has been supposed to be of importance for implantation, early pregnancy, and the maintenance of trophoblast mass during later pregnancy, while disturbed regulation of apoptosis has been suggested to be associated with pregnancy failure (Sakuragi et al., 1994; Marx et al., 1999
; Toki et al., 1999
). While bcl-2 was immunolocalized at the maternalfetal interface in healthy and failing pregnancies, it was shown that bcl-2 immunostaining was greater in viable pregnancies (Lea et al., 1997
, 1999
). Tissue obtained from ectopic pregnancy has always been proposed as an excellent model to identify the mechanism of trophoblast invasion although, according to recent evidence (Marx et al., 1999
) in ectopic pregnancy, fewer apoptotic cell bodies were present as compared with eutopic pregnancy. Unlike in normal pregnancy, apoptosis has been shown to occur in the villi but not in the decidua of tubal ectopic pregnancies (Kokawa et al., 1998b
), which suggests that the implantation site might affect the occurrence of apoptotic changes in early pregnancy of humans. As apoptosis seems to be intensified in cases of spontaneous abortion compared with normal pregnancies (Lea et al., 1997
; Kokawa et al., 1998a
), it is postulated that invasiveness of ectopic trophoblast towards the muscularis zone of the tubal wall, consequently leading to tubal rupture, might also be due to disturbed regulation of apoptosis, such as high antiapoptotic activity and, thus, unlimited cell survival and proliferation of ectopic trophoblast.
This preliminary study was designed to investigate the quantitative immunolocalization of bcl-2 and thus the regulation of apoptosis during first-trimester ruptured and non-ruptured human tubal ectopic pregnancies. We wanted to find out whether bcl-2 protein expression was disturbed in cases of ruptured tubal pregnancies. Knowledge about bcl-2 expression in ruptured and non-ruptured tubal ectopic pregnancies was thought to be a first step in the establishment of a circulating marker for tubal rupture in the future. As efforts towards the identification of a prognostic marker for tubal rupture have already been made, though mainly on retrospective evaluation of patient's characteristics (Job et al., 1999; Mol et al., 1999
), the aim was to elucidate this most threatening complication of ectopic pregnancy on the immunohistochemical level.
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Materials and methods |
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The study was carried out in accordance with the 1975 Helsinki Declaration on Human Experimentation and approved by the Ethics Committee.
Immunohistochemistry
Serial sections (5 µm thick) of paraffin-embedded specimens were cut and then mounted on glass slides. All specimens were cut in one sequence and stained in a single reaction to ensure standard thickness and comparability of colour development. The sections were deparaffinized using xylene (Merck, Germany) and rehydrated in graded alcohol series (100 to 70%). Then they were treated with 0.3% hydrogen peroxide for 20 min to block endogenous peroxidase activity. Afterwards, the slides were placed in a citrate buffer (Antigen Retrieval Citra; Bio Genex, San Ramon, CA, USA) and heated three times for 5 min in the microwave oven (HM 146; Elektra Bregenz, Austria) set to decreasing levels of power (750, 650, 500 W). Bcl-2 expression was detected by using a standard immunohistochemical procedure, incubating the slides at 4°C overnight with monoclonal mouse anti-human bcl-2 [clone 124, isotype immunoglobulin (Ig) G1; Dako, Glostrup, Denmark] diluted 1:40 in phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA, Dako). After two washes with PBS, the slides were incubated with secondary biotinylated rabbit anti-mouse antibodies (Dako) for 30 min. Then they were exposed to avidin-biotin-peroxidase complex (Dako). Sections were stained with Fast Red Chromogen (Bio Genex) for 10 min and then washed twice with distilled water for 5 min. Counterstaining was performed with haematoxylin for 10 s before mounting. The positive control slides were prepared from tonsillar tissue and lymphocytes. For negative controls the mouse antibody HK 119/7M (Bio Genex) was used.
Evaluation of immunostaining by a computerized image analysis
All sections were investigated by light microscopy (Nikon, Mikrophot-FXA, x20 objective). The images were scanned with a video-camera (Hamamatsu, Colour Chilled 3CCD, Hamamatsu City, Japan), and afterwards saved with the Raster OPS-Videograbbercard for McIntosh Power PC 8100. Finally, transformation to a digitized image (Adobe Photoshop 4.0, Mountain View, CA, USA) for computerized image analysis (Lucia 32 G/VGA-Version 4.11 for Optoteam Image Analysis System; Laboratory Imaging, Ltd, Nikon Optoteam, http://www.lim.cz.) and quantification of bcl-2 immunostaining were performed.
First, the syncytiotrophoblast layer of the chorionic villi was identified manually by tracing the lines on each colour image with a computer mouse (Figure 1a: non-ruptured tubal ectopic pregnancy, Figure 1b
: ruptured tubal ectopic pregnancy). The resulting binary image was measured by splitting the red immunostaining of bcl-2 into red-green-blue (RGB) signals. For RGB signals thresholds were determined: red (range 115180), green (range 95160) and blue (range 100155). The measuring frame was 0.45 mm2. To assess bcl-2 immunostaining objectively in the syncytiotrophoblast, five randomly selected fields of vision were measured on the basis of good tissue morphology in the 36 specimens of tubal ectopic pregnancies. For measuring of the five fields of vision, one sample site in the upper left, another one in the upper right, the next two sites right and left at the bottom of the sample, and another one in the middle of the sample were selected. It is suggested that with the selection of five fields of vision, a representative survey regarding bcl-2 immunostaining was obtained.
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Observations were made of signs of apoptosis in the samples morphologically: cellular shrinkage, nuclear condensation and fragmentation were clearly diagnosed by use of light microscopy, although differences between ruptured and non-ruptured samples could only be suspected.
Statistics
Differences regarding patient and specimen specific characteristics such as quantified staining intensity between ruptured and non-ruptured tubal ectopic pregnancies were compared using the Wilcoxon two-sample test. P-values of 0.05 were considered to be statistically significant.
Pearson correlation coefficients were performed for correlation analyses between bcl-2 immunostaining, such as the mean percentage (±SD) of %PA, the variables of the HSI system, and patient characteristics.
The method of stepwise logistic regression was used to evaluate the possible role of bcl-2 immunostaining in ruptured and non-ruptured tubal ectopic pregnancies. The variables age, gestational weeks, size of adnexal mass measured by transvaginal sonography, human chorionic gonadotrophin (ß-HCG), and the variables regarding bcl-2 immunostaining, such as %PA and the variables of the HSI system, were considered as independent variables to model the probability of rupture of the tube. Calculations were done using the SAS statistical software system (SAS Institute Inc., Cary, N.C., USA).
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Results |
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Differences regarding patient and specimen specific characteristics between ruptured and non-ruptured tubal ectopic pregnancies, such as gestational weeks, %PA, S and I, were found to be statistically significant as shown by the Wilcoxon two-sample test (Tables I and II). Statistically significantly higher (P = 0.0009) immunostaining for bcl-2 was found in ruptured tubal ectopic pregnancies (Figure 1b
).
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Using the method of stepwise logistic regression, the variables percentage of bcl-2 immunostained area (%PA) (ß = 0.203, P = 0.0036, odds ratio=1.23) and S (ß = 0.458, P = 0.02, odds ratio = 0.63) entered the model. The intercept of the stepwise logistic regression model was ß = 3.229. For a probability threshold of 0.5 (<0.5 = non-ruptured ectopic pregnancy, >0.5 = ruptured ectopic pregnancy), this logistic regression model yields a sensitivity and specificity of 94.4%. It should be mentioned that the estimation of sensitivity and specificity will be positively biased in the learning sample. Moreover, on account of small sample sizes, the confidence interval for sensitivity and specificity were calculated (72.7%; 99.9%).
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Discussion |
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While other trophoblast-associated apoptotic markers, such as Fas and Fas ligand, have been described in co-occurrence with bcl-2 in term placenta, an apoptotic process mediated by Fas ligand has been suggested to play a role in placental invasion during early trophoblast implantation which underscores similarities between the trophoblast and neoplastic cells (Uckan et al., 1997). It was suggested that the invasiveness of ectopic trophoblast towards and through the muscularis zone of the tubal wall, consequently leading to tubal rupture, might be due to unlimited cell survival and high proliferative activity reflected by elevated bcl-2 expression in the syncytiotrophoblast overlying the cytotrophoblastic layer. There have also been efforts to identify parameters reflecting proliferative cell activity (Klein et al., 1995a
; Klein et al., 1995b
; Kiss et al., 1997
) or biological activity (Kemp et al., 1996
; Kemp et al., 1997
) of ectopic trophoblast. Particularly in cases of asymptomatic ectopic pregnancies, the identification of parameters reflecting the biological activity of ectopic trophoblast could enable selection of those patients who require surgical intervention rather than medical treatment. The results of this study are in agreement with others (Klein et al., 1995a
,b
) in which it was postulated that the degree to which trophoblast proliferation has progressed yields a better basis for therapeutic decisions, as evidence of invasive growth might indicate a decision against medical treatment.
While bcl-2 protein expression has been shown in first trimester chorionic villi of intrauterine pregnancies (Sakuragi et al., 1994), in the current study, bcl-2 was immunolocalized in first trimester ectopic pregnancies. In agreement with other investigators (Lea et al., 1997
), the greater intensity of syncytiotrophoblast immunostaining in the group of ruptured ectopic pregnancies in the current patient group might reflect different local environments, e.g. tumour necrosis factor alpha (TNF-
), which might also affect bcl-2 expression. As the inhibitory effect of bcl-2 on apoptosis may occur through proapoptotic proteins, elevated expression of bax, too, for example, could counter the protective effect of bcl-2. Enhanced bcl-2 expression in the syncytiotrophoblast overlying subtrophoblastic fibrin deposits was suggested to be necessary for the preservation of the placenta during gestation and for reparative processes of the trophoblast (Marzioni et al., 1998
). It was already emphasized that subtrophoblastic intravillous fibrinoid was the result of immunological processes altering the syncytiotrophoblastic barrier, thus increasing bcl-2 expression in the overlying syncytium (Kaufmann et al., 1996
).
As the preliminary data presented here demonstrated statistically significantly elevated bcl-2 immunostaining in ruptured human tubal ectopic pregnancies, further studies are being conducted at our department to reveal whether the localization of other members of the bcl-2 family is involved in tubal rupture. This suggested that the extensive bcl-2 expression reflecting unlimited cell survival of ectopic pregnancies might play a pivotal role in the development of tubal rupture. On the basis of immunohistochemical findings, the establishment of a circulating marker for tubal rupture is the goal of further investigations.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 30, 2000; accepted on February 26, 2001.