Apoptosis of Extravillous Trophoblast Cells Limits the Trophoblast Invasion in Uterine but not in Tubal Pregnancy During FirstTrimester

Placenta (2003), 24, 929–940 doi:10.1016/S0143-4004(03)00168-1

Apoptosis of Extravillous Trophoblast Cells Limits the Trophoblast Invasion in Uterine but not in Tubal Pregnancy During First Trimester U. von Rango a,d, C. A. Krusche a, S. Kertschanska b, J. Alfer c, P. Kaufmann b and H. M. Beier a a

Department of Anatomy and Reproductive Biology, RWTH University of Aachen, Wendlingweg 2, D-52057 Aachen, Germany; b Department of Anatomy II, RWTH University of Aachen, Aachen, Germany; c Department of Pathology, Medical Faculty, RWTH University of Aachen, Aachen, Germany Paper accepted 17 June 2003

During the first trimester of pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua. Invasion normally is reduced from the second trimester onwards and stops in the inner third of the myometrium. By contrast, in extrauterine tubal pregnancy, trophoblast invasion may even penetrate the tubal wall, which ultimately leads to the rupture of the fallopian tube. Induction of apoptosis of EVT cells, by maternal immune competent cells, may be an important mechanism to limit EVT invasion in uterine pregnancy. Tissue specimens from first and second trimester uterine pregnancy and first trimester tubal pregnancy were analyzed for apoptosis by TUNEL- and M30-staining. By immunohistochemical double labelling, maternal leukocyte subtypes were co-localized to apoptotic cells and in this context, the number of CD56+ NK cells was analyzed. Our data show that apoptosis is confined to the decidua basalis. Most apoptotic cells are single cytokeratin-positive epithelial cells residing in the stromal compartment. Consequently these cells can only be EVT cells. Maternal leukocytes are not apoptotic. They are located in close contact to apoptotic cells. The number of apoptotic cells in the second trimester (1.80.7 per cent) is reduced compared to first trimester (5.60.7 per cent) of uterine pregnancy. In parallel, the number of NK cells declines from first (24.42.9) to second (12.41.8) trimester. Furthermore, apoptosis is significantly reduced in ectopic (0.90.3 per cent) compared to eutopic first trimester pregnancies. Consequently, we suggest that in first trimester uterine pregnancy, induction of EVT cell apoptosis by the maternal immune system is one mechanism to limit EVT invasion. During the second trimester, in parallel to declining numbers of NK cells, the mechanism changes. However, in tubal pregnancy due to differing immunological microenvironments at the ectopic implantation site, apoptosis induction fails, which deleteriously may result in uncontrolled invasion and penetration of the tubal wall. Placenta (2003), 24, 929–940  2003 Elsevier Ltd. All rights reserved.

INTRODUCTION During the first trimester of uterine pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua beyond the endometrial-myometrial border (Benirschke and Kaufmann, 2000), normally until the inner third of the myometrium. The extent of this invasion is tightly regulated and declines from first trimester of pregnancy to term (Kemp et al., 2002). The control mechanisms regulating the invasion depth are still unknown. Either a basic genetic program of EVT stem cells, or the maternal decidua should influence the extent of invasion (Morrish, Dakour and Li, 1998; Bischof, Meisser and Campana, 2000). In extrauterine viable tubal pregnancy (EP), the trophoblast invades deeper into the wall and trophoblast proliferation is d

To whom correspondence should be addressed. +49-241-8082508; E-mail: [email protected] 0143-4004/03/$–see front matter

Fax:

more intense than in uterine pregnancy (Kemp et al., 1999). It can be assumed that the genetic nature of the EVT cell is equal in eutopic and ectopic pregnancies. Consequently, we assume that the increased invasive potential of the trophoblast cells in the fallopian tube is the result of the different paracrine and/or immunological environment of the fallopian tube compared to the uterus. In fact, our recent studies on leukocyte pattern show that CD56+ NK cells—the most frequent leukocytes in uterine implantation site—are nearly absent in the tubal implantation site (Marx et al., 1999; von Rango et al., 2001). Hence, we suggest a feto-maternal dialogue between the maternal leukocytes and their cytokines on the one hand and the invading trophoblast cells on the other hand. This idea is supported by the recent finding that LIF, which was shown to be essential for normal implantation in the mouse, is expressed by maternal leukocytes and may be involved in the regulation of human implantation and pregnancy (Sharkey et al., 1999).  2003 Elsevier Ltd. All rights reserved.

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Several mechanisms may regulate EVT invasiveness: reduced motility (Lacey et al., 2002), altered adhesion molecule- or MMP-expression (Huppertz et al., 1998) or mechanisms like polyploidization, syncytial fusion and apoptosis (Kemp et al., 2002; for review see Chakraborty et al., 2002, Goldman-Wohl and Yagel, 2002). Apoptosis of trophoblast cells can be induced by IFNg (Ho et al., 1999; Liu et al., 2002; Smith et al., 2002), which we recently have shown to be predominantly expressed (mRNA and protein) in the decidua basalis (von Rango et al., 2003). Taken together, the mechanism and extent of limiting trophoblast invasion may be dependent on the localization of the implantation site, pregnancy age and maternal microenvironments. Therefore we analyzed trophoblast apoptosis in dependence of the stage of pregnancy (uterine decidua from first vs second trimester pregnancy) and the localization of implantation site [first trimester pregnancy from uterine vs ectopic tubal pregnancy (EP)]. Apoptotic cells were analyzed quantitatively and co-localized to maternal leukocytes subtypes, to give first evidence for the hypothesis that maternal immune cells are involved in induction of trophoblast apoptosis.

MATERIALS AND METHODS Decidual and tubal tissues Human decidual tissue from first trimester (5–12 weeks of pregnancy; n=7) and second trimester (13–17 weeks of pregnancy; n=7) was obtained after elective termination of normal healthy pregnancies by vacuum aspiration. Pregnancy was confirmed by serum hCG-testing and ultrasonography immediately before surgery. Only women with proven fertility (at least one child, no spontaneous abortions) who did not receive any exogenous hormones during three months prior to pregnancy were included in the study. Our research on these specimen was approved by the Ethics Committee of the University of Aachen School of Medicine (permission number 560). Pregnancy was terminated 5–17 weeks after the last menstrual period. The gestational age was established from the duration of amenorrhea, analysis of individual menstrual cycle data, and the crown–rump length (CRL) or bi-parietaldiameter (BPD) as determined by ultrasonography. Tissue samples contain no myometrial part which would allow to orientate the specimen, therefore they show random orientation. Decidual tissue specimen were retrieved directly after vacuum aspiration, fixed with buffered 3.7 per cent formalin for 4 h, and embedded in paraffin using nuclease-free solutions. Decidua basalis and Decidua parietalis were identified by immunostaining of cytokeratin-8, -18 and -19, which are expressed by trophoblast cells (Mu¨hlhauser et al., 1995). All tissue specimen from one patient were stained for cytokeratin and two—randomly chosen—specimen of both decidua basalis and decidua parietalis were included in the study.

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Six specimens of ectopic tubal pregnancies (EP) of 6–9 weeks of pregnancy were retrieved from the archives of the Department of Pathology (RWTH University of Aachen).

Monoclonal antibodies Single and double immunohistochemical staining procedures were performed with seven antibodies suitable for antigen detection in formalin-fixed, paraffin-embedded tissue. For antibody clones, antigen retrieval procedure, incubation conditions for antibody binding, and binding detection see Table 1. Cytokeratin and M30-staining was performed as single label and first label in the double labelling procedure. Bcl-2 was performed as single label. CD8, CD45, CD56, and CD68 were performed as second label in the double labelling procedure.

Immunohistochemistry Briefly, 5 µm thick sections were mounted on 3-amino-propyltri-ethoxy-silane (APES) coated glass slides, deparaffinized with xylol and rehydrated. For antigen retrieval, sections were heated for four times (5 min each) in citric acid buffer (1.8 m citric acid, 8.2 m tri-sodium-citrate-dihydrate, pH 6.0; 600 W) in a microwave oven (in case of cytokeratin- and bcl-2-staining), or were incubated in 0.1 per cent Trypsin/0.1 per cent CaCl2 for 15 min at room temperature (in case of M30-staining; see Table 1). The detection of the different leukocyte subtypes was performed as second label without additional unmasking procedure. After microwave heating sections were allowed to cool down for about 30 min. Afterwards they were placed in 0.3 per cent H2O2/methyl alcohol for 30 min to block endogenous peroxidase activity, washed in PBS/0.1 per cent BSA, and non-specific binding sites were blocked according to the manufacturer’s instructions. After incubation with the specific primary antibody diluted in PBS/1.5 per cent BSA (PBS/0.1 per cent TWEEN in case of M30 staining; see Table 1) the slides were washed in PBS/0.1 per cent BSA and incubated at room temperature with the second antibody provided by the Histostain or Vector Kit according to the manufacturer’s instructions. Following three washes in PBS/0.1 per cent BSA slides were placed in streptavidin-peroxidase conjugate supplied with the kits. Antibody binding was detected by immersion of the sections for 5–10 min in chromogen solution [aminoethylcarbazole (AEC); GENZYME, Cambridge, MA, USA] or Vector Blue (Vector SG substrate Kit, Vector Laboratories). Finally, in case of single labelling, the slides were washed in PBS and deionized water and mounted in glycerolgelatin without additional counterstaining. In case of double labelling, staining was proceeded without additional pretreatment or blocking. First step of second labelling was blocking of non-specific binding sites according to the manufacturers instructions (for details to the second labelling see

Clone

Antigen retrieval

Incubation conditions

Antibody binding detection

Cytokeratin 2,5,6,8,18,19

KL1, Coulter Immunotech, Hamburg, Germany

Microwave, 45 min, 600 W

1 : 50, 1 h; room temperature

bcl-2 apoptosis-specific cytokeratin 18 cleavage product CD8 cytotoxic or suppressor T cells CD45 leukocyte common antigen CD56 natural killer cells

124, DAKO Diagnostica M30, Roche, Mannheim, Germany C8/144B, DAKO Diagnostica, Hamburg, Germany 2B11/PD7/26, DAKO Diagnostica Clone 123C3, Zymed, San Francisco, USA PG-M1, DAKO Diagnostica

Microwave, 45 min, 600 W 0.1% Trypsin/0.1% CaCl2, 15 min; room temperature Used as second label; no further pretreatment necessary Used as second label; no further pretreatment necessary Used as second label; no further pretreatment necessary Used as second label; no further pretreatment necessary

1 : 5, 1.5 h; 37(C 1 : 50 in PBS 0.1% Tween, 1.5 h; 37(C 1 : 20, 1 h; room temperature

single label: Histostain-SPe, Zymed, USA; AEC: double label: Vectastain Universal Elite, Vector Laboratories, Burlingame, USA; Vector Blue Histostaine PLUS, Zymed, USA; AEC Vectastain Universal Elite, Vector Laboratories, Burlingame, USA; Vector Blue Histostaine PLUS, Zymed, USA; AEC

1 : 100, 16 h; 4(C

Histostain-SPe, Zymed, USA; AEC

1 : 75, 1 h; 37(C plus 16 h; 4(C 1 : 500, 16 h; 4(C

Histostain-SPe, Zymed, USA; AEC

CD68 macrophages

von Rango et al.: Apoptosis of Extravillous Trophoblast Cells

Table 1. Immunohistochemistry

Histostain-SPe, Zymed, USA

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Table 1). Color development, and final embedding of the sections were the same as described above. For negative controls, normal mouse IgG was used instead of primary antibody at the same protein concentration as the specific antibody.

Detection of apoptotic cell death in situ using the TUNEL method To detect the fragmentation of the DNA in the nucleus the TUNEL (Terminal desoxy-UTP transferase-end-labelling) method (Gavrieli, Sherman and Ben-Sasson, 1992) was used as described earlier (von Rango et al., 1998). TUNEL-reaction was performed on serial sections to Cytokeratin- and M30staining. Slight modifications of the pretreatment were necessary to adapt the TUNEL reaction to the requirements of human decidual tissue. Briefly, the 5 µm thick sections were mounted on 3-aminopropyl-tri-ethoxy-silane coated glass slides, deparaffinized and rehydrated. After endogenous peroxidase blocking in 0.3 per cent H2O2/methanol and microwaving (600 W) for 60 sec in citric acid buffer (1.8 m citric acid, 8.2 m tri-sodiumcitrate-dihydrate, pH 6.0) slides were incubated in 0.1 per cent Triton X-100 (in PBS) for 10–15 min at 37(C. The labelling reaction was performed as described earlier (von Rango et al., 1998). Detection of the digoxigenin-labelled deoxynucleotides was performed using an anti-DIG-antibody conjugated with alkaline phosphatase (1 : 300; 45 min RT). Color staining was developed for 5–10 min using 0.16 mg/ml BCIP (5-bromo-4chloro-3-indolyl-phosphate) and 0.33 mg/ml NBT (nitro blue tetrazolium chloride) as substrates. Positive controls were treated with 3U DNAse I/µl (Roche; 15 min 37(C) prior to the tailing reaction. As negative control either the DIG-dUTP or TdT was omitted during the tailing reaction. In case of combined TUNEL- and immunohistochemical-labelling, TUNEL staining was followed by immunohistochemical staining beginning from the retrieval procedure.

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trophoblast) and weakly invaded areas (5–45 per cent trophoblast). Total number of apoptotic cells was determined by counting TUNEL-positive cells. Apoptotic epithelial cells were detected by M30-staining. Apoptotic cell numbers finally were referred to total cell number or the trophoblast cell number (per cent apoptotic cells related to total cells or to trophoblast cells; see Figure 2). The statistical analysis was performed with GraphPad PRISM Version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). For all data mean and SEM were calculated. Apoptotic cell numbers showed a normal distribution as assessed by Kolmogorov–Smirnov test. The six groups (strongly and weakly invaded areas from: first trimester and second trimester uterine pregnancy, first trimester tubal pregnancy) were defined by two factors: implantation site/age on the one hand and invasion intensity on the other hand. Therefore differences with respect to both factors were tested using the two way analysis of variance (ANOVA). Differences between the numbers of TUNEL-positive cells and M30-positive cells within the same tissue, were tested by the paired Student’s two-tailed t-test. Resulting P-values <0.05 were accepted as statistically significant. Quantitative evaluation and statistical analysis of CD56+ natural killer cells. Sections of first (n=7) and second (n=7) trimester uterine decidua were double-stained for cytokeratin and CD56. Stained cells were counted within random areas of 0.085 mm2 using a high power magnification (400) in strong invaded decidual areas (>45 per cent trophoblast) vs weaker invaded areas (5–45 per cent trophoblast) and decidua parietalis (0 per cent trophoblast). For all data the mean and SEM were calculated. As the data showed a normal distribution as assessed by Kolmogorov– Smirnov test, numbers of CD56+ NK cells were compared in corresponding areas between first and second trimester decidua by the paired Student’s two-tailed t-test. Resulting P-values <0.05 were accepted as statistically significant.

Quantitative evaluation and statistical analysis

RESULTS

Apoptosis detection. Serial sections of first (n=7) and second (n=7) trimester uterine decidua and ectopic tubal pregnancies (n=6) were stained for cytokeratin, DNA-fragmentation (TUNEL), and caspase specific cytokeratin cleavage (M30). Quantitative evaluation of all TUNEL- and immunohistochemical staining was performed within randomly chosen areas of 0.085 mm2 using a high power magnification (400). Firstly, total number of cells was counted by cellular profile, then trophoblast cell number was determined by cytokeratin immunoreactivity giving the relative proportion of trophoblast cells. Further quantitative analysis was performed discriminating between strongly invaded decidual areas (>45 per cent

Apoptosis is found in eutopic uterine and ectopic tubal implantation sites Three different sites of implantation were analyzed: first trimester uterine implantation site (n=7), second trimester uterine implantation site (n=7), and first trimester ectopic tubal implantation site (n=6). Decidua basalis (invaded by EVT; Figure 1D) was distinguished from decidua parietalis (without invasion; Figure 1A) by cytokeratin staining. Apoptotic cells were found in the stromal compartment of decidua basalis of the first and second trimester and in ectopic tubal implantation site (Figure 1F, I, L). In the decidua

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Figure 1. Detection of apoptotic cells in first (A–F) and second (G–I) trimester uterine and first trimester tubal (J–L) pregnancy. Paraffin sections of decidual tissue were stained for cytokeratin (A, D, G, J) to identify decidua basalis and decidua parietalis. On serial sections, apoptotic cells were detected via M30-staining (B, E, H, K) or TUNEL-labelling (C, F, I, L). Decidua parietalis from the first trimester of pregnancy (A: cytokeratin staining) shows no apoptotic cells neither by B: M30 staining nor by C: TUNEL detection. Insert C: Sections pretreated with DNAse I (positive control) show TUNEL-staining in nearly all nuclei. Decidua basalis from the first trimester of pregnancy (D: cytokeratin staining) shows nearly equal amounts of E: M30-staining and F: TUNEL staining. Decidua basalis from the second trimester of pregnancy (G: cytokeratin staining) shows significantly reduced amounts of H: M30-staining and I: TUNEL staining compared to first trimester. In first trimester ectopic tubal pregnancy (J: cytokeratin staining) only few apoptotic cells are found by K: M30-staining and L: TUNEL staining, whereas in fetal villi (Insert L), frequently apoptotic cell death is found. Bar represents 50 µm.

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(5–45 per cent trophoblast), and strongly invaded areas (>45 per cent trophoblast). Apoptotic cells were detected exclusively in areas invaded by trophoblast cells. In case of first and second trimester uterine implantation site we found more TUNELand M30-positive apoptotic cells in strong invaded areas than in weaker invaded areas (Figure 2, Table 2). Apoptotic cell numbers [given as percentage of total cells (Figure 2A) or trophoblast cells (Figure 2B)] decreased significantly from first trimester [1.60.1 per cent (per cent of total cells; weak invaded area) and 5.60.7 per cent (per cent of total cells; strong invaded area)] to second trimester (0.50.01 per cent and 1.80.7 per cent). In first trimester ectopic tubal pregnancies (0.50.2 per cent and 0.50.1 per cent) significantly less apoptotic cells were detected than in first trimester uterine pregnancies. Differences between weakly and strongly invaded areas were only significant when apoptotic cells numbers were related to total cell number (Table 2). In case of EP but not in uterine pregnancies significant differences between apoptotic cell numbers detected by TUNEL- vs M30-staining were shown (EP: weakly invaded area P=0.0089; strongly invaded area P=0.0253).

Apoptotic cells are predominantly CD45 negative

Figure 2. Quantitative analysis of M30-staining and TUNEL-staining of apoptotic cells. A: Percentage of apoptotic cells from total cell number is the highest in strong invaded areas from the first trimester (5.60.7 per cent) and second trimester (1.80.7 per cent) decidua. In first trimester decidua more apoptotic cells were found than in second trimester decidua. In contrast, in first trimester ectopic tubal implantation site the extent of apoptosis is reduced compared to first trimester uterine implantation site. B: Assuming that nearly all apoptotic cells are EVT cells, the percentage of apoptotic trophoblast cells was determined by quantitative analysis of M30-staining and TUNELstaining cells. Most apoptotic EVT cells were found during the first trimester in strong (10.1–7.0 per cent1.6 per cent) and weak (7.00.9 per cent) invaded areas. In first trimester uterine pregnancy more EVT cells are apoptotic than in second trimester uterine pregnancy or first trimester ectopic pregnancy.

parietalis apoptotic cells were neither detected by the TUNEL method nor by staining for apoptosis-specific cytokeratin degradation (Figure 1B, C). Negative controls did not show any cytokeratin-, M30-, or TUNEL-staining. Nearly all nuclei were stained in sections pretreated with DNAse I (positive control for TUNEL) (Figure 1 C insert).

Apoptosis shows spatial and temporal distribution patterns Three decidual areas were analyzed according to the degree of trophoblast invasion related to all cells counted within the area: decidua parietalis (0 per cent trophoblast), weakly invaded

Whereas M30-staining detects exclusively epithelial apoptotic cells, TUNEL-staining is able to detect all types of apoptotic cells. To analyze if there are apoptotic decidual leukocytes we stained TUNEL-positive cells in double labelling with the common leukocyte marker CD45. No double staining was obtained (Figure 3A). In contrast, decidual leukocytes showed strong protein expression of the apoptosis inhibitor bcl-2 as shown by immunostaining (Figure 3B).

Potential decidual effector cells and macrophages are closely related to the invading EVT Apoptosis induction (e.g. by the FasR/FasL system, granzyme/perforin, or paracrine mediators as TNFa or soluble FasL) needs at least a transient contact between effector and target cell. We performed double labelling of M30- or TUNEL combined with staining for CD56 (NK cells) or CD8 (cytotoxic T cells) to co-localize dying cells to potential effector cells. In a semiquantitative analysis apoptotic cells located near (distance smaller that one leukocyte diameter of about 7 µm) potential effector cells (T-cells, NK-cells) were counted in 3–4 randomly chosen areas (0.085 mm2) for first trimester uterine pregnancies. Thus, 9.72.3 per cent apoptotic cells were co-localized to CD8+ cells and 26.02.5 per cent apoptotic cells were co-localized to CD56+ cells (Figure 4A, B, D). Macrophages neighboring apoptotic cells showed strong lysosomal staining (Figure 4C).

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Figure 3. Analysis of apoptotic activity of decidual leukocytes. A: Double labelling for CD45 (red) and TUNEL-stain (blue; arrow). No CD45+ leukocytes are TUNEL-positive. B: Immunostaining of Bcl-2. Besides the glandular epithelial cells showing weak staining, decidual leukocytes stain positive for the anti-apoptotic protein Bcl-2. Bar represents 50 µm.

Figure 4. Co-localization of leukocyte subtypes to apoptotic cells. A: Double-labelling for CD8 (red) and TUNEL (blue), B: CD56 (red) and TUNEL (blue), C: CD68 (red) and TUNEL (blue), and D: Quantitative analysis of co-localization of CD8 T cells and CD56 NK cells to apoptotic cells (stained by M30 or TUNEL). Bar represents 25 µm.

Numbers of CD56+ NK cells and apoptotic cells decrease in parallel By double immunohistochemical staining for cytokeratin and CD56, the numbers of NK cells were analyzed in areas with

different extent of trophoblast invasion from first (n=7) and second (n=7) trimester decidua (Figure 5). In general strongly invaded areas (>45 per cent trophoblast) contained fewer CD56+ NK cells. This observation confirms our own and other earlier data (Loke and King, 1995; von Rango et al.,

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Figure 5. Quantitative analysis of CD56+ NK cells in the first and second trimester of uterine pregnancy. (A). Immunohistochemical double labelling of CD56+ (red) and Cytokeratin (blue) showed that number of CD56+ NK cells is higher in first trimester (B) than in second trimester (C) uterine pregnancy. This relation was found in all areas of the decidual tissue. In decidua parietalis and in weakly invaded areas most NK cells were found. Bar represents 50 µm.

2001). Comparing corresponding areas, significantly less CD56+ NK cells were found in the second trimester vs first trimester in decidua parietalis (P=0.0038) and decidua basalis invaded by 5–45 per cent trophoblast (P=0.0241) (Table 2).

DISCUSSION In successful uterine pregnancies, invasion of extravillous trophoblast cells (EVT) is well-controlled, whereas in ectopic tubal pregnancy (EP) this control seems to fail, resulting in over-invasion and tubal rupture (for review see Benirschke and Kaufmann, 2000). In the present study we show that the extent of apoptosis at different implantation sites correlates with

trophoblast invasiveness and CD56+ NK cell numbers. In detail it was found: (1) Apoptotic cells are confined to areas containing trophoblast, namely the decidua basalis. (2) Decidual CD45+ leukocytes did not show DNAfragmentation, but in contrast, stain positive for bcl-2. (3) The number of apoptotic cells decreases from first to second trimester of uterine pregnancy. (4) Apoptosis in first trimester ectopic tubal pregnancies is reduced compared to first trimester uterine pregnancy. (5) Potential effector cells (e.g. T cells and NK cells) are co-localized near apoptotic cells. (6) Macrophages—able to phagocytose apoptotic cells—were found near the dying cells. (7) Numbers of CD56+ NK cells and apoptotic cells decline in parallel. From these data the following suggestions were drawn.

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Table 2. Statistical analysis of apoptosis in 1st and 2nd trimester uterine and 1st trimester ectopic tubal pregnancy (EP) Variable

TUNELEVT M30EVT ELtotal cells M30total cells

Significance with respect to implantation Significance with respect to degree of Interaction site and age trophoblast invasion Per cent1

df2 F3

P4

Per cent df F

P

Per cent df F

P

64.19 61.27 30.02 30.52

2 2 2 2

<0.0001 <0.0001 <0.0001 <0.0001

1.48 2.91 18.56 25.23

0.2363 0.1012 0.0002 <0.0001

3.60 4.39 11.30 12.62

0.1870 0.1332 0.0100 0.0008

31.72 30.29 14.42 22.59

1 1 1 1

8.68 2.872 17.83 37.35

2 2 2 2

21.11 2.169 5.425 9.338

1

Of total variation. Degree of freedom. 3 Test variable for calculating variation of the group averages with regard to the degree of freedom, number of sample types, and number of tests. 4 Significance level resulting from the comparison between the calculated F-value and given F-values for each significance level. Statistically significant values (P<0.05) appear in bold type. 2

Apoptotic cells found in decidua basalis and ectopic tubal implantation sites are predominantly EVT cells As most pathways to apoptosis converges in fragmentation of the DNA (Bortner, Oldenburg and Cidlowsky, 1995), TUNEL labelling is the most common method to show apoptosis of all types of cells within tissue sections although there may be problems as detection of necrotic cells death in addition to apoptosis as discussed earlier (von Rango et al., 1998). For these reason, only single TUNEL-stained cells were included in our analysis and we confirmed our TUNEL data by M30-staining of apoptosis-specific cytokeratin 18 cleavage products which is an established marker in endometrial and placental tissue (Austgulen et al., 2002; Jordan and Butchko, 2002; Mourits et al., 2002; Huppertz et al., 2003). Apoptotic cells numbers and localization obtained by these two markers were comparable indicating that there was no falsepositive TUNEL-staining. M30 detects apoptosis especially in epithelial cells and their derivates (e.g. EVT). Neither decidual fibroblasts nor leukocytes express cytokeratin. Consequently cells staining positive for apoptosis-specific cytokeratin fragments which were especially found in the stromal compartment of the decidua basalis (see Figure 1E, H, K), can only be EVT cells. This conclusion is in accordance with other studies showing apoptosis in villous cytotrophoblast cells (Kadyrov, Kaufmann and Huppertz, 2001, in human tissue; Mochizuki et al., 1998, in human tissue), in cytotrophoblast cells of the cell column differentiating into EVT (Fei et al., 2001, in the rhesus monkey), and under pathological conditions (Reister et al., 2001 in the human; DiFederico, Genbacev and Fisher, 1999 in the human). Furthermore, it was shown that invasive EVT does not express bcl-2 indicating high incidence for apoptosis (Quenby et al., 1998). Apoptosis of decidual leukocytes—as suggested by other groups (Hammer and Dohr, 1999, first trimester; Coumans et al., 1999, in vitro studies from term placentae) however, are unlikely, as we found apoptotic cells exclusively in the decidua

basalis (leukocytes are present in the decidua parietalis too) and apoptotic cells did not stain positive for CD45. The positive staining of leukocytes for bcl-2 shown here and confirming earlier data (Lea et al., 1997; von Rango et al., 1998; King, Gardner and Loke, 1999) gives another hint that most of the leukocytes should be protected against apoptosis, although additional studies on other members of the numerous bcl-2 family would be necessary for further conclusions. Nevertheless, apoptosis of decidual leukocytes is most probably the exception.

Apoptosis of EVT may be one important mechanism to limit its invasiveness in first trimester uterine pregnancy From first to third trimester the differentiation program of cytotrophoblast cells changes from a highly invasive to a less invasive phenotype preferring syncytial fusion (Morrish, Dakour and Li, 1998). Already during the second trimester invasiveness and proliferative activity of the trophoblast decline (Benirschke and Kaufmann, 2000). Several mechanisms were proposed to limit EVT invasion: reduced motility (Lacey et al., 2002), altered expression of adhesion molecules or MMPs (Huppertz et al., 1998), syncytial fusion, and apoptosis (Kemp et al., 2002). Our data give evidence for a massive reduction of trophoblast cells by apoptosis during first trimester of pregnancy (7–11 per cent see Figure 2B) which can be compensated by high proliferative activity of trophoblast stem cells at the basement membrane and the cell columns. In parallel to declining invasiveness and proliferative activity of the EVT from the first to second trimester onwards, the number of apoptotic cells decreases. From these data we suggest that during the first trimester of uterine pregnancy apoptosis is an important mechanism to limit EVT invasion, whereas in the second trimester its significance declines and other mechanisms may dominate the regulation of trophoblast invasion.

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In first trimester extrauterine tubal pregnancies number of apoptotic cells is lower than in uterine pregnancies. This is in accordance with other studies (Kokawa, Shikone and Nakano, 1998; Marx et al., 1999), which however did not compare apoptosis in first and second trimester uterine pregnancy and did not determine the cell types undergoing apoptosis. Trophoblast cells have the potential to enter the invasive, syncytial or apoptotic differentiation pathway. Which of these three possible pathways is chosen, is thought to be tightly regulated by autocrine factors from the trophoblast itself and by paracrine factors from the maternal decidua (Lala and Hamilton, 1996; Morrish, Dakour and Li, 1998; Bischof, Meisser and Campana, 2000). In ectopic tubal pregnancy paracrine factors-inducing apoptosis of EVT in uterine pregnancy-may lack. Consequently, induction of apoptosis may fail, resulting in uncontrolled invasion of highly invasive first trimester EVT cells. Indeed our finding that NK cells, the predominant leukocyte subtype in the uterine pregnancy, are absent from the tubal implantation site (von Rango et al., 2001) supports this hypothesis.

Apoptosis of EVT is influenced by the local maternal immune system, and itself modulates maternal immunological function Apoptosis of EVT cells may be induced by several ways. CD56+ NK cells, the predominant uterine leukocyte population, are cytotoxic to their target cells by inducing apoptosis via direct cell–cell interaction or by expression of cytokines such as IFN
which activates cytotoxic T cells and upregulates apoptosis related molecules such as caspases (Ahn et al., 2002). IFN
was already shown to promote trophoblast apoptosis (Yui et al., 1994; Ho et al., 1999; Liu et al., 2002; Smith et al., 2002). We have shown recently that IFN
is expressed predominantly in the decidua basalis compared to the decidua parietalis (von Rango et al., 2003). CD56+ NK cells and CD8+ T cells accumulate at the invasion front (von Rango et al., 2001) and may act as apoptosis inducing effector cells towards invading EVT. These cytotoxic cells induce apoptosis e.g. via membrane bound or soluble forms of death ligands like FasL or TNF. We have recently shown that FasL is expressed in decidual leukocytes (von Rango et al., 2002). Prerequisite for such apoptosis induction is a transient contact between effector sand target cell. In this study we show that z26 per cent (10 per cent) of CD56+ NK cells (CD8+ T cells) are in close contact with TUNEL- or M30-positive trophoblast cells by immunohistochemical double-staining (Figure 4). Taken together, these data support the hypothesis that maternal immune cells and their cytokines regulate trophoblast apoptosis. Vice versa, apoptotic cells influence the immune system when being phagocytozed by macrophages and dendritic cells (Savill et al., 2002 for review). The presence of potent dendritic cells in the human decidua was recently shown

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(Ka¨mmerer et al., 2000), and we have co-localized macrophages to apoptotic cells in this study. Besides the phagocytosis of apoptotic cells, both cell types influence the maternal (immuno-) reaction on the fetus. Activated macrophages promote trophoblast invasion by secreting IL-1 by increasing metalloproteinase activity of EVT (Librach et al., 1994). On the other hand IL-1 (e.g. secreted by macrophages) and IFN
(e.g. secreted by T cells) induce the expression of the tryptophan degrading enzyme, indole-amine 2,3-dioxygenase (IDO) in macrophages (Carlin et al., 1989; Currier et al., 2000). Catabolism of tryptophan by this enzyme induces apoptosis in certain tumour cell lines (Konan and Taylor, 1996). Recent in vitro studies demonstrate an analogous effect on human trophoblast cells in the pathological case of pre-eclampsia (Reister et al., 2001). Dendritic cells present peptides from degraded proteins of phagocytosed cells on their surface (Bellone et al., 1997; Albert, Sauter and Bhardwaj, 1998; Currier et al., 2000; Savill et al., 2002). Since apoptosis does not evoke an inflammatory response, these peptides are presented in absence of costimulatory signals (Bellone et al., 1997; Steinman et al., 2000). Therefore, T cells recognizing these peptides are anergized (Van Parijs and Abbas, 1998). This way to induce T-cell anergy is suggested to establish peripheral self tolerance (Levine and Koh, 1999). The same mechanism may be used by fetal trophoblast cells to escape from the maternal immune system. The idea that the establishment of fetal tolerance and peripheral self-tolerance use a similar pathway is supported by the finding that women with auto-immune diseases suffer more often from reproductive failure than normal healthy women (Gleicher, Vidali and Karande, 2002). According to this hypothesis, trophoblast apoptosis at the beginning of pregnancy (e.g. induced by IFN
-expression of NK cells or tryptophan depletion as discussed above), may be necessary to further establish maternal tolerance to the fetus by induction of T-cell anergy (Figure 6). This hypothesis is supported by findings that tryptophan catabolism prevented fetal rejection by inducing T-cell tolerance (Munn et al., 1998; Mellor and Munn, 1999). In conclusion, we have demonstrated that apoptosis of EVTs declines from first to second trimester in human pregnancy. In parallel the number of NK cells decreases. Extrauterine tubal implantation sites—lacking maternal NK cells—show reduced amount of EVT apoptosis. Possible effector cells (as T cells and NK cells) are in contact to apoptotic EVTs, and macrophages adjacent to the dying cells show strong lysosomal staining as sign of their phagocytotic activity. These data suggest that apoptosis of EVT, induced by cellular or cytokine components of the maternal immune system, may be an important mechanism to limit invasiveness during the first trimester of uterine pregnancy. In ectopic tubal pregnancy, this regulation of apoptosis may be disturbed leading to an uncontrolled invasion. Apoptotic EVT cells themselves may induce, by mechanisms similar to those preserving peripheral self-tolerance, T-cell anergy and consequently fetal tolerance.

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Figure 6. Schematic sequence of events, which may result in T-cell anergy as the result of trophoblast apoptosis. CD56+ NK cells, present at the feto-maternal interface, interact with trophoblast cells via several receptors (for review see Moffet-King, 2002). In addition they release cytokines, which influence several types of immune competent cells. NK cells may recognize trophoblast cells as ‘non-self’ and induce apoptosis. Via IFN- g release they activate macrophages and T cells. Phagocytosis of apoptotic bodies by macrophages leads to peptide presentation without costimulatory signals . Consequently specific T cells, which recognize these peptides, are anergized.

ACKNOWLEDGEMENTS The authors wish to acknowledge the excellent technical assistance of Diana Seelis, Rick Kamps, and Kerstin Ziob. We thank Gabie Raven, Frans Bocken and all colleagues at the Bourgognekliniek Maastricht for the excellent cooperation. This work was supported by the START program (Project 24/2001/2002 of the School of Medicine, RWTH Aachen).

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