Molecular Human Reproduction, Vol. 7, No. 7, 681-689,
July 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Apoptosis in the normal human amnion at term, independent of Bcl-2 regulation and onset of labour
1 Department of Obstetrics and Gynecology and 2 Department of Anatomy and Biology, Osaka Medical College, 2–7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan
Abstract
This study was designed to detect apoptosis in the human amnion and to elucidate the signalling pathway involved in its regulation. Samples of human amnion were obtained from 34 women (weeks 11–42 of gestation) and studied using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) method with light microscopy (LM) and transmission electron microscopy (TEM). Apoptotic regulators in the samples were studied by immunohistochemistry and caspase activity assay. The TUNEL method with LM demonstrated that the percentage of TUNEL-positive cells in the amniotic epithelium was the highest in weeks 40–41 of gestation (P < 0.05) independent of the onset of labour, and the cells were often detached from the epithelium into the amniotic cavity at term. The TUNEL method with TEM clearly showed the characteristic features of apoptosis such as the nuclear condensed chromatin with abundant free 3'-OH DNA ends, cell shrinkage and a decrease in the number of desmosomes, except for the presence of apoptotic bodies. Fas and Fas ligand (FasL) were constantly expressed on apical membranes of amniotic epithelial cells from weeks 16–27 through to 40–41 of gestation, while no Bcl-2 expression was observed throughout the gestational periods. Activities of caspase-3 and caspase-8, but not of caspase-9, were higher in weeks 40–41 than those from weeks 16–27 of gestation (P < 0.01). We conclude that apoptosis in term amniotic epithelium is independent of Bcl-2 regulation and onset of labour, and may play an important role in the fragility and rupture of human fetal membranes at term.
amnion/apoptosis/caspase/Fas antigen/Fas ligand
Introduction
Determining the mechanism of fetal membrane rupture at term and preterm is a significant clinical problem. It is generally accepted that the rupture of fetal membranes is caused by the increase in physical stress such as labour and by the fragility of the membrane itself due to inflammation. Recent studies have suggested that the rupture of fetal membranes is related not only to physical stress but also the biochemical processes, including extracellular matrix remodelling and cell death in fetal membranes (Bryant-Greenwood, 1998
; Parry and Strauss, 1998).
Much focus has been given to apoptosis in various reproductive tissues, such as the endometrium (Otsuki et al., 1994; Kokawa et al., 1996; Yamashita et al., 1999), ovarian atretic follicles (Tilly et al., 1991) and the placenta (Levy and Nelson, 2000). More recently, apoptosis was reported in the studies of fetal membranes in rat (Paavola et al., 1995; Lei et al., 1996) and in human (Runic et al., 1998; McLaren et al., 1999). However, it is open to question as to how apoptosis in fetal membranes is regulated and how apoptosis contributes to the fragility of fetal membranes.
Apoptosis is a type of cell death controlled by a series of molecular events and is executed by a family of cystein-containing aspartate-specific proteases called the caspase family. A recent study (Hengartner, 2000) has summarized two major apoptotic pathways in mammalian cells: the death receptor and mitochondrial pathways. The death receptor pathway involves Fas, a type I membrane protein that belongs to the tumour necrosis factor (TNF) receptor/nerve growth factor receptor family (Nagata and Golstein, 1995), and Fas ligand (FasL), a type II membrane protein that belongs to the TNF family. Binding of FasL to Fas induces receptor clustering and formation of a death-inducing signalling complex. This complex recruits, via the Fas-associated death domain protein, multiple procaspase-8 molecules, resulting in caspase-8 activation. The activated caspase-8 cleaves procaspase-3 to obtain the active form. Downstream of caspase-3, the apoptotic programme branches into multiple subprogrammes in the ordered cell dismantling and production of degradation products of actin, DNase, and lamins (Cohen, 1997). Fas and FasL expression in both term and preterm human fetal membranes has been reported (Runic et al., 1996, 1998; Uckan et al., 1997), accompanying the appearance of apoptotic cells. However, the co-expression of Fas and FasL on the cell membrane does not necessarily ensure the activation of the downstream signalling of the death receptor pathway. To our knowledge, the expression and activation of caspase-8 and caspase-3, which are downstream signal transducers, have not yet been reported in human fetal membranes, except in human villous trophoblast (Huppertz et al., 1998, 1999). The mitochondrial pathway is used extensively for responding to extracellular cues and internal insults such as DNA damage. The release of cytochrome c from the mitochondria to the cytosol is a critical step in this pathway (Mignotte and Vayssiere, 1998). This step is inhibited by Bcl-2 present in the internal mitochondrial membranes (Akao et al., 1994). Cytochrome c is associated with Apaf-1 and then with procaspase-9 to form the apoptosome, which, in turn, cleaves procaspase-3 to obtain the active form (Li et al., 1997). Whether or not the mitochondrial pathway involving Bcl-2 is the main apoptotic signalling in the fetal membranes is unknown.
This study was designed to quantify the number of apoptotic cells at different weeks of gestation and to elucidate the relationship between apoptosis in the amnion and the fragility of the fetal membrane and the onset of labour. In addition, the involvement of the death-receptor and mitochondrial pathways of apoptosis in the human fetal membranes is discussed.
Materials and methods
Patients and tissue preparation
Tissue samples of human fetal membranes were collected after obtaining informed consent from 34 subjects who underwent elective abortion (n = 9), vaginal delivery (n = 15), or Caesarean section (n = 10) immediately after delivery. The study was approved by the ethical board of the Osaka Medical College. Fresh tissue samples were collected as near as possible to the rupture site by trimming into 2-cm-wide strips using double-edged safety razors. The clinical characteristics of the 34 subjects analysed in this study are presented in Table I. The gestational period and the due date were determined based either on the last menstrual period of each subject or the crown–rump length of the fetus measured by ultrasonography. Cases with an abnormal amount of amniotic fluid, chorioamnionitis, rupture of the membranes more than 24 h previous to the delivery or obvious fetal disease such as molar pregnancies were excluded from this study. The 34 subjects were then divided into five groups according to the gestational period: weeks 11–15 (n = 6), weeks 16–27 (n = 6), weeks 28–39 (n = 11), weeks 40–41 (n = 8) and week 42 (n = 3) of gestation.
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Fresh tissue samples were rinsed with phosphate-buffered saline (PBS) and immediately frozen in acetone using dry ice and embedded in a tissue mount (Chiba Medical Inc., Saitama, Japan). Serial cryosections, placed on poly-L-lysine-treated glass slides, were lightly fixed in 4% paraformaldehyde for 10 min and postfixed in a 2:1 mixture of methanol and acetone for 5 min at –20°C. After washing with 0.01 mol/l PBS (pH 7.4), endogenous peroxidase activity was blocked by 1% H2O2 in PBS for 10 min at room temperature. The sections were subjected to the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin-nick end-labelling (TUNEL) method and immunostaining. Some of the samples were immediately fixed in 0.1 mol/l phosphate buffer (PB) containing 2.5% glutaraldehyde and 2% paraformaldehyde for 4 h at room temperature. The tissues were then postfixed with 1% osmium tetroxide in 0.1 mol/l PB for 2 h and then routinely dehydrated through a graded ethanol series and embedded in epoxy resin for the TEM study. The remaining fresh samples were used for DNA fragmental assay and caspase activity assay.
TUNEL by light microscopy
Free 3'-OH DNA ends were detected in situ by TUNEL as previously described (Gavrieli et al., 1992) with some modifications. The ApopTag kit (Oncor, Inc., Gaithersburg, MD, USA) was used according to the manufacturer's protocol. Briefly, a dideoxynucleotide labelled with exogenous digoxigenin was bound to the 3'-OH DNA ends by a TdT enzyme on the cryosections. The sections were then incubated with an anti-digoxigenin antibody conjugated with horseradish peroxidase. Peroxidase activity was analysed by exposure of sections to a solution containing 0.05% 3,3'-diaminobenzidine tetrahydroxychloride and 0.01% H2O2 in Tris–HCl buffer (DAB solution) at pH 7.6 for 5 min. Sections were finally counterstained with 1% methyl green. For negative controls, distilled water was used instead of the TdT enzyme solution. Human lymph node tissues were used as positive controls. For quantitative analysis, the number of TUNEL-positive cells among 500–1000 amniotic epithelial cells per slide was counted by two independent observers under 100–400-fold magnification by light microscopy (LM) and expressed as a percentage.
TUNEL by transmission electron microscopy
Ultrathin sections (60–80 nm thick) collected on nickel grids were subjected to the etching step using drops of saturated sodium metaperiodate for 2 min, and rinsed with drops of distilled water for 10 min. The procedure was fundamentally the same as that for TUNEL/LM, using the same kit, except with the incorporation of immunogold particles (instead of immunoperoxidase) by an anti-digoxigenin antibody conjugated to 10 nm colloid gold particles (British BioCell International, Golden Gate, Cardiff, UK). The ultrathin sections processed by the TUNEL method were stained with uranyl acetate and lead citrate for transmission electron microscopy (TEM) observation. Image analysis for DNA fragmentation was performed using the NIH Image Program. Briefly, images of the nuclei in amniotic epithelial cells were captured by a computer. The labelling density for free 3'-OH DNA ends was evaluated based on the number of immunogold particles per nuclear area (µm2). For TEM analysis, samples from 16 subjects were randomly selected from the following four groups: weeks 16–27 (n = 4), weeks 28–39 (n = 5), weeks 40–41 (n = 4) and week 42 (n = 3) of gestation.
Immunohistochemistry
The immunohistochemistry procedure for Bcl-2, Fas and FasL was performed according to a previous study using human placenta (Steele et al., 1998). Antibodies used in this study were the following: a mouse monoclonal anti-human Bcl-2 antibody (MAb 100; a kind gift from Professor D.Y.Mason, Oxford, UK) at a dilution of 1:100, a rabbit polyclonal anti-human Fas antibody (N-18; Santa Cruz Biotechnology, CA, USA) at a dilution of 1:300, and a rabbit polyclonal anti-human FasL antibody (Q-20; Santa Cruz Biotechnology) at a dilution of 1:300. The appropriate dilutions for antibodies were determined in preliminary experiments. After incubation with the primary antibody for 1 h at room temperature, the fixed cryosections were rinsed with PBS and incubated with goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (Dako, Glostrup, Denmark) at a dilution of 1:60 for 1 h at room temperature. Peroxidase activity was detected by exposing the sections to a DAB solution at pH 7.6 for 5 min. The slides were then rinsed and counterstained with 1% methyl green. Mouse immunoglobulin (Ig) G1 for Bcl-2 and rabbit IgG1 for Fas and FasL, diluted at 1:100 and 1:200 respectively, were used as negative controls for immunohistochemical analyses. Moreover, a FasL peptide (Santa Cruz Biotechnology) was co-incubated with an anti-FasL antibody for neutralization, demonstrating the total loss of staining reactivity. Trophoblasts of human placenta were used as positive controls for Bcl-2, Fas and FasL. The immunostaining was independently confirmed by two observers and assigned to one of three subgroups: –, <10%; +, 10–50%; ++, >50%.
DNA agarose gel electrophoresis
The tissues were rinsed with PBS, homogenized with RIPA buffer (1.0% NP-40/50 mmol/l Tris–HCl, pH 8.0/150 mmol/l NaCl/0.1% sodium deoxycholate/0.1% sodium dodecyl sulphate) containing 100 mg/ml PMSF and 1 mg/ml aprotinin, and incubated on ice for 45 min. The homogenized tissues were centrifuged, and the supernatant was collected, treated with 400 mg/ml RNase A at 37°C for 15 min, treated with 400 mg/ml proteinase K at 37°C for 1 h, and then precipitated with isopropyl alcohol containing NaCl. The precipitated DNA pellets were redissolved in distilled water and fractionated on 1.8% agarose gels. The gels were stained with ethidium bromide and then photographed under UV light.
Caspase activity assay
Activities of caspase-3, -8 and -9 in amnion tissues were determined using caspase assay kits (MBL, Nagoya, Japan), according to the manufacturer's protocol with minor modifications. Briefly, human amnion samples and HHUA cells (derived from human endometrial carcinoma cell line) treated with a mouse anti-human Fas IgM antibody (MBL, Nagoya, Japan) were immediately rinsed with PBS, and the tissues were then homogenized with RIPA buffer containing 100 mg/ml PMSF and 1 mg/ml aprotinin. The homogenized tissues were incubated on ice for 45 min and then cleared by centrifugation. Protein concentrations were determined by the Bradford assay (Bio-Rad, CA, USA). Whole cell lysates (100 µg) were incubated with substrates (DEVD-p-nitroanilide for caspase-3, IETD-p-nitroanilide for caspase-8, and LEHD-p-nitroanilide for caspase-9) at 37°C for 90 min. The p-nitroanilide light emission from the substrates digested by caspase was quantified at 405 nm. Activities of caspases were expressed as optical density at 405 nm.
Statistical analysis
Data are shown as mean ± SD. Significant differences were assessed by a two-sided analysis of variance and subsequently by Scheffé's test. In the case of comparison between two groups, a two-sided Student's t-test was used (software, StatView; Abacus Concepts Inc., CA, USA).
Results
TUNEL/LM study
The TUNEL/LM study showed only a few TUNEL-positive cells in the amniotic epithelium through weeks 11–39 of gestation (Figure 1A). In contrast, the TUNEL-positive cells abruptly increased in number at term (Figure 1B). In the TUNEL-positive cells, DAB reaction products indicating the sites of free 3'-OH DNA ends were consistent with the localization of the nuclear heterochromatin, showing a peripheral granular staining pattern. One noteworthy finding was that the epithelial cells with intense TUNEL reactivity were superimposed on the cells with weak reactivity, and were often detached from the epithelium. The mean percentages of TUNEL-positive epithelial cells in the five groups during pregnancy are shown in Figure 2. The percentages of TUNEL-positive cells (%) at weeks 11–15, 16–27 and 28–39 were similar to each other. However, there was a statistically significant difference in percentages of TUNEL-positive cells between the weeks 40–41 and preterm groups (P < 0.01), and between the weeks 40–41 and post-term group (P < 0.05).
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There was no statistically significant difference in the percentage of TUNEL-positive cells between the subjects that underwent labour (n = 15; 8.4 ± 17.2%) and those that did not (n = 19; 6.3 ± 15.2%), and between the vaginal delivery group (n = 13, 9.6 ± 18.2%) and the Caesarean section group (n = 9, 12.5 ± 21.0%) from the weeks 28–42 of gestation.
TUNEL/TEM study
The ultrastructural features of human amniotic epithelial cells during pregnancy in this study agreed well with those reported by previous investigators (Bourne and Lacy, 1960). In brief, the normal amniotic epithelial cells before term were characterized by the presence of numerous microvilli on their surface, many granules in the cytoplasm and well-developed desmosomes between the adjacent cells.
In this study, only a few immunogold particles indicating the sites of free 3'-OH DNA ends were distributed on the peripheral nuclear chromatin amniotic epithelial cells before term (Figure 3A). At term, cells with various nuclear changes were observed in the amniotic epithelium. As the peripheral nuclear chromatin increased and became confluent, the nuclei exhibited a dappled cloth-like appearance (Figure 3B) and chromatin condensation, together with an increase in the number of immunogold particles (Figure 3C). Associated with the nuclear changes, some apoptotic cells exhibited poorly developed microvilli and reduced cytoplasm volume, and therefore changed from cuboidal to spherical shape and were detached from the epithelium, losing their junctional apparatus such as desmosomes. Cytoplasmic organelles were well preserved morphologically compared to the nuclei. No typical apoptotic bodies were observed at any time during the course of gestation.
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At post-term, some amniotic epithelial cells showed dissolution of nuclear chromatin and cytoplasmic organelles could not be detected (data not shown).
Image analysis combined with TUNEL/TEM
The average TUNEL/TEM labelling density of free 3'-OH DNA ends in the nuclei of amniotic epithelial cells is presented in Figure 4. The labelling density was the highest in the nuclei in weeks 40–41 of gestation and declined thereafter.
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Immunohistochemistry
The results of immunohistochemistry for Bcl-2, Fas, and FasL in the human amnion are summarized in Table II
. Bcl-2 was expressed in the syncytiotrophoblasts of the human placenta at term (Figure 5A). Immunoreactivity of Bcl-2 was not noted in amniotic epithelial or mesenchymal cells throughout the gestational period (Figure 5B). Fas expression was observed in both amniotic epithelial and mesenchymal cells throughout the gestational period, showing increased immunoreactivity of amniotic epithelial cells from weeks 16–27 to 40–41 of gestation. FasL was expressed in both epithelial and mesenchymal cells from weeks 11–15 to 40–41 of gestation, while both cell types did not exhibit FasL expression in week 42 of gestation. The most intense expression of FasL in the amniotic epithelial cells was observed in weeks 40–41 of gestation. Interestingly, the strong immunoreactivities of Fas and FasL were observed on the apical membrane of amniotic epithelial cells (Figure 5C, D).
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There was no significant difference between Caesarean and vaginal deliveries, as well as no association with the presence or absence of labour, in terms of the expression of Bcl-2, Fas and FasL during the gestational periods.
DNA ladders
Agarose gel electrophoresis of extracted DNA did not show typical DNA ladders in all amniotic samples examined, though faint bands corresponding to DNA ladders were detected in one amnion sample in week 40 of gestation (case no. 27: data not shown).
Caspase activity
Activities of caspase-3 and caspase-8 in the human amnion samples were higher in weeks 40–41 than in weeks 16–27 of gestation (P < 0.01). No statistically significant difference in the activity of caspase-9 was observed between the two groups. There was no significant difference in the activities of caspase-3, -8 and -9 between weeks 16–27 and 28–39 of gestation (data not shown). Activities of caspase-3, -8 and -9 in apoptotic HHUA cells induced by anti-Fas IgM antibody were higher than those in control HHUA cells (P < 0.01) (Figure 6).
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Discussion
The TUNEL method, along with DNA agarose gel electrophoresis, is widely used for distinguishing apoptotic cells from necrotic cells. This study using the TUNEL/LM method clearly demonstrated that the percentage of apoptotic cells in the human amniotic epithelium was highest in weeks 40–41 compared with other gestational periods. This finding is supported by a previous finding of an increase in the percentage of apoptotic cells in rat amniotic epithelium before the onset of active labour (Lei et al., 1996). In contrast, it has been reported (Runic et al., 1998) that the percentage of apoptotic cells (range 8–29%; the same as that of the apoptotic index) in the human amniotic epithelium was the highest in weeks 23–30. This discrepancy may be explained by the difference in the samples used; the tissue samples of Runic et al. (1998) were obtained from subjects with uncomplicated pregnancies or pregnancies complicated by diabetes, chorioamnionitis, or premature rupture of membranes, whereas our samples were collected only from subjects with uncomplicated pregnancies.
In this study, DNA agarose gel electrophoresis using fetal membranes revealed some faint bands in only one case at term (case no. 27), whereas many TUNEL-positive cells in the amniotic epithelium were observed in the same case. This discrepancy may be due to the different targets detected by the two methods: extracted DNA for agarose gel electrophoresis and cells with free 3'-OH DNA ends for the TUNEL/LM method. It is well documented that DNA agarose gel electrophoresis does not always show DNA laddering in apoptosis when the extracted DNA contains only a low level of DNA fragmentation in multiples of 180–200 bp or high mol. wt DNA fragmentation (Walker et al., 1993). It was actually difficult to obtain an adequate amount of DNA for agarose gel electrophoresis from the fetal membranes used, while cryosections for the TUNEL/LM method contained sufficient cells for the detection of apoptosis. If DNA agarose gel electrophoresis was performed on cytocentrifuged cells using the amniotic fluid containing many apoptotic cells at term, the typical DNA ladder pattern might have been obtained from the samples. However, it was very difficult to collect sufficient amniotic fluid to analyse DNA fragmentation on delivery, particularly in cases with ruptured membranes. Several studies (Ansari et al., 1993; Migheli et al., 1995; Hayashi et al., 1998) have indicated that the use of the TUNEL/LM method stains not only apoptotic cells but also necrotic ones, because free 3'-OH DNA ends can also be produced during random DNA digestion in necrosis. Thus, we employed the TUNEL/TEM method to specifically identify apoptotic cells because of the disadvantages of both the DNA agarose gel electrophoresis and the TUNEL/LM methods.
The TUNEL/TEM method enabled us to detect quantitative changes in the number of free 3'-OH DNA ends and to simultaneously observe the ultrastructural features of apoptosis in human amniotic epithelial cells. Apoptotic cells in the amniotic epithelium at term showed the highest labelling density of free 3'-OH DNA ends throughout the gestational period, accompanied by some of the characteristics of apoptosis other than the presence of apoptotic bodies. In recent years, a TEM study (Paavola et al., 1995) demonstrated that rat amniotic epithelial cells exhibited signs of impending cell death characterized by the loss of desmosomes between adjacent epithelial cells and nuclear changes such as the increase in heterochromatin level prior to the complete dissolution of chromatin. The ultrastructural changes of the human amniotic epithelium observed in our present study are comparable to those observed by Paavola et al. (1995).
The biological roles of apoptosis in the female reproductive system are well known. Several reports concerning human endometrial apoptosis have already shown that the co-expression of Fas and FasL (Yamashita et al., 1999) and the cyclic expression of Bcl-2 peaking in the proliferative phase (Otsuki et al., 1994; Tabibzadeh et al., 1995) were both strongly related to apoptosis in the human endometrium. Bcl-2 expression in the human chorion, in relation to fibrin deposition, has been reported (Marzioni et al., 1998). Contrary to our expectations, Bcl-2 was not expressed in human amniotic epithelial cells at any period during the course of gestation. This finding supports the recent study of apoptosis in fetal membranes at term (McLaren et al., 1999), showing that the fetal membrane failed to exhibit significant immunoreactivity for Bcl-2 but exhibited strong immunoreactivity for Bax. Therefore, Bcl-2 expression patterns in the human endometrium and the chorion are clearly different from that in the human amniotic epithelium. In contrast, there are similar expression patterns for Fas and FasL among the three tissues. Fas/FasL signalling can actually promote the operation of the mitochondrial pathway via Bid, although its main pathway is the death receptor pathway. Caspase-8-mediated cleavage of Bid is translated from the cytosol to the mitochondria where it promotes the exit of cytochrome c. Cytochrome c is then associated with Apaf-1 and procaspase-9 activates caspase-9 and caspase-3 (Hengartner, 2000). Therefore, the death-receptor and mitochondrial pathways converge at the level of caspase-3 activation. However, cross-talk between the two pathways is minimal under most conditions, and they operate largely independently of each other (Earnshaw et al., 1999; Yin, et al., 1999). In this study, activities of caspase-3 and caspase-8, but not that of caspase-9, were increased in fetal membrane at term, while HHUA cells constantly expressing Fas on their surfaces exhibited high activities of caspase-3, -8 and -9 after treatment with an anti-Fas antibody. These findings suggest that apoptosis in fetal membranes at term is induced by the death receptor pathway independently of the mitochondrial pathway involving Bcl-2 regulation, while both pathways may be effective for HHUA cells treated with an anti-Fas antibody.
In the present study, Fas and FasL were consistently co-expressed in both term and preterm amniotic epithelial cells. This finding leads to the question of why preterm amniotic epithelial cells scarcely undergo apoptosis. The answer to this question may concern the localization of FasL in the fetal membrane. It is well known that there are two forms of FasL: membrane-bound FasL and soluble FasL. The membrane-bound FasL is converted to the soluble FasL by the action of certain matrix metalloproteinases (MMP) (Tanaka et al., 1996). Recent studies have reported that the activities of MMP, such as MMP-1, -3 and -9, are increased in fetal membranes at term prior to parturition (Vadillo-Ortega et al., 1995; Bryant-Greenwood, 1998; Lei et al., 1999; MacLaren et al., 2000). The immunohistochemistry of Fas and FasL in this study clearly showed that the localization of Fas and FasL was limited to the apical membrane of amniotic epithelial cells. Based on these findings, we can speculate that the membrane-bound FasL is cleaved by the action of MMP at term, resulting in an increase in the amount of soluble FasL in the amniotic fluid and the activation of Fas/FasL signalling on the cell surface of amniotic epithelial cells (Figure 7). These epithelial cells can then undergo apoptotsis and are shed from the amniotic epithelium into the amniotic cavity at term, independent of the onset of labour.
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We conclude that apoptosis in the amniotic epithelium at term is induced by the death-receptor pathway independent of Bcl-2 regulation and the onset of labour, and may play an important role in the fragility and rupture of human fetal membranes at term.
Acknowledgements
We are grateful to our colleagues who collected the clinical materials for the study at the Department of Obstetrics and Gynecology, Osaka Medical College. We also thank E.Shintani and H.Hiyama for their secretarial assistance. This work was supported in part by a Grant-in-Aid for General Scientific Research from the Ministry of Education, Science, Sports and Culture in Japan (No. 10671576).
Notes
3 To whom correspondence should be addressed. E-mail: an1001{at}art.osaka-med.ac.jp 
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Submitted on January 3, 2001; accepted on May 8, 2001.
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