Preterm Birth Research Group, Department of Obstetrics & Gynaecology, University of Leicester, Clinical Sciences Building, Leicester Royal Infirmary, P. O. Box 65, Leicester LE2 7LX, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: apoptosis/fetal membrane/membrane rupture/preterm birth
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanism responsible for the reduction in the cellularity of the cytotrophoblastic and decidual layers is unclear. Since the fetal membranes cease to grow during the latter half of pregnancy (Parry-Jones and Priya, 1976) it could be anticipated that this is a result of either decreased proliferation with a constant rate of cell death or increased rate of cell death with a constant rate of proliferation. Although microscopical examination does not support the process of widespread necrosis (McLaren et al., 1999
), apoptosis, which is the histological manifestation of programmed cell death, could provide a mechanism for the selective regional removal of these cells. Apoptosis is critical to a number of physiological and pathological processes (Williams, 1994
) and is a distinct form of cell death, being an active energy-requiring process in which single cells die and are removed by phagocytosis without producing an inflammatory reaction (Savill, 1994
). It is characterized by specific morphological features, including DNA fragmentation and the condensation of nuclear heterochromatin and the appearance of dense `apoptotic bodies' (Kerr and Winterford, 1993
; Smith et al., 1997
), which are identified by light and electron microscopy. The `gold standard' for the detection of apoptosis is its characterization by morphological criteria, with the most common sign of apoptosis within tissue sections being the presence of apoptotic bodies (Hall, 1999
).
Apoptosis is controlled by the expression of a number of regulatory genes, including c-myc, p53 and apo-1/fas (Osborne and Schwartz, 1994; White, 1993
). One of the most important advances in our understanding of apoptotic cell death has come from studies of the oncogenic Bcl-2 family. BCL-2 protein promotes cell survival by blocking apoptosis induced by a range of stimuli including growth factor withdrawal (Nunez and Clarke, 1994
). It is now clear that the anti-apoptotic properties of BCL-2 depend acutely on its interaction with a number of potent antagonistic proteins (Boise et al., 1993
; Kiefer et al., 1995
; Yang et al., 1995
). In particular, studies have shown that a related protein, BAX, antagonizes the survival-promoting activity of BCL-2 (Oltvai et al., 1993
). BCL-2 needs to form heterodimers with BAX in order to function, and it is the ratio of BCL-2 to BAX which predetermines the cell's susceptibility to a given apoptotic stimulus (Oltvai and Korsmeyer, 1994
).
Apoptosis is known to occur within both rat amnion (Lei et al., 1996, 1999
), human fetal membrane (Runic et al., 1998
) and placental trophoblasts (Smith et al., 1997
; Uckan et al., 1997
), the incidence of which has been determined using the method of terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) to detect DNA fragmentation (Runic et al., 1998
). However, the high incidence of TUNEL-positive cells, in the absence of membrane destruction, has cast doubt on the suitability of this method to detect the true incidence of apoptosis in fetal membranes. In addition, the authors failed to determine whether this phenomenon exhibits regional differences in its incidence within fetal membranes, prior to labour. Therefore, in this study we sought to: (i) determine if apoptosis could account for the selective regional reduction of the cellularity of the cytotrophoblastic and decidual cells within the fetal membrane, prior to labour; and (ii) determine the protein localization of the BCL-2/BAX regulatory proteins in fetal membranes at term, prior to labour.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue sampling
Fetal membranes were regionally biopsied from two principal sites according to our previous protocol (Malak and Bell, 1994, 1996
). (i) Cervical area: following the delivery of the baby, the fetal membranes overlying the cervix were located and marked by the application of a Babcock clip. The area within the jaws of the clip was not used since light microscopy revealed that this area was too damaged to be included. An area of 23 cm2 around the clip was sampled. (ii) Mid-zone area: half-way between the cervical area and the placental edge. This was usually 1012 cm from the cervical area. Specimens were then washed briefly in phosphate-buffered saline (PBS) (pH 7.4). Fetal membrane strips were rolled with the amnion innermost and then fixed in buffered (pH 7.6) formalin for 24 h before processing and mounting in paraffin wax.
Apoptosis identification and quantification
Light microscopy
Tissue sections (68 µm) were cut from paraffin blocks and then stained with haematoxylin and eosin. Apoptotic cells were identified as those having the presence of characteristic apoptotic bodies (Kerr and Winterford, 1993). These were clearly visible under oil (x1000) magnification and appeared as either single or multiple dense bodies, which maintained a consistent density when viewed through planes. Identification of these as apoptotic bodies was confirmed following electron microscopy of a selection of light microscopic identified cells (see below).
Electron microscopy
Fetal membrane rolls were initially fixed in 2.5% glutaraldehyde in sodium cacodylate buffer pH 7.2, overnight. After fixation the tissue was post-fixed in 1% osmium tetroxide, dehydrated through ascending grades of alcohol, embedded in Epon and sectioned on an ultamicrotome. Thin sections (60 nm) were stained with saturated uranyl acetate and aqueous lead citrate for eventual electron microscopy work.
Apoptotic cell quantification
Quantification of apoptotic cells, within the fetal membrane layers, was achieved following cell counting using the Apple Macintosh Centris Running Image System, version 1.49. For each fetal membrane roll 1020 fields were viewed for both the amniotic epithelium, connective tissue layer, cytotrophoblast and decidual layers, at a magnification of x1000. Routinely between 500 and 1000 cells were counted per roll. Each single or group of dense bodies was counted as a single apoptotic cell. The number of apoptotic cells was expressed as a percentage of the total number of cells counted within layer.
Immunohistochemical staining for BCL-2, BAX and Ki-67
Assessment of BCL-2, BAX and Ki-67 proteins was performed on formalin-fixed, paraffin-embedded tissue sections for each antibody in the different regions sampled prior to labour. Serial tissue sections were de-waxed in xylene and hydrated gradually through graded alcohols. These were then microwaved (300 W) in 0.01 mol/l citrate buffer for 30 min, before washing in PBS. Endogenous peroxidase activity was quenched following a 10 min incubation in a 6% solution of hydrogen peroxide. The slides were then treated with a 20% solution of rabbit serum before this was removed and replaced with either 1/40 dilution of mouse monoclonal antibody to BCL-2 (Biogenesis, Poole, UK), BAX (Santa Cruz Biotechnology, Santa Cruz, USA), or Ki-67 (Dako). Positive controls for each antigen were included as well as negative controls in which the primary antibody was omitted and replaced with an irrelevant mouse IgG (Dako). These controls were run in parallel with the fetal membrane samples. The slides were incubated at room temperature for 2 h and then washed in PBS, pH 7.5 for 15 min. A 1/200 dilution of rabbit anti-mouse secondary antibody (Dako) was then added and left for 1 h at room temperature. Following this the slides were washed three times for 15 min and complexed with avidinbiotin peroxidase (Vector, Peterborough, UK) for 30 min. The complex was detected with diaminobenzidine and hydrogen peroxide in 0.1 mol/l TrisHCl, pH 7.5, before the slides were washed and the colour developed using DAB. Blue/black colouration was achieved following the addition of a Nickel solution.
Statistics
Data were given as means ± SD and were normally distributed, therefore parametric paired Student's t-tests were undertaken to determine statistical differences between cervical and mid-zone regions; this was performed for apoptotic-body-positive cells.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The lack of immunoreactive BCL-2 in the fetal membranes and any regional variations suggests that it is not involved in the protection of the cells in this tissue from apoptotic stimuli. The strong immunoreactive BAX staining found within the decidual layer may provide evidence for its potential role in decidual cell apoptosis. However, we observed a lack of apoptosis in this layer suggesting that other anti-apoptotic mechanisms could be involved. The absence of BAX staining within the cytrophoblast layer suggests its non-participation in the mechanisms controlling apoptosis in this layer. These results imply that other factors may be involved in the control of this process. Candidates include the other members of the now extensive Bcl-2 gene family, which can have either pro- or anti-apoptotic regulating activity (Brown, 1996). Alternatively, other potential regulators include Fas receptor and tumour necrosis factor (TNF) receptor 1 (TNFR1) which are cell surface receptors and which mediate apoptosis after binding of Fas ligand and TNF
, respectively. Indeed the presence of Fas ligand and its receptor has been identified throughout the fetal membrane (Runic et al., 1996
, 1998
) as well as TNF
and TNFR1 (Fortunato et al., 1994
). However, their significance as regulators of apoptosis under these conditions is unclear since relative levels of their expression both within the different layers of the fetal membrane and between different regions is unknown. Moreover their presence alone does not guarantee activation of this apoptotic pathway. It is clear that further work is required to clarify the mechanisms involved in the selective regional increase in apoptosis within the cytotrophoblastic populations.
The mechanism(s) responsible for the increased apoptosis in the cytotrophoblastic layer of the membranes overlying the cervix remains to be examined. It is possible that it is the result of a direct para/autocrine induction. Alternatively it may be an indirect effect of extracellular matrix degradation caused by the action of the extracellular matrix degrading matrix metalloproteinase enzymes which also exhibit higher levels in the fetal membranes overlying the cervix prior to labour (McLaren et al., 1998). Indeed recent work, using a rat amnion culture model and cultures of human amnion cells (WISH), has demonstrated that increasing matrix metalloproteinase-9 expression results in an increase in apoptosis and cell detachment (Lei et al., 1999
).
The reduction of the cytotrophoblast layer of the fetal membranes overlying the cervix is associated with the swelling and dissociation of the overlying strength-giving connective tissue layers, which are suggested to be associated with susceptibility for membrane rupture at this site (Malak and Bell, 1994; McLaren et al., 1999
). It is possible that the cytotrophoblastic cells produce factors favouring extracellular matrix synthesis (So et al., 1992
; Vadillo-Ortega et al., 1995
), which are lost during its involution, or indeed apoptosis itself could be associated with factors which induce activity of extracellular matrix-degrading enzymes by its constituent cells (Kakinuma et al., 1997
). The reduction of the cytotrophoblast layer also occurs in parallel with the preparatory changes in the cervix and myometrium during phase 0 of parturition (Casey and MacDonald, 1993
) and therefore may have important implications not only for susceptibility of the membrane to rupture but also for the initiation of parturition. It is well established that membrane stripping from the uterine wall is an effective method of labour induction (Berghella et al., 1996
) and may indicate a potential causal relationship between undefined biophysical/biochemical states of the fetal membranes and labour (Malak and Bell, 1994
). Indeed the amnion of the fetal membranes is a potent source of uterotonins, e.g. prostaglandins (PG) (Okazaki et al., 1981
) and endothelin-1 (Sagawa et al., 1994
). The increased importance of this layer in determining the eventual bioavaliability of uterotonins such as PG is highlighted by recent studies which show a lack of regionality in the synthesis of PGE2 (Sawdy et al., 1999
). Yet paradoxically the cells of the cytotrophoblastic layer possess the capacity to degrade these uterotonins by virtue of its expression of an endopeptidase which degrades endothelins (Germain et al., 1994
), and 15-hydroxyprostaglandin dehydrogenase (PGDH), the principle enzyme responsible for the metabolism of prostaglandins (Sangha et al., 1994
).
We have previously proposed that the regional loss of the cytotrophoblast layer of the fetal membranes overlying the cervix, and hence regional loss of functional capacity, before labour, may allow these factors to escape degradation and to act locally to stimulate the maturation of the cervix and myometrium of the lower segment in preparation for labour (Malak and Bell, 1996). Furthermore the premature and extensive loss of this layer, which we have demonstrated in cases of preterm birth (Malak et al., 1993
), raises the possibility of the premature action of uterotonins to induce idiopathic preterm labour. This is supported by, and provides a rationale for, the reports of a subset of patients presenting with preterm labour who have a reduced expression of PGDH in their chorionic cytotrophoblast (Sangha et al., 1994
) and the demonstration of reduced prostaglandin catabolism in the fetal membranes from the lower uterine segment (Van Meir et al., 1996
, 1997
).
Elucidation of the mechanisms involved in the control of apoptosis in the cytotrophoblastic layer therefore may provide insight into the control of both the preparatory phases of parturition, in terms of the cervix and myometrium, and for fetal membrane rupture in term and preterm birth.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boise, L.H., Gonzalez-Garcia, M., Postema, C.E. et al. (1993) Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell, 74, 597608.[ISI][Medline]
Brown, R. (1996) The bcl-2 family of proteins. Br. Med. Bull., 53, 466477.[Abstract]
Casey, M.L. and MacDonald, P.C. (1993) Human parturition: distinction between the initiation of parturition and the onset of labour. Semin. Reprod. Endocrinol., 11, 272284.[ISI]
Fortunato, S.J., Menon, R. and Swan, K.E. (1994) Expression of TNF-alpha and TNFR p55 in cultured amniochorion. Am. J. Reprod. Immunol., 32, 188193.[ISI][Medline]
Germain, A.M., Smith, J., Casey, M.L. and MacDonald, P.C. (1994) Human fetal membrane contribution to the prevention of parturition: uterotonin degradation. J. Clin. Endocrinol. Metab., 78, 463470.[Abstract]
Hall, M.H., Danielian, P. and Lamont, R. F. (1997) The importance of preterm birth. In Elder, M.G., Lamont, R.F. and Romero, R. (eds), Preterm Labor, Churchill Livingstone, pp. 128.
Hall, P. A. (1999) Assessing apoptosis: a critical survey. Endocrine-Related Cancer, 6, 38
Kakinuma, C., Kuwayama, C., Kaga, N. et al. (1997) Trophoblastic apoptosis in mice with preterm delivery and its suppression by urinary trypsin inhibitor. Obstet. Gynecol., 90, 117124.
Keirse, M.J., Ohlsson, A. and Treffers, P. (1989) Prelabour rupture of the membranes at preterm. In Chalmers, I., Enkin, M. and Keirse, M.J. (eds), Effective Care in Pregnancy and Childbirth. Oxford University Press, Oxford, pp. 666686.
Kerr, J.M., Johnstone, R. and Phillips, M. (1954) In Historical review of British obstetrics and gynaecology. E. and S. Livingston, pp. 180195.
Kerr, J.F.R. and Winterford, C.M. (1993) Apoptosis. Cancer, 73, 20132026.[ISI]
Kiefer, M.C., Brauer, M.J., Powers, V.C. et al. (1995) Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak. Nature, 374, 736739.[ISI][Medline]
Lei, H., Furth, E., Kalluri, R. et al. (1996) A program of cell death and extracellular matrix degradation is activated in the amnion before the onset of labor. J. Clin. Invest., 98, 19711978.
Lei, H., Kalluri, R., Furth, E.E. et al. (1999) Rat amnion type IV collagen composition and metabolism: implications for membrane breakdown. Biol. Reprod., 60, 176182.
Malak, T.M. and Bell, S.C. (1994) Structural characteristics of term human fetal membranes: a novel zone of extreme morphological alteration within the rupture site. Br. J. Obstet. Gynaecol., 101, 375386.[ISI][Medline]
Malak, T.M. and Bell, S.C. (1996) Fetal membranes structure and prelabour rupture. Fetal Maternal Med. Rev., 8, 143164.
Malak, T., Mulholland, G. and Bell, S. (1993) Structural and morphometric characteristics of the fetal membranes in preterm birth. J. Reprod. Fertil., 12, 48
McLaren, J., Taylor, D.J and Bell, S.C. (1998) MMP-9 activity is increased in a structurally altered region of non-labour affected fetal membrane; implications for pre-labour rupture of the fetal membranes. J. Soc. Gynecol. Invest., 5, 59A (abstract 82).
McLaren, J., Malak, T.M. and Bell, S.C. (1999) Structural characteristics of term human fetal membranes, prior to labour: identification of an area of altered morphology overlying the cervix. Hum. Reprod., 14, 237241.
Nunez, G. and Clarke, M.F. (1994) The bcl-2 family of proteins: regulators of cell death and survival. Trends Cell. Biol., 4, 399403.
Okazaki, T., Casey, M.L., Okita, J. et al. (1981) Initiation of parturition XII. Biosynthesis and metabolism of prostaglandins in human fetal membranes and uterine decidua. Am. J. Obstet. Gynecol., 139, 373381.[ISI][Medline]
Oltvai, Z.N. and Korsmeyer, S.J. (1994) Checkpoints of duelling dimers foil death wishes. Cell, 79, 189192.[ISI][Medline]
Oltvai, Z.N., Milliman, C.L. and Korsmeyer, S.J. (1993) Bcl-2 heterodimerises in vivo with a conserved homologue, Bax that accelerates programmed cell death. Cell, 74, 609619.[ISI][Medline]
Osborne, B.A. and Schwartz, L.M. (1994) Essential genes that regulate apoptosis. Trends Cell. Biol., 4, 394403.
Parry-Jones, E. and Priya, S. (1976) A study of the elasticity and tension of fetal membrane and the relation of the area of the gestation sac to the area of uterine cavity. Br. J. Obstet. Gynaecol., 83, 205212.[ISI][Medline]
Runic, R., Lockwood, C.L., Ma, Y. et al. (1996) Expression of Fas ligand by human cytotrophoblasts: implication and fetal survival. J. Clin. Endocrinol. Metab., 81, 31193122.[Abstract]
Runic, R., Lockwood, C.J., Lachapelle, L. et al. (1998) Apoptosis and fas expression in human fetal membranes. J. Clin. Endocrinol. Metab., 83, 660666.
Sagawa, N., Hasegawa, M., Itoh, H. et al. (1994) Secretion of endothelin by avascular amnion tissue: possible roles in human pregnancy. Endocrine. J., 41, s117s125.[ISI]
Sangha, R.K., Walton, J.C., Ensor, C.M. et al. (1994) Immunohistochemical localisation, messenger ribonucleic acid abundance, and activity if 15-hydroxyprostaglandin dehydrogenase in placenta and fetal membranes during term and preterm labor. J. Clin. Endocrin. Metabol., 78, 982989.[Abstract]
Savill, J. (1994) Apoptosis in disease. Eur. J. Clin. Invest., 24, 715723.[ISI][Medline]
Sawdy, R.J., Dennes, W.J.B., Allport, V. et al. (1999) Region and labour-dependent synthesis of prostaglandin E2 by human fetal membrane. Placenta, 20, 181184.[ISI][Medline]
Smith, S. C., Baker, P. N., Symonds, E. M. (1997) Increased placental apoptosis in intrauterine growth restriction. Am. J. Obstet. Gynecol., 177, 13951401.[ISI][Medline]
So, T., Ito, A., Sato, T., Mori, Y. and Hirakawa, S. (1992) Tumor necrosis factor alpha stimulates the biosynthesis of matrix metalloproteinases and plasminogen activator in cultured human chorionic cells. Biol. Reprod., 46, 772778.[Abstract]
Uckan, D., Steele, A., Cherry, B. et al. (1997) Trophoblasts express Fas ligand: a proposed mechanism for immune privilege in placenta and maternal invasion. Mol. Hum. Reprod., 3, 655662.[Abstract]
Vadillo-Ortega, F., Gonzalez-avila, G., Furth, E.E. et al. (1995) 92kd-type IV collagenase (matrix metalloproteinase-9) activity in human amniochorion increases with labour. Am. J. Pathol., 146, 148156.[Abstract]
Van Meir, C.A., Sangha, R.K., Walton, J.C. et al. (1996) Immunoreactive 15-hydroxyprostaglandin dehydrogenase (PGDH) is reduced in fetal membranes from patients at preterm delivery in the presence of infection. Placenta, 17, 291297.[ISI][Medline]
Van Meir, C.A., Ramirez, M.M., Matthews, S.G. et al. (1997) Chorionic prostaglandin catabolism in decreased in the lower uterine segment with term labour. Placenta, 18, 109114.[ISI][Medline]
White, E. (1993) Death-defying acts: a meeting review on apoptosis. Genes Dev., 7, 22772284.[ISI][Medline]
Williams, G.T. (1994) Apoptosis in the immune system. J. Pathol., 173, 14.[ISI][Medline]
Yang, E., Zha, J., Jockel, J. et al. (1995) Bad, a heterodimeric partner for bcl-xl and bcl-2, displaces bax and promotes cell death. Cell, 80, 285291.[ISI][Medline]
Submitted on May 11, 1999; accepted on August 4, 1999.