1 Department of Obstetrics and Gynaecology, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, P.O. Box 65, Leicester LE2 7LX, UK
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Abstract |
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Key words: amnion/chorion/fetal membranes/preterm birth/PROM
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Introduction |
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To account for the phenomenon of fetal membrane rupture, many studies have attempted to identify features of the fetal membranes that may be uniquely associated with the site of rupture after their spontaneous rupture; unfortunately these studies have been contradictory (Bou-Resli et al., 1981; Halburt et al., 1989). However, the comprehensive mapping of fetal membranes, obtained as frozen sections after their spontaneous rupture at term, detected an area of the fetal membranes that exhibited unique morphological features which were only found within a restricted area along the line of rupture (Malak and Bell, 1994
). This restricted area has been termed the `zone of altered morphology' (ZAM) and the features described were consistent with its potential structural weakness (Malak and Bell, 1994
, 1996
). These included marked disruption of the connective tissue layers and marked reduction of the thickness, and hence cellularity, of both the cytotrophoblast and decidual layers. Given the structural features of the ZAM and its restricted localization to an area within the rupture line, it has been proposed that the ZAM may have represented the site of initial fetal membrane rupture in response to the increased intra-amniotic pressures experienced during labour (Malak and Bell, 1993
; Bell and Malak, 1997
).
The aim of the present study was to determine if the structural changes characteristic of the ZAM were present prior to labour. A biopsy procedure was designed with reference to the fetal membranes overlying the cervix since preliminary studies employing frozen sections indicated that this area exhibited altered structural characteristics (Abdel-Malak et al., 1993). It had also been demonstrated that the area of the fetal membranes over the cervix was always detected within the final rupture line after their spontaneous rupture after term (Bourne, 1962
). Confirmation of these regional structural differences in the fetal membranes before labour and rupture may indicate that an area of the fetal membranes is generated prior to labour providing a region of structural weakness and hence a programmed susceptibility to rupture.
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Materials and methods |
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Mid-zone area
This was half-way between the cervical area and the placental edge, more than 1012 cm from the cervical area. At least three specimens were taken from each area per patient. Specimens were then washed briefly in phosphate-buffered saline (pH 7.4). Fetal membrane strips were rolled with the amnion innermost and then fixed.
Tissue fixation
Formalin-fixed tissue sections
Fetal membrane rolls were placed in buffered (pH 7.6) formalin and fixed for 24 h before processing and mounting in paraffin wax.
Cryosat tissue sections
Rolls of fetal membranes were placed in plastic cups filled with tissue embedding compound; Tissue-Tek, OCT (BDH, Poole, UK) tissue blocks were snap-frozen in liquid hexane and dry-ice mixture (70°C), then stored at 80°C until used. Samples for formalin fixation and frozen tissue were not taken from the same patient, since correct specific localized sampling would only allow for the assessment of one tissue treatment per patient. Thin tissue sections were cut and then stained with haematoxylin and eosin. Stained tissue sections were examined under light microscopy connected to an image capture system. This system comprised a Apple Macintosh Centris Running Image (TM) version 1.49. Each section of roll was divided into quadrants and areas of thickness were measured along the intersections of 12 to 6 o'clock and 3 to 9 o'clock. This was done for each roll with 810 intersections counted per roll.. Each captured image was measured following calibration of the image with an internal scale slide. The thicknesses (µm) of the fetal membrane connective tissue, trophoblast and decidual layers were determined. Measurements were taken only from sections cut vertically, showing a single layer of amniotic epithelium. Separation of the amnion and chorion layers occasionally occurred in the formalin-fixed paraffin-embedded tissue; the spaces were subtracted from the final measurement in order to obtain the final connective tissue layer. Thickness measurements were represented as mean ± SD.
Statistics
Data values from formalin-fixed paraffin-embedded tissue were normally distributed and comparisons were made between the two different membrane zones using Student's t-tests. Frozen tissue samples, which were normally distributed, had originally included additional membrane areas to those fixed in formalin, therefore two-way analysis of variance was carried out. Scheffé's test was used for subsequent comparisons between different membrane zones.
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Results |
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Discussion |
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Previous work on spontaneously ruptured fetal membranes obtained at term and after vaginal delivery identified an area of structurally altered morphology, termed the ZAM (Malak and Bell, 1994). The ZAM was always detected as a restricted area within the post-delivery rupture tear and was most frequently located at one end of the tear. The alterations we describe in the present study in the cervical zone were similar, but less extreme than those observed in the ZAM. Previous workers have alluded to the possibility that the area directly over the cervix is associated with the rupture site and the studies of Bourne et al. (1962) directly supports the hypothesis. These workers, by staining the area of the fetal membranes overlying the cervix with a trypan blue dye accessed through the cervix prior to rupture and delivery, demonstrated that this area was included in the post-delivery tear and was located at one end of the tear. Given the similarities, the ZAM most likely arises from the cervical area of the fetal membranes we have detected prior to labour, with the increased structural alterations resulting from the combined effects of the process of labour and delivery.
The temporal development of these changes in the cervical membranes during normal pregnancy is not known, but indirect evidence may help to characterize its development during the latter weeks. Oncofetal fibronectin, which is present at high concentrations in fetal membranes and in amniotic fluid, is detected in cervico-vaginal secretions up to 34 weeks prior to labour (Lockwood et al., 1991, 1993
). The structural features of the fetal membranes overlying the cervix suggests that this fibronectin could be released from such a disrupted extracellular matrix or could leak from the amniotic fluid through such a matrix; this may indicate that the kinetics of appearance in the cervico-vaginal secretions reflects the generation of changes in the cervical zone.
Given the degradative nature of the connective tissue layer disruption and that these morphological changes are consistent with the disruption of the major components of the connective tissue, the collagens, this would suggest the involvement of proteolytic enzymes, in particular matrix metalloproteinases. These are a family of zinc-dependent enzymes responsible for the degradation of most of the components of the extracellular matrix (for review see Birkedal-Hansen et al., 1993). There is accumulating evidence to suggest that labour and delivery induces the synthesis of these enzymes (Draper et al., 1995
; Vadillo-Ortega et al., 1995
; Qin et al., 1997
); however, whether they are involved in the development of the structural alterations seen in the fetal membranes overlying the cervix needs to be determined.
The thinning of the trophoblast layer and the absence or dramatic thinning of the decidua could be the result of a number of processes. First, in the region of the lower segment there is a limited blood supply to the decidua (MacDonald et al., 1991), which may result in the selective removal of cells due to the processes of either necrosis or apoptosis. Second, it could have occurred by progressive stretch. The significant reduction of the cytotrophoblast layer in the cervical membranes could have implications for the local action of uterotonins such as prostaglandins and endothelin. Uterotonins are produced in the amnion and are present in amniotic fluid, yet the cytotrophoblast layer possesses the capacity to degrade these factors and prevent their action upon the myometrium and cervix. It has been proposed that local involution of the cytotrophoblast layer could allow uterotonins to escape metabolism and act locally (Malak and Bell, 1996
) and the demonstration of its decreased thickness prior to labour could be causally involved in uterotonin action.
The investigation of the mechanisms responsible for the development of this region of structural alteration may have important implications for our understanding of the mechanisms of pre-labour fetal membrane rupture and preterm birth. Whatever the causes of the preterm pre-labour rupture of the fetal membranes in the absence of infection, their common end effect must be the production of a structurally weakened fetal membrane that predisposes it to subsequent pre-labour rupture. Whether mediated by infection or not, this condition may be the result of the premature activation of the processes involved in the generation of an area of structural weakness equivalent to that in the cervical area in normal pregnancy (Malak and Bell, 1996). Indeed, studies have reported that pre-labour ruptured membranes in both term and preterm births exhibit the extensive structural changes of ZAM, and that membranes from preterm births exhibit more extensive structural changes compared to those in spontaneously ruptured fetal membranes of either term or preterm births (Malak et al., 1993
, 1994
). The appearance of ZAM in the fetal membranes from preterm births also raises the possibility that its premature appearance with the loss of the cytotrophoblast layer will allow premature action of uterotonins to induce idiopathic preterm labour.
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Acknowledgments |
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Notes |
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3 To whom correspondence should be addressed
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References |
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Submitted on May 8, 1998; accepted on October 1, 1998.