Tensile strain increased COX-2 expression and PGE2 release leading to weakening of the human amniotic membrane
Introduction
Pre-term premature rupture of the fetal membrane (PPROM) is a major cause of preterm birth and accounts for 40% of early infant death with National Health Service (NHS) costs £3 billion annually. The causes of spontaneous PPROM are multifactorial and vary according to gestational age. Infection, blood and uterine stretch weaken the membrane but little is known about the factors which initiate the damaging process [1], [2], [3], [4]. Furthermore, techniques that are used to treat fetal anomalies lead to iatrogenic PPROM and occur commonly after amniocentesis, cordocentesis, fetoscopic or open fetal surgery [5]. Spontaneous wound healing of the amniotic membrane is limited after invasive fetal therapy and leaves a visible membrane defect in the majority of cases [6]. The mechanisms which promote fetal membrane healing are poorly understood, which makes development of new therapies to heal the amniotic membrane defects challenging.
The primary signals which initiate the inflammatory process and lead to amniotic membrane weakening are unclear. Studies in animal models have shown that tensile stretch increased myometrial expression of cyclo-oxygenase-2 (COX-2), the oxytocin receptor and the gap junction protein connexin-43 (Cx43) [7], [8], [9]. In human amniotic cells, application of 11% static stretch activates NF-κB and induced expression of COX-2 and prostaglandin E2 (PGE2) production [10]. Cytokines such as interleukin-1β (IL-1β) activate NF-κB and increase production of matrix metalloproteinase-9 (MMP-9) and PGE2 in human amniochorion [11], [12], [13], [14], [15]. The enhanced PGE2 reduced collagen levels and increased tissue softening mediated by increased proteoglycans in human amniotic membrane [16], [17], [18]. The changes in the matrix network are characterised by abnormal weakening of amniotic membrane due to loss in tensile strength and mechanical resistance leading to PPROM [19], [20], [21]. Furthermore, as gestation advances to term, collagen and elastin levels are reduced in the amniotic membrane, making it difficult to assess the effect of matrix tissue architecture on mechanical properties [22]. Previous studies showed substantially greater mechanical integrity in preterm specimens compared to term by biaxial puncture testing with tissue biomechanics dependent on region [20], [23], [24]. For example, amniotic membrane specimens near the placenta were shown to be stronger than tissues from the cervix, and contradict a previous study which observed no difference in tissue region [23], [25]. However, the biomechanical techniques used in previous studies did not take into consideration the orientation of the fibre network in relation to the direction of applied tensile strain. This is an important variable since tissue mechanics is well known to be dependent on fibre architecture and alignment [26], [27]. Taken together, the previous findings highlight the complex nature of mechanical and catabolic factors that control inflammation and tissue biomechanics.
The present study compared the mechanical properties of term amniotic membrane tissues derived close to the cervix or placenta with differing collagen fibre alignment. Using an ex-vivo bioreactor approach, the effect of cyclic tensile strain on the expression levels of Cx43/COX-2 and PGE2 release was examined on tissue composition. We speculate that a combination of inflammatory and mechanical factors disrupt amniotic membrane tissue composition, mediated by abnormal expression of Cx43/COX-2 and PGE2 release.
Section snippets
Amniotic membrane tissue isolation
Term human placentas were collected with informed consent from 30 deliveries from women undergoing elective caesarean section (37–42 weeks of gestation) at University College Hospital London. Ethical approval was given by the Joint UCL/UCLH committees and the Ethics of Human Research Central Office (05/Q0505/82). Women with placenta praevia, multiple pregnancy, antepartum haemorrhage, PPROM and fetal growth restriction were excluded from the study. At Caesarean section after delivery of the
SHG imaging of collagen and effect of orientation on mechanical properties
Collagen fiber organisation in the amniotic membrane as identified by SHG imaging was observed to vary with tissue region and orientation (Fig. 2). In CAM⊥ and PAM⊥ specimens, the collagen fibre bundle orientation appeared to resemble an “open basket weave” with fibres running at approximately ±45° angles to normal specimen axis (Fig. 2, top panel, thick white arrows). In contrast in CAMǁ or PAMǁ specimens, the collagen fibre bundle orientation is approximately parallel with the specimen axis
Discussion
The amniotic membrane has poor spontaneous regenerative capabilities and complete healing is never achieved despite extensive remodelling mechanisms. It is clear that a combination of over-stretch and pathological inflammatory processes will influence the healing capacity and lead to abnormalities in tissue composition, biomechanics and PPROM. In the present study, we demonstrated that the mechanical properties of the amniotic membrane are highly dependent on fibre orientation and location of
Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Acknowledgements
This project was funded by the Wellbeing of Women (ELS052), the Rosetrees Trust (M400) and the Department of Health’s NIHR Biomedical Research Centres funding scheme. The authors would like to thank Dr Chauvanne Thorpe and Dr Nick Peake for supporting BC with ex-vivo bioreactor experiments and biochemical analysis, respectively.
References (43)
- et al.
Epidemiology and causes of preterm birth
Lancet
(2008) - et al.
A histological study of fetoscopic membrane defects to document membrane healing
Placenta
(2006) - et al.
Cytokines of the placenta and extra-placental membranes: roles and regulation during human pregnancy and parturition
Placenta
(2002) - et al.
Changes in the synthesis and metabolism of prostaglandins by human fetal membranes and decidua at labour
Am J Obstet Gynaecol
(1985) - et al.
MMP-9 and tensile strength of fetal membranes in uncomplicated labour
Obstet Gynecol
(2000) The extracellular matrix of the human fetal membranes: structure and function
Placenta
(1998)- et al.
Relation between mechanical properties and microstructure of human fetal membranes: an attempt towards a quantitative analysis
Eur J Obstet Gynecol Reprod Biol
(2009) - et al.
Premature rupture of the fetal membranes: is the amnion the major determinant?
Am J Obstet Gynecol
(2006) - et al.
Different biomechanical properties of human fetal membranes obtained before and after delivery
Eur J Obstet Gynecol Reprod Biol
(1993) - et al.
Detection of elastin in the human fetal membranes: proposed molecular basis for elasticity
Placenta
(1997)