[Show abstract][Hide abstract] ABSTRACT: Generation of reactive oxygen species (ROS) has been suggested as a mechanism of fetal membrane (FM) weakening leading to rupture, particularly with preterm premature rupture of the fetal membranes (PROM). In vitro, FM incubation with tumor necrosis factor (TNF) mimics physiological FM weakening, concomitant with generation of ROS and collagen remodeling. Proinflammatory cytokines are also postulated to have a role in the development of the FM physiological weak zone where rupture normally initiates in-term gestations. We hypothesized that antioxidant treatment may block ROS development and resultant FM weakening. Two studies examining antioxidant effects upon FM strength were conducted, one in vivo and the other in vitro. Fetal membrane of patients enrolled in a multicenter placebo-controlled trial to determine the effect of vitamin C (1 g/day) and vitamin E (400 IU/day) upon complications of pre-eclampsia were examined for FM biomechanical properties and biochemical remodeling at birth. Separately, biomechanics and biochemical markers of remodeling were determined in FM fragments incubated with TNF with or without vitamin C preincubation. Supplemental dietary vitamin C in combination with vitamin E did not modify rupture strength, work to rupture, or matrix metalloproteinase-9 (MMP9; protein or activity) either within or outside the term FM physiological weak zone. In vitro, TNF decreased FM rupture strength by 50% while increasing MMP9 protein. Vitamin C did not inhibit these TNF-induced effects. Vitamin C alone had a weakening effect on FM in vitro. We speculate that vitamin C supplementation during pregnancy will not be useful in the prevention of preterm PROM.
[Show abstract][Hide abstract] ABSTRACT: Human fetal membranes (FM) at term have been shown to contain a weak zone in the region overlying the cervix which exhibits characteristics of increased collagen remodeling and apoptosis. It has been hypothesized that the FM rupture initiation site is within this weak zone. Although the FM weak zone has been partially characterized, it is unclear what structural differences in the extracellular matrix result in its decreased rupture strength. A screen for differentially expressed proteins in the amnion of the weak zone versus other FM areas demonstrated that fibulin 1 was decreased. We investigated potential regional differences in all fibulin protein family members.
FM fibulins were localized by immunohistochemistry. Detected fibulins were screened by Western blot for differences in abundance in the amnion of the weak zone versus non-weak zone FM regions. Amnion epithelial and mesenchymal cells were also screened for fibulin production.
Fibulins 1 and 5 were detected in the cytoplasm of and in a pericellular pattern surrounding all FM cells, and in a dense extracellular pattern in the amniotic compact zone. Fibulin 3 was detected within the cytoplasm of amnion epithelial and chorion trophoblast cells. Fibulins 2 and 4 were not detected. Fibulins 1, 3 and 5 demonstrated decreased abundance of 33%, 63% and 58% (all P<0.01) in amnion of the weak zone relative to other FM regions. Amnion cells produced all three detected fibulins. Furthermore, TNF inhibited amnion cell fibulin production in a dose dependent manner.
Fibulins 1, 3 and 5 were localized coincident with major microfibrillar networks in amnion. Each showed decreased abundance in the amnion component of the FM weak zone. Amnion epithelial and mesenchymal cells produced all three fibulins and their abundance was inhibited by TNF. We speculate that the amnion microfibrillar layer undergoes significant remodeling with the development of the FM weak zone.
[Show abstract][Hide abstract] ABSTRACT: Untimely rupture of the fetal membranes (FM), the amnion and choriodecidua, which normally surround and protect the fetus prior to delivery, is a major cause of preterm birth and results in significant infant mortality and morbidity. The physiological mechanism which normally leads the FM to weaken and fail prior to birth is not known. Conventional thinking that FM rupture is precipitated by the stress of uterine contractions during labor fails to explain the 10% of term deliveries and 40% of preterm deliveries in which FM rupture is the sentinel event, preceding any uterine contractions. Recent studies from several laboratories indicate that the FM undergo a genetically-programmed, biochemically-mediated, maturation process, near term, which is characterized by collagen remodeling and apoptosis. In human FM, in contrast to rat membranes, these changes are limited to the region of the FM overlying the cervix . In a series of publications, our group has demonstrated that human FM have a zone of physical weakness (decreased force to rupture and work to rupture relative to the other areas of the same FM) overlying the cervical opening of the uterus. We further demonstrate that this same zone is characterized by specific markers of increased collagen remodeling and apoptosis [1–3]. These regional characteristics develop prior to the onset of contractions of labor and persist until delivery. Furthermore, the rupture tear line of the FM intersects this weak zone and thus the rupture process is hypothesized to initiate in this weak zone . In order to investigate how differences in the biochemical composition of the extra-cellular matrix of the weak and the strong zones of FM reflect their different biomechanical properties, we utilized a proteomics approach to identify differences in the abundance of specific proteins in weak and strong FM fragments. Initial 2-DIGE screening resolved differences in Fibulin 5 protein expression. This prompted further analysis of additional members of the Fibulin protein family.