[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: The fetal membrane (FM) layers, amnion and choriodecidua, are frequently noted to have varying degrees of separation following delivery. FM layers normally separate prior to rupture during in vitro biomechanical testing. We hypothesized that the adherence between amnion and choriodecidua decreases prior to delivery resulting in separation of the FM layers and facilitating FM rupture.
FM from 232 consecutively delivered patients were examined to determine the extent of spontaneous separation of the FM layers at delivery. Percent separation was determined by the weight of separated FM tissue divided by the total FM weight. Separately, the adherence between intact FM layers was determined. FM adherence was tested following term vaginal delivery (13), term unlabored cesarean section (10), and preterm delivery (6).
Subjects enrolled in the two studies had similar demographic and clinical characteristics. FM separation was present in 92.1% of membranes. Only 4.3% of FM delivered following spontaneous rupture of the fetal membranes (SROM) had no detectable separation. 64.7% of FM had greater than 10% separation. FM from term vaginal deliveries had significantly more separation and were less adherent than FM of term unlabored, elective cesarean section (39.0+/-34.4% vs 22.5+/-30.9%, p=.046 and 0.041+/-0.018N/cm vs 0.048+/-0.019N/cm, p<.005). Preterm FM had less separation and were more adherent than term FM (9.95+/-17.7% vs 37.5+/-34.4% and 0.070+/-0.040N/cm vs 0.044+/-0.020N/cm; both p<.001).
Separation of the amnion from choriodecidua at delivery is almost universal. Increased separation is associated with decreased adherence as measured in vitro. Increased separation and decreased adherence are seen both with increasing gestation and with labor suggesting both biochemical and mechanical etiologies. The data are consistent with the hypothesis that FM layer separation is part of the FM weakening process during normal parturition.
[Show abstract][Hide abstract] ABSTRACT: Untimely rupture of the fetal membranes (FMs) is a major precipitant of preterm birth. Although the mechanism of FM weakening leading to rupture is not completely understood, proinflammatory cytokines, including tumor necrosis factor (TNF) and interleukin 1 beta (IL1B), have been shown to weaken FMs concomitant with the induction of reactive oxygen species, collagen remodeling, and prostaglandin release. We hypothesized that alpha-lipoic acid, a dietary antioxidant, may block the effect of inflammatory mediators and thereby inhibit FM weakening. Full-thickness FM fragments were incubated with control media or TNF, with or without alpha-lipoic acid pretreatment. Fetal membrane rupture strength and the release of matrix metalloproteinase 9 (MMP9) and prostaglandin E(2) (PGE(2)) from the full-thickness FM fragments were determined. The two constituent cell populations in amnion, the mechanically strongest FM component, were similarly examined. Amnion epithelial and mesenchymal cells were treated with TNF or IL1B, with or without alpha-lipoic acid pretreatment. MMP9 and PGE(2) were analyzed by ELISA, Western blot, and zymography. TNF decreased FM rupture strength 50% while increasing MMP9 and PGE(2) release. Lipoic acid inhibited these TNF-induced effects. Lipoic acid pretreatment also inhibited TNF- and IL1B-induced increases in MMP9 protein activity and release in amnion epithelial cells, as well as PGE(2) increases in both amnion epithelial and mesenchymal cells. In summary, lipoic acid pretreatment inhibited TNF-induced weakening of FM and cytokine-induced MMP9 and PGE(2) in both intact FM and amnion cells. We speculate that dietary supplementation with alpha-lipoic acid might prove clinically useful in prevention of preterm premature rupture of fetal membranes.
Biology of Reproduction 04/2009; 80(4):781-7. DOI:10.1095/biolreprod.108.073205 · 3.32 Impact Factor
[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.