Differential Expression of Fibulin Family Proteins in the Para-cervical Weak Zone and Other Areas of Human Fetal Membranes

Oct. 8, 2009

Untimely rupture of the fetal membranes (FM), the amnion and choriodecidua, is a major cause of preterm birth and results in significant infant mortality and morbidity. The physiological mechanisms which normally lead the FM to weaken and fail priorto birth are not known. Recent studies indicate that in 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.

In an attempt to investigate how differences between the biomechanical properties of the FM weak zone and that of the remaining stronger FM areas are reflected in their extracellular matrix proteins a proteomics approach was utilized. The initial proteomics 2D-DIGE screening demonstrated decreases in fibulin 1 protein abundance in the weak zone of the FM compared to the remaining areas. As very little has been known and nothing has been reported in amnion, we expanded our investigation to include additional fibulin family members. Thus, a subsequent immunohistochemical analysis revealed localization of fibulin 1, fibulin 3 and fibulin 5 but not fibulins 2 and 4 in the amnion for the first time (Figure 1). Besides, western blot validation analysis of the expressed fibulin 1 and additional fibulin family members identifies the differential abundance in the weak zone when compares with other FM regions. All the three detected fibulins showed deceased abundance in the amnion component of the FM weak zone (Figure 2) and mainly produced by both amnion epithelial and mesenchymal cells.

In summary, the fibulin family proteins have decreased abundance in the para-cervical weak zone compared with the other areas of the FM. Their location is coincident with an extensive microfibrillar network that is integral to the epithelial cell basement membrane and the amniotic compact layer suggesting that fibulins may be integral proteins of these very important strength maintaining, extracellular matrix structures. Degradation or remodeling of the microcellular network in the amnion may be a critical element of the weakening process leading to FM rupture.

Figure 1.

Immunohistochemical localization of fibulin proteins in intact FM: Localization of immuno-reactive (brown staining) fibulin family members 1.5 are shown in representative cross-sections of fetal membranes (left panel). Pre-treatment of sections with blocking peptides against fibulin antibodies prior to incubation (right panel). Abbreviations: AE, amnion epithelial layer; BM, basement membrane; CL, compact layer; D, decidua; FL, fibroblast layer; RL, reticular layer; T, trophoblast.

Figure 2. Fibulin protein expression in amnion from para-cervical weak zone and other FM areas: Panels indicate fibulin 1.5 protein expression, as assessed by Western blot analysis, of amnion from FM fragments from the para-cervical weak zone and other FM areas from three representative patients. Actin expression is shown for comparison of protein loading. Rupture force in Newtons (N) for each fragment is indicated along the bottom.

Results from: Moore, R.M., Redline, R.W., Kumar, D., Mercer, B.M., Mansour, J.M., Yohannes, E., Novak, J.B., Chance. M.R., Moore, J.J. .Differential expression of fibulin family proteins in the para-cervical weak zone and other areas of human fetal membranes., Placenta, 30(4):335-41, 2009.