Investigation of correlation of biochemical marker expression and mechanical properties of preterm membranes

The fetal membrane surrounds and supports the developing fetus during the pregnancy. The cellular structure of the fetal membrane and the proteins synthesized by the cells of amniotic and chorionic membranes, maintain the mechanical integrity of the tissue membrane. Rupture of the fetal membrane is a fundamental stage in normal term delivery. However, preterm premature rupture of membranes (PPROM) leads to preterm birth, which remains a major concern worldwide as the largest contributor of perinatal and neonatal mortality and morbidity. The underlying mechanism of PPROM is complicated and requires further investigation. The aim of this project is to newly reveal the underlying mechanisms of PPROM through the study of mechanical and biochemical properties of fetal membranes in both full term and preterm via new experimental protocols. With ethical approval, 42 fetal membranes were collected for this study from women who had full term normal vaginal delivery, preterm (less than 37 weeks) vaginal delivery (PPROM), full term and preterm delivery via caesarean sections. Mechanical characterisation was conducted for fetal membrane from full term and PPROM normal vaginal deliveries. Two new mechanical test techniques were established. Ball indentation test and Optical Coherence Elastography, were applied for the first time to study fetal membrane comprehensively in ruptured, non-ruptured areas and at different sublayers using optical coherence tomography for the thickness study. Biochemical assays; histological and immunological staining, plus Western Blotting were performed to study the microstructural and biochemical molecular (Collagen, sGAG, MMP 9 and 13, fibronectin, IL-1β & progesterone receptors) changes on the same categories of samples. Furthermore, this study investigated the changing biochemical molecule expression levels of the membranes in response to the applied external mechanical stimulation and maternal risk factors (smoking and maternal hypoglycaemia). In addition, mechanotransduction signalling molecules (such as, nifedipine) and anti-inflammatory agent (dexamethasone) response was also studied in a new biomimetic experimental set up which was designed and implemented in full term and preterm fetal membranes from caesarean section deliveries. The mechanical test results revealed new evidence of noticeable difference between full term and preterm membranes. Preterm membranes showed great heterogeneity between ruptured and non-ruptured sites (creep property: 36%, modulus: 55%, thickness: 48%, collagen content: 59%, sGAG: 48%, and MMP 9: 57%) in comparison to their full term counterparts (creep property: 15%, modulus: 33%, thickness: 23%, collagen content: 24%, sGAG: 27% and MMP9: 23%). For the first time, a clear correlation between biomechanical and biochemical relations in full term and preterm was drawn. The in vitro loading model for the study of biochemical behaviours of fetal membrane in the presence of external force implied that cyclic force, either from fetal movement or fetal fluid can accelerate the synthesis of matrix degradation enzymes (MMPs) or trigger inflammation marker expression, leading to the weakening of the membranes. Preterm membrane was more sensitive to the force and biochemical environment than full term membrane, which might lead to premature rupture accidently. Downregulation of these biomarkers’ expressions by addition of nifedipine during culture suggested that the calcium signalling pathway may play an important role in mechanotransduction in fetal membrane cells. In addition, maternal smoking correlated with membrane weakness; however, diabetes did not show any direct correlation with it under the influence of external force in the current experimental model. Finally, the addition of dexamethasone in the cyclic loading environment decreased expressions of membrane weakening biomarkers, which reinstated dexamethasone’s beneficial effects on the prevention of fetal membrane rupture

New approaches suggest term and preterm human fetal membranes may have distinct biomechanical properties

Preterm prelabour rupture of membranes is the leading cause of preterm birth and its associated infant mortality and morbidity. However, its underlying mechanism remains unknown. We utilized two novel biomechanical assessment techniques, ball indentation and Optical Coherence Elastography (OCE), to compare the mechanical properties and behaviours of term (≥ 37 weeks) and preterm (33-36 weeks) human fetal membranes from ruptured and non-ruptured regions. We defined the expression levels of collagen, sulfated glycosaminoglycans (sGAG), matrix metalloproteinase (MMP-9, MMP-13), fibronectin, and interleukin-1β (IL-1β) within membranes by biochemical analysis, immunohistochemical staining and Western blotting, both with and without simulated fetal movement forces on membrane rupture with a new loading system. Preterm membranes showed greater heterogeneity in mechanical properties/behaviours between ruptured and non-ruptured regions compared with their term counterparts (displacement rate: 36% vs. 15%; modulus: 125% vs. 34%; thickness: 93% vs. 30%; collagen content: 98% vs. 29%; sGAG: 85% vs 25%). Furthermore, simulated fetal movement forces triggered higher MMP-9, MMP-13 and IL-1β expression in preterm than term membranes, while nifedipine attenuated the observed increases in expression. In conclusion, the distinct biomechanical profiles of term and preterm membranes and the abnormal biochemical expression and activation by external forces in preterm membranes may provide insights into mechanisms of preterm rupture of membranes

Effect of Cold Stress on Pyridostigmine Pretreated Rats Exposed to an Organophosphorous Compound

Context: Pyridostigmine bromide (PB) is a quaternary ammonium compound and has been approved as a pretreatment drug against toxic organophosphorous (OP) compounds. The stressful demands of modern military activity include a broad range of activities at extreme cold temperatures along with various physical activities. Objective: The effect of “sign free” dose of PB (0.075 mg/kg body weight) against a toxic OP compound diisopropyl fluorophosphate (DFP) was reassessed in rats. Electrocardiographic (ECG) studies in hypothermic and pretreatment conditions were undertaken to assess the cardioprotective role of PB. Total Antioxidant Status (TAS) was quantified to assess the degree of oxidative stress imposed under such conditions. Possible protective role of pyridostigmine in rat lymphocytes was also determined.Materials& Methods: TAS was estimated spectrophotometrically and the expression of interferon-γ (IFNγ) was measured by Fluorescence Activated Cell Sorting. ECG was monitored by standard protocol.Results: ECG recording showed that the PR and QT interval progressively increased along with widening of QRS complex. There was a progressive fall in heart rate as the body temperature decreased. TAS significantly decreased (p≤0.001) in hypothermic conditions and when pretreated with sign free dose of PB before cold induction (p≤0.001). Following immunostaining of lymphocytes by FITC conjugated mouse anti-rat IFNγ monoclonal antibody, 9.1% of lipopolysaccharide elicited parent cells showed positive IFNγ expression. Hypothermic stress inhibited IFNγ expression (3.6% of parent cells) which was recovered to 6.8% upon pre-treatment with sign-free dose of pyridostigmine. Conclusion: This study is indicative of a possible protective role of PB against hypothermic stress