Myofibroblasts accumulate at the rupture site in preterm amniotic membrane after spontaneous PPROM.

INTRODUCTION: Fetal membranes do not spontaneously heal after trauma, fetal surgery, fetoscopy or amniocentesis leaving a visible tissue defect which is prone to rupture, leading to preterm premature rupture of the fetal membrane (PPROM). We examined the morphological features of myofibroblasts and collagen organization at the amniotic membrane (AM) rupture site after spontaneous PPROM and whether myofibroblasts regulate Cx43 expression. METHODS: Myofibroblast morphology and collagen microstructure were investigated by confocal microscopy and second harmonic generation imaging of AM specimens from three women who spontaneously ruptured the membranes preterm. AM specimens were dissected near the rupture site and immunostained to detect αSMA and Cx43 in myofibroblasts and counterstained with DAPI to quantify nuclei shape. The direction of collagen fibrils in the fibroblast layer at the ruptured region were examined by SHG imaging. RESULTS: In ruptured AM, the diameter of the rupture site ranged from approximately 1.5 cm to 4.0 cm. At the edge of the rupture site, αSMA positive myofibroblasts had deformed nuclei and showed abundant Cx43 localised in the cytoplasm or had formed plaques between cell-to-cell contacts. In the fibroblast layer, collagen had degenerated in some regions of the fibroblast layer or had polarity near the edge of the rupture site. CONCLUSION: In ruptured membranes, contractile myofibroblasts were deformed with collagen fibrils that have polarity. Whilst contractile myofibroblasts expressed intracellular Cx43, cells also formed bulbuous regions near the epithelial edge of the rupture site, indicating re-epithelilisation and attempted healing. It is unclear how myofibroblasts promote healing mechanisms at the site of rupture for cell migration, integrity and repair pathways.