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Because of long-term incorporation of amniotic membrane (AM) into corneal stroma after transplantation as a scaffold for stem cell delivery, the variation in haziness is a major factor that influences visual quality. The aim of this study was to evaluate probable sources of transparency variation in fresh and freeze-dried AM and compare the obtained results with transparency of rabbit corneas.Amnions were extracted from placental and reflected regions of placentas from elective Cesarean sections. The effects of removing epithelial cells and spongy layer on transparency and thickness of fresh and freeze-dried AMs and rabbit cornea were evaluated. The epithelial surface of AMs was evaluated with histological analysis and scanning electron microscopy.The reflected region of intact AM was thinner and more transparent than the placental region. From histological analysis, the main source of difference between placental and reflected regions of amnion is related to epithelial cells. The process of acellularization improved light transmission of the AM in both placental and reflected regions and also omitted variation between transparency of reflected and placental regions of AM. Freeze-drying of intact AM did not improve transparency because of scattering of light by cellular debris; however, removing the epithelial layer before freeze-drying resulted in optimized light transmission similar to transparency of rabbit cornea.The amniotic epithelial cells play a major role as a source of variation in light transmission properties of amnion. From the results, epithelial-denuded freeze-dried AM was found to be a suitable scaffold to be applied in corneal tissue engineering.