Histoanalysis and sterility assay of fresh human amniotic membrane for use in urogynecology

Abstract

Human amniotic membrane has long been used as a biomaterial for wound/burn and varicose/diabetic ulcer dressing to prevent endometrial adhesion, as a graft for the cornea, periosteum, and bladder, or as a material to generate a neovagina. Several clinics in Indonesia opt for fresh amniotic membrane for grafts because it is widely available and easy to obtain. This study investigated the reliability of using fresh amniotic membrane as a biomaterial. Placenta samples were obtained from patients who underwent cesarean without complications, and human amniotic membrane was separated from the chorion and cut into a 3 × 3 cm2 size. Specimens were categorized into two groups: before (0.9% NaCl) and after (2 g.L−1 kanamycin) antibiotic wash as the control and fresh amniotic membrane groups, respectively. Cells in the amniotic membrane matrix were visualized using hematoxylin and eosin and DAPI staining. DNA in the matrix was quantified using Quick-DNA™ Miniprep Plus Kit, measured using a BioDrop nanospectrophotometer. DNA length was determined using gel electrophoresis. Sterility assay was performed using nutrient broth, followed by plating on agarose to identify any strain. Sections of both groups showed an intact cuboidal epithelial monolayer at the fetal surface. DNA residue in the matrix after NaCl and kanamycin washes was 41.36 ± 8.48 ng.mg−1 and 42.53 ± 9.57 ng.mg−1 tissue weight, respectively. All samples were sterile. Fresh amniotic membrane for clinical use might not be ideal because nuclei were identified across the membrane and the dsDNA residue length was >200bp.