A novel approach to quantifying ovarian cell lipid content and lipid accumulation in vitro by confocal microscopy in lean women undergoing ovarian stimulation for in vitro fertilization (IVF)

Purpose To quantify intracellular lipid levels in cumulus cells (CCs) and mural granulosa cells (MGCs) of lean women undergoing gonadotropin therapy for in vitro fertilization (IVF), based upon different cell preparation methods. Methods CCs and MGCs from 16 lean women undergoing ovarian stimulation for IVF were studied. Cells were pooled by cell type, with each type of cell separated into two groups for determination of initial lipid content (Method 1) and subsequent lipid accumulation in vitro (Method 2). Cells for initial lipid content were immediately fixed at the time of the oocyte retrieval with 4 % paraformaldehyde in suspension, while those for subsequent lipid accumulation in vitro were cultured for 4 h with 5 % fetal calf serum and then fixed. Cells were treated with lipid fluorescent dye BODIPY® FL C 16 and nuclear marker DAPI. Intracellular lipid was quantified by confocal microscopy, using ImageJ software analysis. Results There was no significant effect of cell type ( P = 0.2) or cell type-cell preparation method interaction ( P = 0.8) on cell area (Method 1: CC 99.7 ± 5.1, MGC 132.8 ± 5.8; Method 2: CC 221.9 ± 30.4, MGC 265.1 ± 48.5 μm 2 ). The mean area of all cells combined was significantly less for cells prepared by Method 1 (116.2 ± 4.9 μm 2 ) vs. Method 2 (243.5 ± 22.5 μm 2 , P