Fluorescent probes allow rapid and precise recording of follicle density and staging in human ovarian cortical biopsy samples

Objective: To develop rapid and reliable techniques for determining follicle content, viability, and morphologic features in an ovarian cortical biopsy specimen. Design: Prospective laboratory study. Setting: Academic research institution. Patient(s): Consenting patients undergoing laparoscopic surgery for nonovarian disease. Intervention(s): Ovarian cortical biopsy specimens were stained by using fluorescent probes and conventional histologic stains. Observations were made by using light, fluorescent, and confocal laser microscopy. Main Outcome Measure(s): Density, cellular viability, and morphologic features of ovarian follicles. Result(s): After 45 minutes of incubation of ovarian cortical tissue with calcein AM, follicles were visualized by fluorescent microscopy and counted. Ten minutes of exposure is sufficient to stain all cell nuclei with the PicoGreen probe. Subsequent confocal microscopy allows quantification of follicle density and reveals follicle morphology in the optical sections. Follicle density as assessed by using these rapid methods was concordant with the number of units counted in conventionally stained histologic sections. Conclusion(s): Use of fluoroprobes for mitochondrial and DNA staining allows rapid evaluation of follicle density in ovarian tissue. (C) 2001 by American Society for Reproductive.