A simple and objective approach to identifying human round spermatids

Abstract

Introduction: Round spermatids have been studied extensively in stained and electron microscopy preparations. However, little information is available about their appearance in fresh specimens. We describe a method to identify and collect unstained round spermalids from ejaculates and testicular biopsies. Material and methods: Ejaculates (n=115) and testicular biopsies (n=7) were assessed for the presence of round cells by routine semen analysis and Testsimplets staining. With the aid of a micromanipulator, cells considered to be round spermatids according to cell size and shape, nuclear characteristics, presence of the acrosome, and cytoplasmic characteristics were collected in groups of identical diameter. They were then placed on separate microslides and analyzed by multiprobe fluorescence in situ hybridization (FISH) to test the validity of the selection process and in order to determine the range of spermatid diameter. When no spermatozoa were present in a testicular biopsy, the dissected tissue was smeared and processed by FISH to examine for hapioid cells (spermatids). If hapioid cells were observed selection of individual round cells and FISH analysis were performed. Results: Routine semen analysis and Testsimplets identified >1 × 106 round cells in 33 ejaculates: three of five azoospermic ejaculates had spermatids. Five biopsies contained spermatozoa; one had spermatids and the other showed spermatocytic arrest. The results of the smears examined by FISH confirmed the observations by the Testsimplets staining. Four ejaculates and six biopsies were processed with micromanipulation. Spermatoids chosen with the micromanipulator were 5-7.5 μ, in diameter with a spherical and homogeneous nucleus 4-6.5 μ. in diameter. An acrosome granule was occasionally observed as a light-gray spot; cytoplasm surrounded the nuclear as a thin halo. The surface of the spermalid was smooth or slightly irregular. The 1000 cells (10 slides containing 10 cells each) individually selected were analyzed by FISH. Only 49 cells filmed on sided after hybridization; all of which showed hybridization signals. Of these 41 (83.7%) were hapioid. Conclusions: Using morphologic criteria we harvested a population of round spermatids. The reliability and reproducibility of this collection procedure was established by using FISH to verify the presence of hapioid cells. These procedures provide basic inflammation necessary before considering the routine implementation of round spermatid injection for treatment of severe male factor infertility.