Methods for isolation and purification of post-ejaculate human round spermatids for possible use in intracytoplasmic round spermatid injection*

Objective: To develop methods for the isolation and purification of round spermatids (RS) from ejaculated semen specimens recovered from patients suffering from nonobstructive azoospermia for the purpose of performing intracytoplasmic round spermatid injections (ROSI) into human oocytes. Design: Prospective study. Setting: Audrology Institute of Lexington, Lexington, Kentucky. Patients: Six men with nonobstructive azoospermia. Interventions: Semen specimens were washed via centrimgation prior to selection and purification of RS. The RS were isolated via a multi-layer Percoll(90, 80, 70, 60 and 40% Percoll) centrimgation procedure. The isolated RS were purified via a second multi-layer Percoll (70, 40, 30 and 20% Percoll) centrimgation procedure. Purified RS were washed twice, resuspended in polyvinylpyrrolidone (PVP) and identified via microscopy. Main Outcome Measures: Isolation and purification of RS from other round cells (RC) and seminal debris. Results: High recovery of RS (66 to 76% of the original RS population) with minimal background (round cells and debris) after Percoll isolation and the various purification steps. Conclusions: ‘Large quantities of RS were detected in the original ejaculates and subsequently isolated and purified. Intracytoplasmic injection techniques with ejaculated and purified spermatids can be beneficial for patients with defective spermatogenesis such as nonobstructive azoospermia, without any required urologic intervention.