In vitro differentiation of germ cells from nonobstructive azoospermic patients using three-dimensional culture in a collagen gel matrix

Abstract

Objective: To assess the effectiveness of the three-dimensional culture of spermatogenic cells in a collagen gel matrix from nonobstructive azoospermic patients and examine the relation between the success rate of in vitro spermatogenesis and serum FSH level as a diagnostic prediction. Design: Prospective study using radioimmunoassay, immunocytochemistry, and flow cytometry with primary cultured cells. Setting: Gynecologic clinics and human reproduction research laboratory. Patient(s): Primary culture of spermatogenic cells established from 18 nonobstructive azoospermic patients who underwent histologic diagnoses. Intervention(s): Primary culture of spermatogenic cells in a collagen-based gel matrix, subjected to immunological and flow cytometric analyses. Main Outcome Measure(s): In vitro culture of spermatogenic cells was established in an extracellular milieu that more closely resembled the in vivo condition. The number of chromosomes in newly generated cells during culture was determined by fluorescence-activated cell sorter (FACS) and immunocytochemical analysis. Effects of FSH on the differentiation of the spermatogenic cells were measured. Result(s): Results of histologic studies indicated that 8 of 18 patients showed the spermatocyte arrest. Immunocytochemical and FACS analysis indicated that after 12 days in culture, haploid cells comprised 11%-37% of the cultured cell population with a characteristic expression of a cellular marker for spermatids. The serum level of FSH appeared to be closely correlated with an increase in the number of haploid cells in culture. Conclusion(s): The present three-dimensional culture in a collagen gel matrix provides a suitable means by which spermatocytes could be induced to differentiate into presumptive spermatids in vitro. In addition, the plasma FSH level could be a good indicator for the success of differentiation of cultured spermatogenic cells obtained from patients with spermatogenic arrest.