JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):67
Oral Presentation
29^th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263629

P-55. Culture of Spermatogonial Stem Cells from Cryopreserved Testicular Tissue for Male Fertility Preservation

André Lucas Caldeira-Brant¹, Júnia Figueiredo Moraes¹, Elisa Mello Gazzinelli¹, Vitor Andrade Fortunato², Reginaldo Andre Martello², Augusto Barbosa Reis², Fernando Marcos Reis²

¹ Pontifícia Universidade Católica de Minas Gerais - Belo Horizonte - MG - Brasil ² Universidade Federal de Minas Gerais - Belo Horizonte - Minas Gerais - Brasil

Objective: Prepubertal boys undergoing cancer treatment and non-obstructive azoospermic men both face a risk of permanent infertility, as they do not have sperm for cryopreservation. In this context, the cryopreservation of testicular tissue for the future use of immature germ cells, such as spermatogonial stem cells (SSCs), is essential for male fertility preservation. However, the successful in vitro cultivation of these cells is challenging due to frequent maturation or cell death. Therefore, the objective of the present study is to develop a culture method for spermatogonial stem cells derived from cryopreserved adult testicular tissue, aiming to promote their proliferation for future use in fertility preservation for both patient groups.
Methods: Blood and testicular samples were collected from five prostate cancer patients. The patients' mean age was 65±3 years. Blood samples were analyzed to measure basal levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and total testosterone. Testicular tissue samples, obtained via total bilateral orchiectomy, were divided for two purposes. A portion was fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin for histological assessment. The remaining samples were cryopreserved in MEM-α medium supplemented with 10% fetal bovine serum (FBS) and 5% dimethyl sulfoxide (DMSO). For cell culture, samples were thawed at 37°C and digested with Collagenase I and DNAse. Following differential plating on 0.1% gelatin-treated plates, floating germ cells were transferred to wells coated with basement membrane factors (Geltrex). Cells were then cultured for 7 days at 37°C under three different conditions in DMEM-F12 medium supplemented with self-renewal factors GDNF and FGF. To inhibit differentiation, cells were divided into three groups treated with varying concentrations of WIN 18,446: 0 μM (control), 1 μM (W1), and 5 μM (W5). On day 7, cells were counted using a Neubauer chamber. Subsequently, they were analyzed by immunocytochemistry (ICC) and fluorescence-activated cell sorting (FACS) to assess cell viability (Ghost Dye780), the presence of spermatogonial stem cells (SSEA4-488), and differentiated spermatogonia (cKIT-APC). Values were tested for normality and analyzed using ANOVA. Data are presented as mean±standard error of the means.
Results: Hormonal assays revealed normal values for FSH (3.95±1.97), LH (4.96±1.65), and basal total testosterone (216.6±154.57). Histological assessment through Johnsen’s Score demonstrated the presence of germ cells in all patients (7.8±0.9) prior to testicular tissue cryopreservation. After 7 days of culture, the percentage of total cells relative to day 0 was 57.48±10.66% (control), 62.48±7.47% (W1), and 57.88±12.69% (W5). The fraction of SSEA4-positive spermatogonial stem cells, relative to day 0, was 70.16±20.55% (control), 84.16±30.51% (W1), and 94.62±41.94% (W5). In contrast, the fraction of differentiated cKIT-positive cells, relative to day 0, was 201±100% (control), 223.2±112.4% (W1), and 208±105% (W5). The presence of both spermatogonial stem cells and differentiated spermatogonia on day 7 was confirmed by ICC. However, no significant differences were observed between the culture groups (p>0.05).
Conclusion: These results indicate that culturing free germ cells on plates with basement membrane proteins and supplemented with self-renewal factors such as GDNF and FGF, along with a differentiation inhibitor (WIN 18,446), can promote the long-term survival of SSEA4-positive spermatogonial stem cells. Although not statistically significant (p>0.05), a trend toward an increase in SSEA4-positive cell numbers was observed in the groups treated with the differentiation inhibitor. This suggests a potential for future therapies involving the transplantation of cryopreserved spermatogonial stem cells.