Establishment of a controlled slow freezing-based approach for experimental clinical cryopreservation of human prepubertal testicular tissues

Abstract

Objective: To develop an efficient, clinical-grade, freezing protocol toward experimental clinical cryopreservation of testicular tissues in prepubertal boys suffering from cancer.

Design: Experimental cryopreservation of testicular tissue.

Setting: University Medical Center.

Patients: Adult patients undergoing orchiectomy for various tumors and prepubertal boys scheduled for gonadotoxic treatment.

Interventions: None.

Main outcome measures: Histopathological analysis of tissue architecture, structural integrity, and cellular morphology was performed for control and frozen-thawed cryopreserved tissues.The number of seminiferous tubules per testicular section was calculated. The survival of spermatogonial stem cells (SSCs) and Sertoli cells of the control and frozen-thawed cryopreserved tissues was analyzed by immunofluorescence staining.

Results: Uncontrolled Slow Freezing, Controlled slow freezing, and vitrification similarly preserved the integrity of the adult testicular tissues and the survival of SSCs and Sertoli cells. Controlled slow freezing of prepubertal testicular tissues effectively preserved their architecture, the number of tubules, SSCs, and Sertoli cells. In addition, we observed SSC loss after chemotherapy in prepubertal boys, reemphasizing the importance of fertility preservation before gonadotoxic treatment.

Conclusions: Future fertility restoration for male survivors of pediatric cancers depends on the development of an optimal prepubertal testicular tissue cryopreservation method. Our findings demonstrate the effectiveness of controlled slow freezing for cryopreservation of human prepubertal testicular tissues and may contribute to more effective banking of these tissues and potential fertility restoration.

Clinical trial registration number: NIH research clinical trials number: NCT02529826.

Keywords: Fertility preservation; controlled slow freezing; human testicular cryopreservation; male infertility; prepubertal boys.

© 2021 The Authors.

Figures

Figure 1

Histological analysis of control and cryopreserved adult human testicular tissues. Representative hematoxylin-eosin stained sections of control and frozen-thawed testicular tissues from donor 3 cryopreserved by controlled slow freezing (CSF), uncontrolled slow freezing (USF), and vitrification. Bar = 100 μm; Bar for insets = 200 μm (A). Boxplots displaying the distribution of intact tubules numbers (per 1,000 × 1,500 μm, ×100 magnification) in control, CSF, USF, and vitrification cryopreserved testicular tissues from donors 3, 4, and 2, respectively (Supplemental Table 1). The line in the center of each box represents the median. The top and bottom parts of each box represent 75% and 25% percentiles, respectively. The lines extending from each box show the maximum and minimum values of the data. P value was determined by the Kruskal-Wallis H test (nonpaired) (B). n = number of analyzed tubule sections.

Figure 2

Immunohistochemical analysis of control and cryopreserved adult human testicular tissues from donor 3. The control and thawed testicular tissues sections are cryopreserved by uncontrolled slow freezing (USF), controlled slow freezing (CSF), and vitrification, stained for MAGE-A4, Oct4, and vimentin. Bar = 100 μm (A). Boxplots displaying the distribution of positively stained cell numbers per tubule section in control and cryopreserved testicular tissues. P values were determined by the Kruskal-Wallis H test (nonpaired). For Oct4, P values in multiple comparisons were determined by post hoc pairwise Mann-Whitney U test (nonpaired) with Bonferroni corrections (B). n = number of analyzed tubule sections.

Figure 3

Histological and immunohistochemical analysis of control and controlled slow freezing (CSF)-cryopreserved prepubertal human testicular tissues from patients with primary cancer. Hematoxylin-eosin stained sections of control and frozen-thawed testicular tissues cryopreserved by CSF from patients 1, 2, and 5. Bar = 100 μm (A). Boxplots displaying the distribution of intact tubules numbers (per 1,000 × 1,500 μm, ×200 magnification, in control and CSF-cryopreserved testicular tissues. P values were determined by the Mann-Whitney U test (nonpaired). (B). MAGE-A4 stained sections of control and CSF-cryopreserved testicular tissues. Bar = 100 μm (C). Boxplots displaying the distribution of MAGE-A4 positively stained cell numbers per tubule section, control, and CSF-cryopreserved testicular tissues (D) vimentin stained sections of control, and CSF-cryopreserved testicular tissues. Bar = 100 μm P values were determined by the Mann-Whitney U test (nonpaired).

Figure 4

Histological and immunohistochemical analysis of control and controlled slow freezing (CSF)-cryopreserved prepubertal human testicular tissues from patients with recurrent cancer. Hematoxylin-eosin stained sections of control and frozen-thawed testicular tissues cryopreserved by CSF from patients 3 and 4. Bar = 100 μm (A). Boxplots displaying the distribution of intact tubules numbers (per 1,000 × 1,500 μm, ×200 magnification, in control and CSF-cryopreserved testicular tissues. P values were determined by the Mann-Whitney U test (nonpaired) (B). MAGE-A4. Because MAGE-4 positive cells were not demonstrated in fresh testicular tissues of patient 3 (control), immunohistochemical analysis post-thawing was not performed. (C) and vimentin (D) stained sections of control and frozen-thawed testicular tissues. Bar = 100 μm (C–D).