Impact of chemotherapy received prior to testicular tissue cryopreservation on somatic cells from prepubertal to postpubertal males.

Abstract

Study question: Does exposure to first-line chemotherapies affect the density and maturity status of testicular somatic cells from prepubertal to postpubertal males at the time of cryopreservation?

Summary answer: Chemotherapy exposures, particularly high doses of alkylating agents, are associated with alterations in immunohistochemical markers of testicular somatic cell density and maturation, including SOX9 for Sertoli cell density, COUP-TFII for peritubular myoid cell maturation, and CYP11A1 and INSL3 for Leydig cell maturation, with the related effects becoming more nuanced when age and underlying pathology are considered.

What is known already: The increased survival rates following cancer treatment highlight the importance of integrating fertility preservation, whether through sperm cryopreservation in pubertal and postpubertal males or testicular tissue freezing (TTF), which is now established for prepubertal and peripubertal boys. Although induction chemotherapy is known to impair germ cells, its effects on somatic cells (Sertoli, peritubular myoid, and Leydig cells) within testicular tissue collected after exposure to chemotherapeutic drugs remain largely unexplored.

Study design, size, duration: This retrospective study included testicular tissue samples from 51 patients from prepubertal to postpubertal males diagnosed with cancer, aged between 6 months and 33 years. Their medical diagnoses and previous chemotherapy exposures were recorded. We examined histological sections of testicular tissue to assess the impact of chemotherapy on somatic cell density and maturity status.

Participants/materials, setting, methods: The density and maturity status of testicular somatic cells were assessed by immunofluorescent staining. The analyses were performed on fresh testicular tissue fragments from prepubertal and postpubertal males with cancer who had undergone TTF prior to conditioning for hematopoietic stem cell transplantation. All had received first-line chemotherapies. In addition, the functionality of somatic cells was assessed using hormone serum levels that reflect their endocrine activity, to provide a better understanding of the functional integrity of the testicular environment.

Main results and the role of chance: This study investigated the impact of chemotherapy drug exposure and cancer type on testicular somatic cells in boys. Patients were categorized based on their exposure to specific chemotherapeutic drugs, including carboplatin, other alkylating agents, anthracyclines, vincristine, and cytarabine. High cumulative exposure to alkylating agents, defined as a cyclophosphamide equivalent dose (CED) ≥ 8000 mg/m², led to a decrease in COUP-TFII expression in peritubular myoid cells and CYP11A1 in Leydig cells (P = 0.038 and P = 0.013, respectively). Additionally, INSL3 expression in Leydig cells declined with increasing CED (r = -0.33, P = 0.019), independently of the patients' age. Exposure to anthracyclines was also associated with reduced INSL3 (P = 0.027), although this association did not persist after CED adjustment. Combined exposure to alkylating agents and anthracyclines further decreased SOX9+ Sertoli cells (P = 0.021), whereas vincristine showed no additive deleterious effect. An alteration in Leydig cell maturation was observed in patients treated for neuroblastoma, highlighting the impact of the disease itself on testicular tissue. Hormonal analyses indicated no significant impact of induction chemotherapy on somatic cell functionality.

Limitations, reasons for caution: Histological data were obtained from testicular tissue of patients exposed to chemotherapy treatments, mostly prepubertal fragments, with no healthy controls being available. Analyses included histology and hormonal profiling; molecular studies were not performed due to limited tissue cryopreserved for future fertility restoration.

Wider implications of the findings: This study provides valuable information on the impact of chemotherapy on testicular somatic cells in patients. Because somatic cells are essential for supporting spermatogenesis, the preservation of the testicular microenvironment after chemotherapy is a reassuring prospect for patients considering future fertility restoration approaches.

Funding: This work was funded by a PhD grant by La Ligue Nationale contre le Cancer [to V.R.] as well as financial contributions from University of Rouen Normandy, Rouen University Hospital, and the Institute for Research and Innovation in Biomedicine (IRIB). Additional fundings were provided by French National Research Agency (ANR-21-CE14-0068) [to N.R.], French Biomedicine Agency [to A.F.], GIRCI NO [to A.F.], Laurette Fugain's Association [to L.D.], Rouen Normandy Metropolis [to L.D.], and Priority Research Programs and Equipment (PEPR): Women's Health, Couples' Health (SAFE). The funding sources had no role in the design of the study, collection, analysis and interpretation of data, and writing of the manuscript.

Disclosures: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Trial registration number: N/A.