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Histologic Analysis of Testes from Prepubertal Patients Treated with Chemotherapy Associates Impaired Germ Cell Counts with Cumulative Doses of Cyclophosphamide, Ifosfamide, Cytarabine, and Asparaginase

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Abstract

Cryopreservation of immature testicular tissue is an experimental strategy for the preservation of fertility in prepubertal boys that will be subjected to a gonadotoxic onset, as is the case of oncologic patients. Therefore, the objective of this study was to assess the impact of chemotherapeutic treatments on the testicular histologic phenotype in prepubertal patients. A total of 56 testicular tissue samples from pediatric patients between 0 and 16 years old (28 with at least one previous chemotherapeutic onset and 28 untreated controls) were histologically analyzed and age-matched compared. At least two 5-μm sections from testis per patient separated by a distance of 100 μm were immunostained for the germ cell marker VASA, the spermatogonial markers UTF1, PLZF, UCHL1, and SALL4, the marker for proliferative cells KI67, and the Sertoli cell marker SOX9. The percentage of tubule cross-sections positive for each marker and the number of positive cells per tubule cross-section were determined and association with the cumulative dose received of each chemotherapeutic drug was statistically assessed. Results indicated that alkylating agents, cyclophosphamide and ifosfamide, but also the antimetabolite cytarabine and asparaginase were associated with a decreased percentage of positive tubules and a lower number of positive cells per tubule for the analyzed markers. Our results provide new evidences of the potential of chemotherapeutic agents previously considered to have low gonadotoxic effects such as cytarabine and asparaginase to trigger a severe testicular phenotype, hampering the potential success of future fertility restoration in experimental programs of fertility preservation in prepubertal boys.

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Funding

This work was supported by a private donation of the Celtic Submarí club—Villareal C.F. to Hospital Universitario y Politécnico La Fe intended to promote the scientific research on fertility preservation in child with cancer, and an AES project grant (PI16/00931) conceded by the Instituto de Salud Carlos III.

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Authors and Affiliations

  1. Instituto de Investigación Sanitaria La Fe (IIS La Fe), Av. Fernando Abril Martorell, 106. Tower A, Lab. 6.22, 46026, Valencia, Spain

    Jose V. Medrano, D. Hervás, T. Vilanova-Pérez, A. Navarro-Gomezlechon, A. Pellicer, M. M. Andrés & E. Novella-Maestre

  2. Vrije Universiteit Brussel (VUB), 1090, Brussels, Belgium

    E. Goossens

  3. Fundación IVI, 46026, Valencia, Spain

    A. Pellicer

  4. Hospital Universitario y Politécnico La Fe, 46026, Valencia, Spain

    M. M. Andrés & E. Novella-Maestre

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  1. Jose V. Medrano
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  2. D. Hervás
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  3. T. Vilanova-Pérez
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Contributions

JVM, ENM, AP, and MMA conceived this work. MMA and EG provided samples. JVM, TVP, and ANG conducted the experiments. DH performed statistical analysis of data. JVM analyzed data and wrote the manuscript. All listed authors revised and approved the manuscript.

Corresponding author

Correspondence to Jose V. Medrano.

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Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

Samples used in this study were recruited at Hospital La Fe in Valencia (Spain) (32 samples) and UZ Brussel in Brussels (Belgium) (36 samples) after the approval by the respective Institutional Review Boards of Hospital La Fe (ref: 2013/0457) and UZ Brussel (ref: 2000/149D and 2017/061).

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An informed consent is by parents or legal guardians of the patients.

Code Availability

All statistical analyses were performed using R (version 3.5.3) and the R packages glmnet (version 2.0–16), cluster (version 2.0.7–1) and brms (version 2.8.0).

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Supplemental Table III.

Original data matrix regarding the mean of cell counts for each marker in testicular samples employed for the fuzzy clustering analysis to identify differences between untreated and treated patients. Classification of treated patients as weakly or severely affected resulting from fuzzy clustering analysis has been also included in order to facilitate the identification of patients of each group. (XLSX 19 kb)

Supplemental Table IV.

Original data matrix regarding the cumulative dose of each drug received by patients. Classification of treated patients as weakly or severely affected resulting from fuzzy clustering analysis has been also included in order to facilitate the identification of patients of each group. (XLSX 17 kb)

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Medrano, J.V., Hervás, D., Vilanova-Pérez, T. et al. Histologic Analysis of Testes from Prepubertal Patients Treated with Chemotherapy Associates Impaired Germ Cell Counts with Cumulative Doses of Cyclophosphamide, Ifosfamide, Cytarabine, and Asparaginase. Reprod. Sci. 28, 603–613 (2021). https://doi.org/10.1007/s43032-020-00357-6

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