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Study Question: Is it feasible to disseminate testicular tissue cryopreservation with a standardized protocol through a coordinated network of centers and provide centralized processing/freezing for centers that do not have those capabilities?, Summary Answer: Centralized processing and freezing of testicular tissue from multiple sites is feasible and accelerates recruitment, providing the statistical power to make inferences that may inform fertility preservation practice., What Is Known Already: Several centers in the USA and abroad are preserving testicular biopsies for patients who cannot preserve sperm in anticipation that cell- or tissue-based therapies can be used in the future to generate sperm and offspring., Study Design, Size, Duration: Testicular tissue samples from 189 patients were cryopreserved between January 2011 and November 2018. Medical diagnosis, previous chemotherapy exposure, tissue weight, and presence of germ cells were recorded., Participants/materials, Setting, Methods: Human testicular tissue samples were obtained from patients undergoing treatments likely to cause infertility. Twenty five percent of the patient's tissue was donated to research and 75% was stored for patient's future use. The tissue was weighed, and research tissue was fixed for histological analysis with Periodic acid-Schiff hematoxylin staining and/or immunofluorescence staining for DEAD-box helicase 4, and/or undifferentiated embryonic cell transcription factor 1., Main Results and the Role of Chance: The average age of fertility preservation patients was 7.9 (SD = 5) years and ranged from 5 months to 34 years. The average amount of tissue collected was 411.3 (SD = 837.3) mg and ranged from 14.4 mg-6880.2 mg. Malignancies (n = 118) were the most common indication for testicular tissue freezing, followed by blood disorders (n = 45) and other conditions (n = 26). Thirty nine percent (n = 74) of patients had initiated their chemotherapy prior to undergoing testicular biopsy. Of the 189 patients recruited to date, 137 have been analyzed for the presence of germ cells and germ cells were confirmed in 132., Limitations, Reasons for Caution: This is a descriptive study of testicular tissues obtained from patients who were at risk of infertility. The function of spermatogonia in those biopsies could not be tested by transplantation due limited sample size., Wider Implications of the Findings: Patients and/or guardians are willing to pursue an experimental fertility preservation procedure when no alternatives are available. Our coordinated network of centers found that many patients request fertility preservation after initiating gonadotoxic therapies. This study demonstrates that undifferentiated stem and progenitor spermatogonia may be recovered from the testicular tissues of patients who are in the early stages of their treatment and have not yet received an ablative dose of therapy. The function of those spermatogonia was not tested., Study Funding/competing Interest(s): Support for the research was from the Eunice Kennedy Shriver National Institute for Child Health and Human Development grants HD061289 and HD092084, the Scaife Foundation, the Richard King Mellon Foundation, the Departments of Ob/Gyn & Reproductive Sciences and Urology of the University of Pittsburgh Medical Center, United States-Israel Binational Science Foundation (BSF), and the Kahn Foundation. The authors declare that they do not have competing financial interests., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Purpose: The c-kit receptor is a proto-oncogene important in germ cell migration and maturation and has also been demonstrated on the acrosomal region of mature sperm. The purpose of the present study was to examine the ultrastructural location of the c-kit receptor in mouse testis and sperm., Materials and Methods: Testis and sperm from mature male mice were examined for the c-kit receptor utilizing electron microscopy and Western blot analysis techniques. Thin sections of mouse testis and sperm were stained with immunogold-labeled anti-c-kit antibodies. The protein from these testes and sperm was also utilized for Western blot analysis., Results: The c-kit protein was localized within the mouse testes to the type A spermatogonia, the round spermatids, and the mature testicular spermatozoa. The c-kit receptor was noted to migrate from the lumen of the acrosomal vesicles in the early spermatids to the plasma membrane of the late spermatids. It was also noted in the acrosomal region of the testicular spermatozoa, as well as the sperm from the epididymis. Sperm undergoing the acrosome reaction demonstrated association of the c-kit receptor with the plasma membrane of the acrosome, but not on the acrosomal membrane itself. Western blot analysis demonstrated protein bands of 150 kDa in testis and intact sperm., Conclusions: The present study confirms the presence of the c-kit receptor in mouse testis and sperm. It also demonstrates that this receptor is localized to the region of the developing acrosome.

Objective: To examine the expression of the c-kit receptor and its ligand, stem cell factor, and their possible relation with apoptosis in infertile men., Design: Prospective laboratory study., Setting: Urology laboratory in a university hospital., Patient(s): Men undergoing testicular biopsy during an investigation of subfertility., Intervention(s): None., Main Outcome Measure(s): Expression of the c-kit receptor protein, stem cell factor, and apoptosis in the testes., Result(s): The c-kit receptor was strongly present in Leydig cells and type A spermatogonia of normal testes, with decreased staining in Leydig cells and type A spermatogonia of testes with maturational arrest, and staining in only Leydig cells of Sertoli cell-only specimens. Stem cell factor was demonstrated in Leydig cells and Sertoli cells in all specimens. Western blotting demonstrated the 150-kd c-kit protein in the normal testes and the testes with maturational arrest, but not in the testes with the Sertoli cell-only pattern. Stem cell factor was expressed in all specimens, with a protein size of 45 kd. Increased apoptosis was demonstrated in type A spermatogonia and spermatocytes of tissue with maturational arrest compared with normal testicular tissue., Conclusion(s): C-kit receptor expression is decreased in subfertile testicular tissue compared with normal testicular tissue. Stem cell factor expression is present in Leydig cells and Sertoli cells. Increased apoptosis is seen in tissue with maturational arrest compared with normal tissue.

Objectives: To examine the localization and expression of the c-kit receptor protein in the testes of the mouse, rat, and human, and then compare these among the three species., Methods: Testis tissue from all three species was obtained through biopsy or orchiectomy. Immunohistochemistry was used for the localization, using a monoclonal antibody to the c-kit receptor. The expression of the c-kit receptor protein was examined in the testes and sperm with Western blot analysis., Results: Localization was noted in the early spermatogenic cells, most likely type A spermatogonia, as well as in the acrosomal region of more mature germ cells, such as the round spermatids. The c-kit receptor was localized in analogous sites in all three species. The Western blot data revealed testicular expression of the c-kit receptor protein in all three species as well. Similar bands were recognized on the Western blots of all three species in testes at approximately 75 kDa and approximately 90 kDa, and sperm at approximately 90 kDa only., Conclusions: The c-kit receptor protein is expressed in the early spermatogenic cells, as well as the later stages of spermatogenesis, specifically, the acrosomal granules of the round spermatids, and the acrosomal region of testicular spermatozoa, in the mouse, rat, and human. All three species exhibit similar expression of the c-kit receptor protein in both testis and sperm, although to a varying degree. We believe that these observations allow direct valid comparisons concerning the expression of the c-kit receptor to be made cautiously to the human condition from experimental data obtained from rodents.

The c-KIT proto-oncogene encodes for a transmembrane receptor and is associated with maturation of several cell types, including germ cells. The ligand of the receptor has been identified as stem cell factor (SCF). Loss or alteration of the expression of either of these factors leads to anemia, albinism, and/or sterility in mice. We examined the expression of c-KIT and SCF by immunohistochemistry in specimens from normal and infertile human testis. All specimens were obtained in the evaluation of male subfertility. We were able to demonstrate staining for c-KIT in Leydig cells in all specimens. Normal testis stained for c-KIT in the cytoplasm of early spermatogenic cells, as well as the acrosomal granules of the round spermatids and the acrosome of testicular spermatozoa. However, staining in testis demonstrating maturation arrest failed to demonstrate acrosomal staining, and Sertoli-only specimens demonstrated staining for c-KIT in Leydig cells only. The results for SCF demonstrated an overall uniform staining of Leydig cells in all specimens. The intensity of staining of Sertoli cells increased from normal to maturation arrest to Sertoli-only specimens. Germ cell staining was consistently negative. We hypothesize that these staining patterns for SCF are due to either lack of staining of the receptor-ligand complex or overexpression of the kit ligand in tissue that does not express the kit receptor. It appears that the c-kit receptor is expressed in the acrosome of developing germ cells, as well as in Leydig cells and early spermatogenic cells, suggesting a role in the acrosome reaction, as well as germ cell maturation and differentiation.