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STUDY QUESTION Can a systematic scoring procedure provide crucial information on the status of highly heterogeneous immature human testicular tissues in the context of cryopreservation for fertility preservation? SUMMARY ANSWER We developed a systematic histological score as a novel diagnostic tool which differentiates the patient cohort according to the status of germ cell differentiation and number of spermatogonia (normal, diminished and absent), and which could be relevant in the fertility clinic. WHAT IS KNOWN ALREADY Cryopreservation of testicular tissue of immature boys is currently considered the option for future fertility restoration. However, experimental techniques for the derivation of sperm as well as valid diagnostic scoring of these immature testis tissues are not yet reported. STUDY DESIGN, SIZE, DURATION Testicular tissues of 39 patients (aged 2-20 years) who attended our clinic for cryopreservation between 2010 and 2015 were analyzed to determine the variability of testicular tissue composition, germ cell numbers and differentiation status. PARTICIPANTS/MATERIALS, SETTING, METHODS Human testicular tissue samples were divided into three groups. Group NT included patients suffering from diseases which do not directly affect the testes (n = 6; aged 6-14 years), group AT included patients suffering from diseases that directly affect the testes (n = 14; 2-17 years), and group KS (Klinefelter patients, n = 19; 12-20 years). Based on immunohistochemical stainings for MAGEA4, the differentiation status as well as the numbers of gonocytes, spermatogonia and spermatocytes were determined. MAIN RESULTS AND THE ROLE OF CHANCE Testicular tissue samples from the NT group contained a mean of 100.3 spermatogonia/mm3 (×103). Highly heterogeneous and significantly lower mean numbers of spermatogonia were scored in testes from boys after cytotoxic exposures or with pre-existing disease (AT group: 35.7 spermatogonia/mm3 (×103); KS group: 1.8 spermatogonia/mm3 (×103)). In addition, the germ cell differentiation status was determined and revealed tissues with either spermatogonia and gonocytes, only spermatogonia, spermatogonia and spermatocytes, or all three germ cell types were present. Based on spermatogonial numbers and differentiation status, we developed a germ cell score which we applied to each individual patient sample. LIMITATIONS REASONS FOR CAUTION Normal human testicular tissue samples are difficult to obtain for ethical reasons and the sample numbers were small. However, six such samples provide a valid baseline for the normal situation. WIDER IMPLICATIONS OF THE FINDINGS Fertility preservation of immature male tissues is an emerging field and is currently offered in many specialized centers worldwide. Our diagnostic germ cell score delivers an easily applicable tool, facilitating patient counseling and thus ensuring comparability between the centers with regard to future studies. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Funding Initiative: Translational Research, Ministry of Innovation, Science and Research, Federal State of North Rhine Westphalia (z1403ts006). The authors declare that they do not have competing financial interests.