Abstract STUDY QUESTION Does chemotherapy exposure (with or without alkylating agents) or primary diagnosis affect spermatogonial quantity in human prepubertal testicular tissue? SUMMARY ANSWER Spermatogonial quantity is significantly reduced in testes of prepubertal boys treated with alkylating agent therapies or with hydroxyurea for sickle cell disease. WHAT IS KNOWN ALREADY Cryopreservation of spermatogonial stem cells, followed by transplantation into the testis after treatment, is a proposed clinical option for fertility restoration in children. The key clinical consideration behind this approach is a sufficient quantity of healthy cryopreserved spermatogonia. However, since most boys with malignancies start therapy with agents that are not potentially sterilizing, they will have already received some chemotherapy before testicular tissue cryopreservation is considered. STUDY DESIGN, SIZE, DURATION We examined histological sections of prepubertal testicular tissue to elucidate whether chemotherapy exposure or primary diagnosis affects spermatogonial quantity. Quantity of spermatogonia per transverse tubular cross-section (S/T) was assessed in relation to treatment characteristics and normative reference values in histological sections of paraffin embedded testicular tissue samples collected from 32 consecutive boy patients (aged 6.3 ± 3.8 [mean ± SD] years) between 2014 and 2017, as part of the NORDFERTIL study, and in 14 control samples (from boys aged 5.6 ± 5.0 [mean ± SD] years) from an internal biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS Prepubertal boys in Sweden, Finland and Iceland who were facing treatments associated with a very high risk of infertility, were offered the experimental procedure of testicular cryopreservation. Exclusion criteria were testicular volumes >10 ml and high bleeding or infection risk. There were 18 patients with a diagnosis of malignancy and 14 patients a non-malignant diagnosis. While 20 patients had the testicular biopsy performed 1–45 days after chemotherapy, 12 patients had not received any chemotherapy. In addition, 14 testicular tissue samples of patients with no reported testicular pathology, obtained from the internal biobank of the Department of Pathology at Karolinska University Hospital, were included as control samples in addition to reference values obtained from a recently published meta-analysis. The quantity of spermatogonia was assessed by both morphological and immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE The main finding was a significant reduction in spermatogonial cell counts in boys treated with alkylating agents or with hydroxyurea for sickle cell disease. The mean S/T values in boys exposed to alkylating agents (0.2 ± 0.3, n = 6) or in boys with sickle cell disease and exposed to hydroxyurea (0.3 ± 0.6, n = 6) were significantly lower (P = 0.003 and P = 0.008, respectively) than in a group exposed to non-alkylating agents or in biobank control samples (1.7 ± 1.0, n = 8 and 4.1 ± 4.6, n = 14, respectively). The mean S/T values of the testicular tissue samples included in the biobank control group and the patient group exposed to non-alkylating agents were within recently published normative reference values. LIMITATIONS, REASONS FOR CAUTION Normal testicular tissue samples included in this study were obtained from the internal biobank of Karolinska University Hospital. Samples were considered normal and included in the study if no testicular pathology was reported in the analysed samples. However, detailed information regarding previous medical treatments and testicular volumes of patients included in this biobank were not available. WIDER IMPLICATIONS OF THE FINDINGS This study summarizes, for the first time, spermatogonial quantity in a prepubertal patient cohort just before and after potentially sterilizing treatments. Boys facing cancer and cytotoxic therapies are regarded as the major group who will benefit from novel fertility preservation techniques. There are no previous reports correlating spermatogonial quantity to cumulative exposure to alkylating agents and anthracyclines (non-alkylating agents) and no information about the timing of cytotoxic exposures among this particular patient cohort. For prepubertal boys in whom fertility preservation is indicated, testicular tissue should be obtained before initiation of chemotherapy with alkylating agents, whilst for those with sickle cell disease and treated with hydroxyurea, this approach to fertility preservation may not be feasible. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from The Swedish Childhood Cancer Foundation (PR2016-0124; TJ2016-0093; PR2015-0073, TJ2015-0046) (J.-B.S. and K.J.), the Jane and Dan Olssons Foundation (2016-33) (J.-B.S.), the Finnish Cancer Society (K.J.), the Foundation for Paediatric Research (J.-B.S.), Kronprinsessan Lovisas Förening För Barnasjukvård/ Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation (J.-B.S.), the Väre Foundation for Paediatric Cancer Research (K.J.) and the Swedish Research Council (2012-6352) (O.S.). R.T.M. was supported by a Wellcome Trust Fellowship (09822). J.P.A.-L. and M.K. were supported by the ITN Marie Curie program ‘Growsperm’ (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

and the Swedish Research Council (2012-6352) (O.S.). R.T.M. was supported by a Wellcome Trust Fellowship (09822). J.P.A.-L. and M.K. were supported by the ITN Marie Curie program ‘ Growsperm ’ (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no con fl icts of interest. t test (normally distributed variables), the Mann – Whitney U test (non-normally distributed variables) and Pearson correlation coef fi cient ( r ) were used as appropriate to determine the relationships between spermatogonial quantity, age and treatment characteristics. Analyses were performed using IBM SPSS Statistics V23.0 software (IBM Corporation, Armonk, NY, USA).

STUDY QUESTION Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA None. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests.