27 28 Study question: Is nuclear quality of in vitro generated spermatozoa from fresh or 29 frozen/thawed pre-pubertal mouse testes similar to that of their in vivo counterparts? 30 Summary answer: The production of spermatozoa with aneuploidy, DNA fragmentation or 31 chromatin condensation defects was not significantly increased in organotypic cultures 32 compared to in vivo controls. 33 What is known already: Although murine spermatozoa have been produced in vitro from 34 pre-pubertal testes, their nuclear DNA integrity has never been investigated. 35 Study design, size, duration: Fresh and frozen/thawed testicular fragments from 6-7 days 36 postpartum (dpp) mice were cultured for 30 days. Testicular tissues were frozen by controlled 37 slow freezing (CSF) or solid surface vitrification (SSV). In total, 30 fresh, 30 CSF, 30 SSV 38 testes were used for in vitro maturation and 6 testes from 36-37 dpp mice were used as in vivo 39 controls. 40 Participants/materials, setting, methods: Murine spermatozoa were extracted from pooled 41 in vitro cultured testicular fragments and from in vivo controls. Sperm aneuploidy was 42 analyzed by fluorescence in situ hybridization (FISH), DNA fragmentation by terminal 43 deoxynucleotidyltransferase mediated dUTP nick end labelling, chromatin condensation by 44 aniline blue staining, telomere length and number by quantitative FISH, DNA oxidation by 45 immunohistochemical detection of 8-hydroxy-2‟-deoxyguanosine (8-OHdG). Because of the 46 low spermatogenic yield in cultures, a hundred spermatozoa extracted from pooled tissues 47 were examined and compared to their in vivo counterparts. 48 Main results and the role of chance: Most of spermatozoa generated in vitro and in vivo 49 were haploid, contained unfragmented DNA and normally condensed chromatin. A similar 50 proportion of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation 51 defects was found in cultures and in vivo. No significant difference in telomere length was 52 found within the nuclei of in vitro and in vivo generated spermatozoa. However, the number 53 of telomere spots was lower in gametes obtained from cultures of fresh, CSF and SSV testes 54 than in their natural counterparts (p<0.01). Moreover, the proportion of spermatozoa 55 containing 8-OHdG was significantly increased in frozen/thawed tissues in comparison to 56 fresh tissues and in vivo controls (p<0.05). 57 Large scale data: none 58 Limitations, reasons for caution: Further studies will be needed to enhance the production 59 of spermatozoa in organotypic cultures while preserving their quality, to investigate 60 epigenetic modifications and embryonic development. 61 Wider implications of the findings: This is the first study comparing the nuclear quality of 62 in vitro and in vivo generated murine spermatozoa. The organotypic culture system will have 63 to be adapted for human tissue and extensive analyses of human gamete quality will have to 64 be performed before potential clinical applications can be envisaged. 65 Study funding and competing interest(s): This work was supported by Rouen University 66 Hospital, Ligue contre le Cancer, Agence de la Biomédecine, Association Laurette Fugain, 67 France Lymphome Espoir, and co-supported by European Union and Région Normandie. 68 Europe gets involved in Normandie with European Régional Development Fund (ERDF). The 69 authors declare that they have no conflict of interest. 70 71