Assessment of fresh and cryopreserved testicular tissues from (pre)pubertal boys during organ culture as a strategy for in vitro spermatogenesis.

Can the organ culture method be applied to both fresh and cryopreserved human (pre)pubertal testicular tissue as a strategy for in vitro spermatogenesis? Although induction of spermatogenesis was not achieved in vitro, testicular architecture, endocrine function and spermatogonial proliferation were maintained in both fresh and cryopreserved testicular tissues. Cryopreservation of a testicular biopsy is increasingly offered as a fertility preservation strategy for prepubertal cancer patients. One of the proposed experimental approaches to restore fertility is the organ culture method, which, in the mouse model, successfully allows for in vitro development of spermatozoa from testicular biopsies. However, complete spermatogenesis from human prepubertal testicular tissue in such an organ culture system has not been demonstrated. Testicular tissue was collected from nine (pre)pubertal boys diagnosed with cancer (ranging from 6 to 14 years of age) admitted for fertility preservation before treatment. Testicular biopsies were either immediately processed for culture or first cryopreserved, using a controlled slow freezing protocol, and thawed before culture. Organ culture of testicular fragments was performed in two different media for a maximum period of 5 weeks, targeting early cellular events (viability, meiosis and somatic differentiation) in vitro. Fresh and cryopreserved-thawed testis fragments (1-2 mm3) were cultured at a gas-liquid interphase (34°C, 5% CO2) in Minimum Essential Medium alpha + 10% knock-out serum replacement medium containing 10-7 M melatonin and 10-6 M retinoic acid, with or without 3 IU/L FSH/LH supplementation. The effect of culture conditions on testicular fragments was weekly assessed by histological evaluation of germ cell development and immunohistochemical identification of spermatogonia (using MAGEA4), proliferative status of spermatogonia and Sertoli cells (using proliferating cell nuclear antigen [PCNA]), intratubular cell apoptosis (by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) and Sertoli cells maturation (using Anti-Müllerian Hormone [AMH] versus Androgen Receptor [AR]). Additionally, Leydig cells' functionality was determined by measuring testosterone concentration in the culture media supernatants. Neither fresh nor cryopreserved human (pre)pubertal testicular fragments were able to initiate spermatogenesis in our organ culture system. Nonetheless, our data suggest that fresh and cryopreserved testicular fragments have comparable functionality in the described organ culture conditions, as reflected by the absence of significant differences in any of the weekly evaluated functional parameters. Additionally, no significant differences were found between the two tested media when culturing fresh and cryopreserved human testicular fragments. Although spermatogonia survived and remained proliferative in all culture conditions, a significant reduction of the spermatogonial population (P ≤ 0.001) was observed over the culture period, justified by a combined reduction of proliferation activity (P ≤ 0.001) and increased intratubular cell apoptosis (P ≤ 0.001). We observed a transient increase in Sertoli cell proliferative activity, loss of AMH expression (P ≤ 0.001) but no induction of AR expression. Leydig cell endocrine function was successfully stimulated in vitro as indicated by increased testosterone production in all conditions throughout the entire culture period (P ≤ 0.02). N/A. Although not noticeable in this study, we cannot exclude that if an optimized culture method ensuring complete spermatogenesis in human testicular fragments is established, differences in functional or spermatogenic efficiency between fresh and cryopreserved tissue might be found. The current inability to initiate spermatogenesis in vitro from cryopreserved human testicular fragments should be included in the counselling of patients who are offered testicular tissue cryopreservation to preserve fertility. This project was funded by EU-FP7-PEOPLE-2013-ITN 603568 `Growsperm'. None of the authors have competing interests. Not applicable.

Portela JMD ，de Winter-Korver CM ，van Daalen SKM ，Meißner A ，de Melker AA ，Repping S ，van Pelt AMM ... -  《-》

Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue.

Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)? Our organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation. Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans. The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm3 tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared. ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3β-hydroxysteroid dehydrogenase (3β-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis. Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either medium. GDNF was expressed until Day 139, demonstrating SC functionality. Moreover, a significant reduction in AMH expression and release indicated SC maturation. Testosterone concentrations in supernatants increased in both culture media, demonstrating LC functionality with paracrine interactions. SG were present up to Day 139, although the ratio between MAGE-A4-positive cells and well-preserved tubules was significantly reduced over the course of culture (P ≤ 0.001). SCs exhibited a decreased proliferation rate over time (P ≤ 0.05). The proliferation rate of SG remained stable until Day 64, but over the total culture period (139 days), it was found to have decreased (P ≤ 0.05). The number of apoptotic cells did not vary during culture, nor was any statistical difference observed between the two culture media for any of the studied parameters. N/A LIMITATIONS, REASONS FOR CAUTION: Loss of SG constitutes a limitation for evaluating full functionality of spermatogonial stem cells and warrants further investigation. The scarcity of human immature material is the reason for the limited amount of tissue available for experiments, precluding more comprehensive analysis. Our culture system, mimicking the peripubertal testicular microenvironment with SC maturation, LC functionality and preserved paracrine interactions, and the first to use human ITT, opens the door to a deeper understanding of niche and culture conditions to obtain sperm from cryostored ITT, with the ultimate goal of restoring fertility after gonadotoxic treatments. This project was supported by a grant from the Fond National de la Recherche Scientifique de Belgique (grant Télevie N° 7.4554.14F and N° 7.4512.15F) and the Fondation Salus Sanguinis. No conflict of interest is declared.

de Michele F ，Poels J ，Weerens L ，Petit C ，Evrard Z ，Ambroise J ，Gruson D ，Wyns C ... -  《-》

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase_9) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.

Dumont L ，Chalmel F ，Oblette A ，Berby B ，Rives A ，Duchesne V ，Rondanino C ，Rives N ... -  《-》

In vitro formation of the blood-testis barrier during long-term organotypic culture of human prepubertal tissue: comparison with a large cohort of pre/peripubertal boys.

de Michele F ，Poels J ，Giudice MG ，De Smedt F ，Ambroise J ，Vermeulen M ，Gruson D ，Wyns C ... -  《-》

Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate.

Can full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously? Intratesticular xenotransplantation supported the differentiation of immature germ cells from marmoset (Callithrix jacchus) into spermatids and spermatozoa at 4 and 9 months post-transplantation, while in subcutaneous transplants, spermatogenic arrest was observed at 4 months and none of the transplants survived at 9 months. Auto-transplantation of cryopreserved immature testis tissue (ITT) could be a potential fertility restoration strategy for patients with complete loss of germ cells due to chemo- and/or radiotherapy at a young age. Before ITT transplantation can be used for clinical application, it is a prerequisite to demonstrate the feasibility of the technique and identify the conditions required for establishing spermatogenesis in primate ITT transplants. Although xenotransplantation of ITT from several species has resulted in complete spermatogenesis, in human and marmoset, ITT has not been successful. In this study, we used marmoset as a pre-clinical animal model. ITT was obtained from two 6-month-old co-twin marmosets. A total of 147 testis tissue pieces (~0.8-1.0 mm3 each) were transplanted into the testicular parenchyma (intratesticular; n = 40) or under the dorsal skin (ectopic; n = 107) of 4-week-old immunodeficient Swiss Nu/Nu mice (n = 20). Each mouse received one single marmoset testis tissue piece in each testis and 4-6 pieces subcutaneously. Xenotransplants were retrieved at 4 and 9 months post-transplantation and evaluations were performed with regards to transplant survival, spermatogonial quantity and germ cell differentiation. Transplant survival was histologically evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Spermatogonia were identified by MAGE-A4 via immunohistochemistry. Germ cell differentiation was assessed by morphological identification of different germ cell types on H/PAS stained sections. Meiotically active germ cells were identified by BOLL expression. CREM immunohistochemistry was performed to confirm the presence of post-meiotic germ cells and ACROSIN was used to determine the presence of round, elongating and elongated spermatids. Four months post-transplantation, 50% of the intratesticular transplants and 21% of the ectopic transplants were recovered (P = 0.019). The number of spermatogonia per tubule did not show any variation. In 33% of the recovered intratesticular transplants, complete spermatogenesis was established. Overall, 78% of the intratesticular transplants showed post-meiotic differentiation (round spermatids, elongating/elongated spermatids and spermatozoa). However, during the same period, spermatocytes (early meiotic germ cells) were the most advanced germ cell type present in the ectopic transplants. Nine months post-transplantation, 50% of the intratesticular transplants survived, whilst none of the ectopic transplants was recovered (P < 0.0001). Transplants contained more spermatogonia per tubule (P = 0.018) than at 4 months. Complete spermatogenesis was observed in all recovered transplants (100%), indicating a progressive spermatogenic development in intratesticular transplants between the two time-points. Nine months post-transplantation, transplants contained more seminiferous tubules with post-meiotic germ cells (37 vs. 5%; P < 0.001) and fewer tubules without germ cells (2 vs. 8%; P = 0.014) compared to 4 months post-transplantation. N/A. Although xenotransplantation of marmoset ITT was successful, it does not fully reflect all aspects of a future clinical setting. Furthermore, due to ethical restrictions, we were not able to prove the functionality of the spermatozoa produced in the marmoset transplants. In this pre-clinical study, we demonstrated that testicular parenchyma provides the required microenvironment for germ cell differentiation and long-term survival of immature marmoset testis tissue, likely due to the favourable temperature regulation, growth factors and hormonal support. These results encourage the design of new experiments on human ITT xenotransplantation and show that intratesticular transplantation is likely to be superior to ectopic transplantation for fertility restoration following gonadotoxic treatment in childhood. This project was funded by the ITN Marie Curie Programme 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568) and the scientific Fund Willy Gepts from the UZ Brussel (ADSI677). D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

Ntemou E ，Kadam P ，Van Saen D ，Wistuba J ，Mitchell RT ，Schlatt S ，Goossens E ... -  《-》