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

STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LIMITATIONS REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): 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.

Vitamin A prevents round spermatid nuclear damage and promotes the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Does vitamin A (retinol, Rol) prevent round spermatid nuclear damage and increase the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue? SUMMARY ANSWER: The supplementation of an in vitro culture of ~0.75 mm3 testicular explants from pre-pubertal mice with Rol enhances spermatogenesis progression during the first spermatogenic wave. WHAT IS KNOWN ALREADY: The production of functional spermatozoa in vitro has only been achieved in the mouse model and remains a rare event. Establishing an efficient culture medium for vitrified pre-pubertal testicular tissue is now a crucial step to improve the spermatic yield obtained in vitro. The role of Rol in promoting the differentiation of spermatogonia and their entry into meiosis is well established; however, it has been postulated that Rol is also required to support their full development into elongated spermatids. STUDY DESIGN, SIZE, DURATION: A total of 60 testes from 6.5 days post-partum (dpp) mice were vitrified/warmed, cut into fragments and cultured for 30 days: 20 testes were used for light microscopy and histological analyses, 20 testes for DNA fragmentation assessment in round spermatids and 20 testes for induced sperm motility assessment. Overall, 16 testes of 6.5 dpp were used as in vitro fresh tissue controls and 12 testes of 36.5 dpp mice as in vivo controls. Testes were vitrified with the optimal solid surface vitrification procedure and cultured with an in vitro organ culture system until Day 30 (D30). Histological analysis, cell death, degenerating round spermatids, DNA fragmentation in round spermatids and induced sperm motility were assessed. Testosterone levels were measured in media throughout the culture by radioimmunoassay. MAIN RESULTS AND THE ROLE OF CHANCE: At D30, better tissue development together with higher differentiation of spermatogonial stem cells, and higher global cell division ability were observed for vitrified/warmed testicular fragments of ~0.75 mm3 with a culture medium supplemented with Rol compared to controls. During in vitro culture of vitrified pre-pubertal testicular tissue, Rol enhanced and maintained the entry of spermatogonia into meiosis and promoted a higher spermatic yield. Furthermore, decreased round spermatid nuclear alterations and DNA damage combined with induced sperm motility comparable to in vivo highlight the crucial role of Rol in the progression of spermatogenesis during the first wave. LIMITATIONS, REASONS FOR CAUTION: Despite our promising results, the culture media will have to be further improved and adapted within the context of a human application. WIDER IMPLICATIONS OF THE FINDINGS: The results have potential implications for the handling of human pre-pubertal testicular tissues cryopreserved for fertility preservation. However, because some alterations in round spermatids persist after in vitro culture with Rol, the procedure needs to be optimized before human application, bearing in mind that the murine and human spermatogenic processes differ in many respects. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by a Ph.D. grant from the Normandy University and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Rouen University Hospital, Institute for Research and Innovation in Biomedicine (IRIB) and Agence de la Biomédecine. The authors declare that there is no conflict of interest.

Fertility preservation in adolescent males: experience over 22 years at Rouen University Hospital.

BACKGROUND: Sperm banking is a suitable procedure to prevent infertility after cancer therapy in male adolescents. We evaluated the feasibility of semen preservation in 156 adolescents aged between 13 and 20 years and then we assessed fertility outcome after treatment. METHODS: Age, urogenital history, indications for cryopreservation, histological diagnosis and semen parameters were recorded. Fertility status after treatment was assessed by a questionnaire addressed to those patients who had utilized sperm storage. Post-treatment semen analysis was performed for 22 patients. RESULTS: Cryopreservation was possible in 88.5% of cases. Azoospermia was detected in 2.6% of the patients at the time of diagnosis. Malignant disease accounted for 84% of our male adolescents. In this type of disease, semen parameters were significantly altered only among patients with metastatic malignant bone tumour. After treatment, nine patients presented azoospermia, five patients achieved pregnancy spontaneously, two achieved it after assisted reproductive technique using fresh ejaculated spermatozoa and one following sperm donation. Three failed with cryopreserved sperm. CONCLUSIONS: Semen cryopreservation is possible for most adolescents and, regardless of disease type, may be a means of preserving fertility prior to gonadotoxic treatment that might impair the spermatogenesis process.

[Chromosome abnormalities of spermatozoa]. / Anomalies chromosomiques du spermatozoïde.

Chromosome meiotic pairing during male meiosis is a major event for chromosome segregation during anaphase I and spermatogenesis normal process. Chromosome non-disjunctions responsible for aneuploidy in male gametes can be observed during the first and the second meiotic divisions. The analysis of sperm nuclei chromosome constitution is a major and indirect tool for assessing male meiotic non-disjunctions and the genesis of chromosomal abnormalities. This evaluation has been performed initially by the human sperm/hamster oocyte fusion assay and more recently by fluorescence in situ hybridisation (FISH). Therefore, male populations with increased risk of aneuploidy for their progeny could be identified before entering an in vitro fertilization procedure, and depending on the potential risk a preimplantation or prenatal genetic diagnosis could be performed. For males with constitutional chromosome abnormalities, a specific genetic counselling could also be proposed.

Fluorescence in situ hybridization with chromosome paint probes: a novel approach to assess aneuploidy in human sperm nuclei.

PURPOSE: Fluorescence in situ hybridization (FISH) using whole-chromosome paint probes was performed to evaluate disomy and diploidy frequency for chromosomes 1, 18, 19, and 22 in human sperm nuclei. METHODS: Ten subjects of proven fertility and normal spermatic parameters were included in the study. A dual-color FISH method was carried out. RESULTS: A total of 157,896 spermatozoa was scored. The mean frequencies of disomic sperm for chromosomes 1, 18, 19, and 22 were 0.22% (range, 0.19 to 0.28%), 0.24% (range, 0.14 to 0.37%), 0.22% (range, 0.17 to 0.30%), and 0.25% (range, 0.21 to 0.29%), respectively. The mean frequency of diploidy was 0.14% (range, 0.09 to 0.18%). No interindividual and interchromosomal variations in the aneuploidy frequency were observed between the different subjects. CONCLUSIONS: FISH with whole-chromosome paint probes provides a novel and efficient approach for disomy assessment in human sperm nuclei.