Recommandations de l’AFU et de la SALF concernant l’évaluation de l’homme infertile

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Paradoxical risk of reduced fertility after exposure of prepubertal mice to vincristine or cyclophosphamide at low gonadotoxic doses in humans

Abstract Cancer treatment can have long-term side effects in cured patients and infertility is one of them. Given the urgency of diagnosis in children with cancer, the toxicity of treatments on the gonad was overshadowed for a long time. In the present study, prepubertal mice were treated by vincristine or cyclophosphamide commonly used in acute leukaemia treatment. The prepubertal exposure to cyclophosphamide, at a low gonadotoxic dose in humans (< 3.5 g/m 2 ), led to morphological alterations of prepubertal testicular tissue. An increased proportion of spermatozoa with hypocondensed chromatin and oxidized DNA associated with decreased fertility were uncovered at adulthood. Short- and long-term morphological alterations of the testicular tissue, disturbed progression of spermatogenesis along with increased proportions of isolated flagella and spermatozoa with fragmented DNA were evidenced in vincristine-treated mice. Moreover, the fertility of mice exposed to vincristine was severely affected despite being considered low-risk for fertility in humans. Paternal exposure to vincristine or cyclophosphamide before puberty had no impact on offspring development. Contrary to the current gonadotoxic risk classification, our results using a mouse model show that vincristine and cyclophosphamide (< 3.5 g/m 2 ) present a high gonadotoxic risk when administered before the initiation of spermatogenesis

Dynamics of epigenetic modifications in ICSI embryos from in vitro‐produced spermatozoa

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Achievement of complete in vitro spermatogenesis in testicular tissues from prepubertal mice exposed to mono- or polychemotherapy

International audienceThe assessment of the impact of chemotherapies on in vitro spermatogenesis in experimental models is required before considering the application of this fertility restoration strategy to prepubertal boys who received these treatments before testicular tissue cryopreservation. The present work investigated the effects of exposure of prepubertal mice to mono- (vincristine or cyclophosphamide) and polychemotherapy (a combination of vincristine and cyclophosphamide) on the first wave of in vitro spermatogenesis. When testicular tissue exposed to monochemotherapy was preserved, polychemotherapy led to severe alterations of the seminiferous epithelium and increased apoptosis in prepubertal testes prior in vitro maturation, suggesting a potential additive gonadotoxic effect. These alterations were also found in the testicular tissues of polychemotherapy-treated mice after 30 days of organotypic culture and were associated with a reduction in the germ cell/Sertoli cell ratio. The different treatments neither altered the ability of spermatogonia to differentiate in vitro into spermatozoa nor the yield of in vitro spermatogenesis. However, more spermatozoa with morphological abnormalities and fragmented DNA were produced after administration of polychemotherapy. This work therefore shows for the first time the possibility to achieve a complete in vitro spermatogenesis after an in vivo exposure of mice to a mono- or polychemotherapy before meiotic entry

Complete meiosis in rat prepubertal testicular tissue under in vitro sequential culture conditions

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IHC_Tool: An open-source Fiji procedure for quantitative evaluation of cross sections of testicular explants

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Sperm Chromatin Condensation Defect Accelerates the Kinetics of Early Embryonic Development but Does Not Modify ICSI Outcome

The origin and quality of gametes are likely to influence the kinetics of embryonic development. The purpose of the study was to assess the impact of sperm nuclear quality, and in particular sperm chromatin condensation, on the kinetics of early embryo development after intracytoplasmic sperm injection (ICSI). Our study included 157 couples who benefitted from ICSI for male factor infertility. Chromatin condensation and DNA fragmentation were assessed in spermatozoa prior to ICSI. Above the 20% threshold of sperm condensation defect, patients were included in the abnormal sperm chromatin condensation (ASCC) group; below the 20% threshold, patients were included in the normal sperm chromatin condensation (NSCC) group. After ICSI, the oocytes were placed in the time-lapse incubator. The kinetics of the cohort’s embryonic development have been modeled. The fading times of pronuclei and the time to two blastomeres (t2, first cleavage) and four blastomeres (t4, third cleavage) differed significantly between the NSCC and ASCC groups, with earlier events occurring in the ASCC group. On the other hand, the state of sperm chromatin condensation did not seem to have an impact on live birth rates or the occurrence of miscarriages. The kinetics of early embryonic development was accelerated in males with a sperm chromatin condensation defect without compromising the chances of pregnancy or promoting miscarriage. However, our study highlights the paternal contribution to early embryonic events and potentially to the future health of the conceptus

Throughout in vitro first spermatogenic wave: Next-generation sequencing gene expression patterns of fresh and cryopreserved prepubertal mice testicular tissue explants

International audienceIntroduction Suitable cryopreservation procedures of pre-pubertal testicular tissue associated with efficient culture conditions are crucial in the fields of fertility preservation and restoration. In vitro spermatogenesis remains a challenging technical procedure to undergo a complete spermatogenesis.The number of haploid cells and more specifically the spermatic yield produced in vitro in mice is still extremely low compared to age-matched in vivo controls and this procedure has never yet been successfully transferred to humans. Methods To evaluate the impact of in vitro culture and freezing procedure, pre-pubertal testicular mice testes were directly cultured until day 4 (D4), D16 and D30 or cryopreserved by controlled slow freezing then cultured until D30. Testes composed of a panel of 6.5 dpp (days postpartum), 10.5 dpp, 22.5 dpp, and 36.5 dpp mice were used as in vivo controls. Testicular tissues were assessed by histological (HES) and immunofluorescence (stimulated by retinoic acid gene 8, STRA8) analyses. Moreover, a detailed transcriptome evaluation study has been carried out to study the gene expression patterns throughout the first in vitro spermatogenic wave. Results Transcriptomic analyses reveal that cultured tissues expression profiles are almost comparable between D16 and D30; highlighting an abnormal kinetic throughout the second half of the first spermatogenesis during in vitro cultures. In addition, testicular explants have shown dysregulation of their transcriptomic profile compared to controls with genes related to inflammation response, insulin-like growth factor and genes involved in steroidogenesis. Discussion The present work first shows that cryopreservation had very little impact on gene expression in testicular tissue, either directly after thawing or after 30 days in culture. Transcriptomic analysis of testis tissue samples is highly informative due to the large number of expressed genes and identified isoforms. This study provides a very valuable basis for future studies concerning in vitro spermatogenesis in mice

Understanding the Underlying Molecular Mechanisms of Meiotic Arrest during In Vitro Spermatogenesis in Rat Prepubertal Testicular Tissue

International audienceIn vitro spermatogenesis appears to be a promising approach to restore the fertility of childhood cancer survivors. The rat model has proven to be challenging, since germ cell maturation is arrested in organotypic cultures. Here, we report that, despite a meiotic entry, abnormal synaptonemal complexes were found in spermatocytes, and in vitro matured rat prepubertal testicular tissues displayed an immature phenotype. RNA-sequencing analyses highlighted up to 600 differentially expressed genes between in vitro and in vivo conditions, including genes involved in blood-testis barrier (BTB) formation and steroidogenesis. BTB integrity, the expression of two steroidogenic enzymes, and androgen receptors were indeed altered in vitro. Moreover, most of the top 10 predicted upstream regulators of deregulated genes were involved in inflammatory processes or immune cell recruitment. However, none of the three anti-inflammatory molecules tested in this study promoted meiotic progression. By analysing for the first time in vitro matured rat prepubertal testicular tissues at the molecular level, we uncovered the deregulation of several genes and revealed that defective BTB function, altered steroidogenic pathway, and probably inflammation, could be at the origin of meiotic arrest

Oxidative Stress Is Associated with Telomere Interaction Impairment and Chromatin Condensation Defects in Spermatozoa of Infertile Males

Telomere length can be influenced by reactive oxygen species (ROS) generated by lifestyle factors or environmental exposure. We sought to determine whether oxidative stress has an impact on sperm nuclear alterations, especially on chromatin organization and telomere interactions in the spermatozoa of infertile males. We performed an observational and prospective study including fifty-two males, allocated in the “case group” (30 infertile males presenting conventional semen parameter alterations) and the “control group” (22 males with normal conventional semen parameters). ROS detection was determined on spermatozoa using CellROX© probes. Sperm nuclear damage was assessed using quantitative fluorescence in situ hybridization (Q-FISH) for relative telomere length and telomere number, aniline blue staining for chromatin condensation, terminal deoxynucleotidyl transferase dUTP nick-end labeling for DNA fragmentation, and FISH for aneuploidy and 8-hydroxy-2′-deoxyguanosine immunostaining for oxidative DNA damages. Infertile males had significantly increased levels of cytoplasmic ROS and chromatin condensation defects as well as a higher mean number of telomere signals per spermatozoon in comparison with controls. In addition, the mean number of sperm telomere signals were positively correlated with the percentage of spermatozoa with chromatin condensation defect. In infertile males with conventional semen parameter alterations, oxidative stress is associated with telomere interaction impairment and chromatin condensation defects