A splice donor variant of GAS8 induces structural disorganization of the axoneme in sperm flagella and leads to nonsyndromic male infertility.

Motile cilia and flagella are closely related organelles structured around a highly conserved axoneme whose formation and maintenance involve proteins from hundreds of genes. Defects in many of these genes have been described to induce primary ciliary dyskinesia (PCD) mainly characterized by chronic respiratory infections, situs inversus and/or infertility. In men, cilia/flagella-related infertility is usually caused by asthenozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we investigated a cohort of 196 infertile men displaying a typical MMAF phenotype without any other PCD symptoms. Analysis of WES data identified a single case carrying a deleterious homozygous GAS8 variant altering a splice donor consensus site. This gene, also known as DRC4, encodes a subunit of the Nexin-Dynein Regulatory Complex (N-DRC), and has been already associated to male infertility and mild PCD. Confirming the deleterious effect of the candidate variant, GAS8 staining by immunofluorescence did not evidence any signal from the patient's spermatozoa whereas a strong signal was present along the whole flagella length in control cells. Concordant with its role in the N-DRC, transmission electron microscopy evidenced peripheral microtubule doublets misalignments. We confirm here the importance of GAS8 in the N-DRC and observed that its absence induces a typical MMAF phenotype not necessarily accompanied by other PCD symptoms.

Steroidogenesis and androgen/estrogen signaling pathways are altered in in vitro matured testicular tissues of prepubertal mice.

Children undergoing cancer treatments are at risk for impaired fertility. Cryopreserved prepubertal testicular biopsies could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from mouse prepubertal testicular tissue, although with low efficiency. Steroid hormones are essential for the progression of spermatogenesis, the aim of this study was to investigate steroidogenesis and steroid signaling in organotypic cultures. Histological, RT-qPCR, western blot analyses, and steroid hormone measurements were performed on in vitro cultured mouse prepubertal testicular tissues and age-matched in vivo controls. Despite a conserved density of Leydig cells after 30 days of culture (D30), transcript levels of adult Leydig cells and steroidogenic markers were decreased. Increased amounts of progesterone and estradiol and reduced androstenedione levels were observed at D30, together with decreased transcript levels of steroid metabolizing genes and steroid target genes. hCG was insufficient to facilitate Leydig cell differentiation, restore steroidogenesis, and improve sperm yield. In conclusion, this study reports the failure of adult Leydig cell development and altered steroid production and signaling in tissue cultures. The organotypic culture system will need to be further improved before it can be translated into clinics for childhood cancer survivors.

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

Introduction: 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.

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

BACKGROUND: Testicular tissue cryopreservation before gonadotoxic treatments allows fertility preservation in children suffering from cancer. Fertility restoration strategies, in particular in vitro maturation of prepubertal testicular tissue, are being developed mainly in animal models. The rat, widely used in biomedical research, including in reproductive biology, is a relevant model. OBJECTIVES: To determine whether sequential two-step culture protocols can improve the efficiency of rat in vitro spermatogenesis. MATERIALS AND METHODS: Rat prepubertal testicular tissues were cultured on agarose gels with either a one-step or two-step protocol with or without polydimethylsiloxane (PDMS) ceiling chips. The progression of spermatogenesis, germ/Sertoli cell ratio, cell proliferation, seminiferous tubule area, and intratubular cell density were assessed by histological and immunohistochemical analyses. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays and Peanut Agglutinin (PNA) lectin labeling were performed to analyze the Deoxyribonucleic Acid (DNA) integrity and differentiation step of in vitro-produced spermatids. RESULTS: Sequential two-step protocols allowed the production of spermatids with a higher efficiency compared with the one-step culture protocol. However, the efficiency was low, as less than 1.5% of tubules contained spermatids. Most of the in vitro-produced spermatids contained unfragmented DNA and were at an early step of differentiation. Rare elongating spermatids could be detected in the cultured explants. Although complete in vitro spermatogenesis could not be obtained with PDMS ceiling chips, entry into meiosis was promoted in one-step organotypic cultures. DISCUSSION AND CONCLUSION: Complete in vitro meiosis and the beginning of the elongation phase of spermiogenesis were obtained in a rat model using sequential culture methods. Because of their low efficiency, further work will be necessary to identify the culture conditions allowing the completion of spermiogenesis. These optimizations could pave the way for future applications, including the development of an in vitro fertility restoration procedure for childhood cancer survivors, which is still far from being clinically available.

Experience, and gynaecological and reproductive health follow-up of young adult women who have undergone ovarian tissue cryopreservation.

RESEARCH QUESTION: What are the experience, and gynaecological and reproductive health outcomes in young adult women who have undergone ovarian tissue cryopreservation (OTC)? DESIGN: A retrospective observational study was conducted at a single institution between May 2019 and February 2021 including 87 women aged over 18 years undergoing OTC. Medical characteristics and questionnaire data collected more than 18 months after OTC were analysed. RESULTS: Close to 74% (n = 64/87) of women had a follow-up consultation and completed the questionnaire. Most women found the information provided on the OTC technique and the strategies proposed to restore fertility with ovarian tissue understandable and useful. The majority of patients thought that OTC had a positive impact on their well-being during disease treatment. Anti-Müllerian hormone serum concentration decreased significantly after treatment (P < 0.0001) and was significantly lower when patients received chemotherapy before OTC (P = 0.0039). The total cyclophosphamide equivalent dose was significantly higher in women with FSH concentrations above 25 IU/l after treatment (P = 0.0004). More than 70% of women who planned a pregnancy after the end of treatment succeeded, with a natural pregnancy rate close to 53%. Only nine patients (8.0%) underwent ovarian tissue transplantation for fertility restoration and six of them became pregnant and delivered at least once. CONCLUSION: Young adult women expressed a good satisfaction rate with OTC and that their experience had been beneficial. The usage rate of cryopreserved ovarian tissue remains low. The gynaecological and reproductive health follow-up consultation should be included in the supportive care provided following OTC.

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

In 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.

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

The 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.

Minimal residual disease detection by multicolor flow cytometry in cryopreserved ovarian tissue from leukemia patients.

BACKGROUND: Cryopreservation of ovarian tissue is a fertility-preservation option for women before gonadotoxic treatments. However, cryopreserved ovarian tissue transplantation must be performed with caution in women with malignancies that may metastasize to the ovaries. For this purpose, detecting minimal residual disease (MRD) in the ovarian cortex using sensitive methods is a crucial step. We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. RESULTS: We assessed MRD by multicolor flow cytometry (MFC) in cryopreserved ovarian cortex of 15 leukemia patients: 6 with B-cell acute lymphoblastic leukemia (B-ALL), 2 with T-cell acute lymphoblastic leukemia (T-ALL) and 7 with acute myeloid leukemia (AML). Ovarian MRD was positive in 5 of the 15 leukemia patients (one T-ALL and 4 AML). No B-ALL patient was positive by MFC. Quantitative reverse-transcribed polymerase chain reaction was performed when a molecular marker was available, and confirmed the MFC results for 3 patients tested. Xenografts into immunodeficient mice were also performed with ovarian cortical tissue from 10 leukemia patients, with no evidence of leukemic cells after the 6-month grafting period. CONCLUSIONS: In conclusion, this is the first study using MFC to detect MRD in ovarian cortical tissue from acute leukemia patients. MFC has been accepted in clinical practice for its ease of use, the large number of parameters available simultaneously, and high throughput analysis. We demonstrate here that MFC is a reliable method to detect MRD in cryopreserved ovarian tissue, with a view to controlling the oncological risk before ovarian tissue transplantation in leukemia patients.

Assessment of the architecture and integrity of frozen-thawed testicular tissue from (pre)pubertal boys with cancer.

BACKGROUND: Testicular tissue freezing is proposed for fertility preservation to (pre)pubertal boys with cancer before highly gonadotoxic treatment. Studies accurately comparing human (pre)pubertal testicular tissue quality before freezing and after thawing are exceptional. No study has reported this approach in a systematic manner and routine care. OBJECTIVES: To assess the impact of a control slow freezing protocol on testicular tissue architecture and integrity of (pre)pubertal boys after thawing. MATERIALS AND METHODS: (Pre)pubertal boys (n = 87) with cancer from 8 Reproductive Biology Laboratories of the French CECOS network benefited from testicular tissue freezing before hematopoietic stem cell transplantation. Seminiferous tubule cryodamage was determined histologically by scoring morphological alterations and by quantifying intratubular spermatogonia and the expression of DNA replication and repair marker in frozen-thawed testicular fragments. RESULTS: A significant increase in nuclear and epithelial score alterations was observed after thawing (p < 0.0001). The global lesional score remained lower than 1.5 and comparable to fresh testicular tissue. The number of intratubular spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells did not vary significantly after thawing. These data showed the good preservation of the seminiferous tubule integrity and architecture after thawing, as previously reported in our studies performed in prepubertal mice and rats. DISCUSSION: The current study reports, for the first time, the development of a semi-quantitative analysis of cryodamage in human (pre)pubertal testicular tissue, using a rapid and useful tool that can be proposed in routine care to develop an internal and external quality control for testicular tissue freezing. This tool can also be used when changing one or several parameters of the freezing-thawing procedure. CONCLUSION: Control slow freezing protocol without seeding maintains the seminiferous tubule architecture and integrity, the concentration of spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells after thawing.

Fertility preservation and sperm donation in transgender individuals: The current situation within the French CECOS network.

BACKGROUND: Many studies reported that reproductive desire could be high among transgender individuals. In France, fertility preservation and sperm donation were very little proposed to transgender individuals until recently, mainly because the Bioethics Law allows the use of assisted reproductive technologies only in infertile couples and prohibits surrogacy. OBJECTIVES: To evaluate the distribution of care on the French territory concerning fertility preservation and sperm donation in transgender individuals. MATERIALS AND METHODS: A multicentric national survey was carried out between January 2019 and October 2020 in 28 assisted reproductive technology centres of the French CECOS (Centres d'Etudes et de Conservation des Oeufs et du Sperme) network. Each centre was questioned to find out how many transgender individuals came, were informed and cared for fertility preservation and sperm donation. RESULTS: Concerning fertility preservation, 71.4% of centres received transgender individuals and performed gamete cryopreservation; 581 transgender individuals consulted for fertility preservation. Transgender women were more likely to desire (p < 0.0001) and achieve (p < 0.0001) fertility preservation than transgender men. Concerning sperm donation in couples including a transgender man, 68% of centres offer the complete course from the first consultation to the completion of the assisted reproductive technology cycles; 122 offsprings have been conceived with sperm donation in couples including a transgender man since 1999. DISCUSSION: Our results showed that even if all centres do not propose fertility preservation or sperm donation in transgender individuals, these assisted reproductive technologies are present throughout the French territory. The major point is that both fertility preservation and sperm donation in transgender individuals have grown significantly and that the care of these patients is improving year after year. CONCLUSION: In France, most of CECOS centres can take care of transgender individuals for fertility preservation and sperm donation. The French Bioethics Law allows these latter, and transgender individuals can benefit from a financial support of the national health care insurance for fertility preservation and sperm donation.

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.

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

Immunohistochemical analysis is a routine procedure for clinical and research studies in male fertility. However, most of the interpretations remain subjective and time-consuming, with inherent intra- and inter-observer variability. Given the prognostic and research implications of testicular assessment, a more objective and less time-consuming method is required. In the current study, we used in vitro matured pre-pubertal murine testes as a model. The main objective was to develop an affordable automated digital immunohistochemistry image analysis tool for an unbiased and quantitative assessment of testicular tissue sections. Testicular explants were fixed, cut, and stained for specific germ cell markers. The classical manual counting procedure was evaluated. Background and noise were reduced on brightfield images. Photomicrographs were stitched (Background_Elimination_Stitching) to create high-quality images. Two procedures were evaluated (IHC_Tool and Stained_Nuclear_Area); then a procedure (Necrotic_Area_Elimination) allowing withdrawal of the necrotic area observed after culture was assessed. Finally, the number of stained nuclei in the unaltered tissue area was extracted. The automated IHC_Tool procedure with images saved as TIFF at a ×200 magnification allowed the most rigorous cell quantification. IHC_Tool developed for testicular sample analysis can be used for various types of tissues. We foresee that this method will minimize inter-observer variations across laboratories and will be helpful for clinical trials and translational initiatives.

Activation of the cannabinoid receptor type 2 by the agonist JWH133 promotes the first wave of in vitro spermatogenesis.

BACKGROUND: Oncological procedures have irreversible side effects on germ cells for childhood cancer survival boys. In vitro culture of prepubertal testicular tissue has been proposed to restore fertility; however, recent data on animal models showed that meiotic and post-meiotic progression was impaired. OBJECTIVES: As potential key inducers of the mitosis-meiosis switch, type 2 cannabinoid receptor (CB2 ) has been proposed to play a central role in the meiotic entry of male germ cells. Herein, the in vitro first spermatogenesis wave in mice was used to understand the impact of CB2 activation on the differentiation of spermatogonia until elongated spermatids. MATERIALS AND METHODS: A first set of cultured testicular explants of 6.5 days post-partum (dpp) mice was performed to assess the impact of a range of JWH133 supplementation (10 nm, 100 nm, 1 µm, 10 µm). Then, the progressive development of germ cells at key timepoints of spermatogenesis was evaluated throughout (i) in vitro culture (day 2 [D2], D3, D6, D10, D18, and D30) coupled with (ii) in vivo counterparts (8.5, 9.5, 12.5, 16.5, 24.5, and 36.5 dpp). RESULTS: CB2 was detected at the plasma membrane of cells, and a successful completion of spermatogenesis was obtained in vitro. One day after the activation of CB2 by 1 µm of the agonist JWH133, percentage of zygotene spermatocyte I increased. CONCLUSION: After 30 days of culture, (i) an enrichment of haploid germ cells detected by flow cytometry, (ii) a reduced necrotic area, and (iii) an increase in the density of post-meiotic germ cells were observed. We showed that the activation of CB2 improves in vitro entry into meiosis and differentiation of spermatogonia, mimicking physiological meiotic transition.

Dynamics of epigenetic modifications in ICSI embryos from in vitro-produced spermatozoa.

BACKGROUND: In prepubertal boys with cancer, fertility preservation relies on testicular tissue freezing before treatment. In vitro maturation of frozen/thawed tissues could be one of the procedures envisaged to restore the fertility of cured patients. It is necessary to ascertain in the mouse model that in vitro-generated spermatozoa are able to ensure embryo development, without altering the epigenetic processes occurring during the pre-implantation period. OBJECTIVES: The aims of the present study were to investigate the fertilizing ability of in vitro-produced spermatozoa and explore several epigenetic marks at different stages of embryo development. MATERIALS AND METHODS: Fresh or controlled slow-frozen (CSF)/thawed testicular tissues from 6 to 7 days post-partum (dpp) mice were cultured for 30 days. Intracytoplasmic sperm injection (ICSI) experiments were performed using in vitro-produced spermatozoa. Testicular spermatozoa from 36 to 37 dpp mice were used as in vivo controls. DNA methylation/hydroxymethylation and histone post-translational modifications (H3K4me3, H3K27me3 and H3K9ac) were analysed by immunofluorescence from the zygote to the blastocyst stages. RESULTS: The spermatozoa generated in cultures of fresh or CSF testicular tissues were able to initiate embryonic development. The freezing of prepubertal testicular tissues limits the production of spermatozoa in vitro and the fertilization rate after ICSI. Similar levels of H3K4me3, H3K27me3 and H3K9ac were found in ICSI embryos derived from in vitro- and in vivo-produced spermatozoa. DNA methylation levels were increased in 4-cell embryos and morula obtained by ICSI with in vitro-produced spermatozoa. DISCUSSION AND CONCLUSION: Our study shows for the first time that the use of in vitro-produced spermatozoa alters DNA methylation/demethylation dynamics but has little impact on H3K4me3, H3K27me3 and H3K9ac levels in mouse early embryos. Further work will have to be performed to determine whether the use of these gametes is not deleterious for embryo development before considering a human application.

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

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/m2), 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/m2) present a high gonadotoxic risk when administered before the initiation of spermatogenesis.