Evaluating testicular tissue for future autotransplantation: focus on cancer cell contamination and presence of spermatogonia in tissue cryobanked for boys diagnosed with a hematological malignancy.

STUDY QUESTION: What is the contamination rate by cancer cells and spermatogonia numbers in immature testicular tissue (ITT) harvested before the start of gonadotoxic therapy in boys with a hematological malignancy? SUMMARY ANSWER: Among our cohort of boys diagnosed with acute lymphoblastic leukemia (ALL) and lymphomas, 39% (n = 11/28) had cancer cells identified in their tissues at the time of diagnosis and all patients appeared to have reduced spermatogonia numbers compared to healthy reference cohorts. WHAT IS KNOWN ALREADY: Young boys affected by a hematological cancer are at risk of contamination of their testes by cancer cells but histological examination is unable to detect the presence of only a few cancer cells, which would preclude autotransplantation of cryobanked ITT for fertility restoration, and more sensitive detection techniques are thus required. Reduced numbers of spermatogonia in ITT in hematological cancer patients have been suggested based on results in a limited number of patients. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 54 pre- and peri-pubertal boys who were diagnosed with a hematological malignancy and who underwent a testicular biopsy for fertility preservation at the time of diagnosis before any gonadotoxic therapy between 2005 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Among the 54 patients eligible in our database, formalin-fixed paraffin-embedded (FFPE) testicular tissue was available for 28 boys diagnosed either with ALL (n = 14) or lymphoma (n = 14) and was used to evaluate malignant cell contamination. Hematoxylin and eosin (H&E) staining was performed for each patient to search for cancer cells in the tissue. Markers specific to each patient's disease were identified at the time of diagnosis on the biopsy of the primary tumor or bone marrow aspiration and an immunohistochemistry (IHC) was performed on the FFPE ITT for each patient to evidence his disease markers. PCR analyses on the FFPE tissue were also conducted when a specific gene rearrangement was available. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age at diagnosis and ITT biopsy of the 28 boys was 7.5 years (age range: 19 months-16 years old). Examination of ITT of the 28 boys on H&E stained sections did not detect malignant cells. Using IHC, we found contamination by cancerous cells using markers specific to the patient's disease in 10 of 28 boys, with a higher rate in patients diagnosed with ALL (57%, n = 8/14) compared with lymphoma (14%, n = 2/14) (P-value < 0.05). PCR showed contamination in three of 15 patients who had specific rearrangements identified on their bone marrow at the time of diagnosis; one of these patients had negative results from the IHC. Compared to age-related reference values of the number of spermatogonia per ST (seminiferous tubule) (Spg/ST) throughout prepuberty of healthy patients from a simulated control cohort, mean spermatogonial numbers appeared to be decreased in all age groups (0-4 years: 1.49 ± 0.54, 4-7 years: 1.08 ± 0.43, 7-11 years: 1.56 ± 0.65, 11-14 years: 3.37, 14-16 years: 5.44 ± 3.14). However, using a cohort independent method based on the Z-score, a decrease in spermatogonia numbers was not confirmed. LIMITATIONS, REASONS FOR CAUTION: The results obtained from the biopsy fragments that were evaluated for contamination by cancer cells may not be representative of the entire cryostored ITT and tumor foci may still be present outside of the biopsy range. WIDER IMPLICATIONS OF THE FINDINGS: ITT from boys diagnosed with a hematological malignancy could bear the risk for cancer cell reseeding in case of autotransplantation of the tissue. Such a high level of cancer cell contamination opens the debate of harvesting the tissue after one or two rounds of chemotherapy. However, as the safety of germ cells can be compromised by gonadotoxic treatments, this strategy warrants for the development of adapted fertility restoration protocols. Finally, the impact of the hematological cancer on spermatogonia numbers should be further explored. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by a grant from the FNRS-Télévie (grant n°. 7.4533.20) and Fondation Contre le Cancer/Foundation Against Cancer (2020-121) for the research project on fertility restoration with testicular tissue from hemato-oncological boys. The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Cancer cell contamination and decontamination methods for ovaries and testes: special focus on prepubertal gonads with a view to safe fertility restoration.

Fertility restoration in patients that survived a hematological cancer during childhood is a core part of their care pathway. Nonetheless, there might be a risk of contamination of the gonads by cancer cells, especially in patients presenting with leukemia and lymphoma. When only a few cancer cells have reached the gonad, they may not be detected by routine histological examination, and therefore more sensitive techniques are required before being confident of the safety of transplanting cryostored testicular and ovarian tissues or cells back to the patient after recovery. Furthermore, if neoplastic cells are identified in the gonadal tissue, methods to eliminate such cells are urgently awaited as the presence of only a few cancer cells may induce disease relapse in these patients. In this review, contamination rates of human gonadal tissue in the case of leukemia or lymphoma as well as decontamination methods applied to both adult and prepubertal testicular and ovarian tissues are presented. Prepubertal gonads will be the main focus as we aim to show how far we have come in establishing safe approaches to fertility restoration. Advances have been made using animal tissue that is usually artificially contaminated by the addition of cancer cell lines to the gonadal cells or tissue, but these techniques need to be improved and still await development in the case of in vivo cancer cell invasion of tissue.

Optimized Recovery of Immature Germ Cells after Prepubertal Testicular Tissue Digestion and Multi-Step Differential Plating: A Step towards Fertility Restoration with Cancer-Cell-Contaminated Tissue.

Undifferentiated germ cells, including the spermatogonial stem cell subpopulation required for fertility restoration using human immature testicular tissue (ITT), are difficult to recover as they do not easily adhere to plastics. Due to the scarcity of human ITT for research, we used neonatal porcine ITT. Strategies for maximizing germ cell recovery, including a comparison of two enzymatic digestion protocols (P1 and P2) of ITT fragment sizes (4 mm3 and 8 mm3) and multi-step differential plating were explored. Cellular viability and yield, as well as numbers and proportions of DDX4+ germ cells, were assessed before incubating the cell suspensions overnight on uncoated plastics. Adherent cells were processed for immunocytochemistry (ICC) and floating cells were further incubated for three days on Poly-D-Lysine-coated plastics. Germ cell yield and cell types using ICC for SOX9, DDX4, ACTA2 and CYP19A1 were assessed at each step of the multi-step differential plating. Directly after digestion, cell suspensions contained >92% viable cells and 4.51% DDX4+ germ cells. Pooled results for fragment sizes revealed that the majority of DDX4+ cells adhere to uncoated plastics (P1; 82.36% vs. P2; 58.24%). Further incubation on Poly-D-Lysine-coated plastics increased germ cell recovery (4.80 ± 11.32 vs. 1.90 ± 2.07 DDX4+ germ cells/mm2, respectively for P1 and P2). The total proportion of DDX4+ germ cells after the complete multi-step differential plating was 3.12%. These results highlight a reduced proportion and number of germ cells lost when compared to data reported with other methods, suggesting that multi-step differential plating should be considered for optimization of immature germ cell recovery. While Poly-D-Lysine-coating increased the proportions of recovered germ cells by 16.18% (P1) and 28.98% (P2), future studies should now focus on less cell stress-inducing enzymatic digestion protocols to maximize the chances of fertility restoration with low amounts of cryo-banked human ITT.

Microfluidic and Static Organotypic Culture Systems to Support Ex Vivo Spermatogenesis From Prepubertal Porcine Testicular Tissue: A Comparative Study.

Background: In vitro maturation of immature testicular tissue (ITT) cryopreserved for fertility preservation is a promising fertility restoration strategy. Organotypic tissue culture proved successful in mice, leading to live births. In larger mammals, including humans, efficiently reproducing spermatogenesis ex vivo remains challenging. With advances in biomaterials technology, culture systems are becoming more complex to better mimic in vivo conditions. Along with improving culture media components, optimizing physical culture conditions (e.g., tissue perfusion, oxygen diffusion) also needs to be considered. Recent studies in mice showed that by using silicone-based hybrid culture systems, the efficiency of spermatogenesis can be improved. Such systems have not been reported for ITT of large mammals. Methods: Four different organotypic tissue culture systems were compared: static i.e., polytetrafluoroethylene membrane inserts (OT), agarose gel (AG) and agarose gel with polydimethylsiloxane chamber (AGPC), and dynamic i.e., microfluidic (MF). OT served as control. Porcine ITT fragments were cultured over a 30-day period using a single culture medium. Analyses were performed at days (d) 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity, diameters, and tissue core integrity were evaluated on histology. Immunohistochemistry was used to identify germ cells (PGP9.5, VASA, SYCP3, CREM), somatic cells (SOX9, INSL3) and proliferating cells (Ki67), and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured in supernatants using ELISA. Results: ITT fragments survived and grew in all systems. ST diameters, and Sertoli cell (SOX9) numbers increased, meiotic (SYCP3) and post-meiotic (CREM) germ cells were generated, and testosterone was secreted. When compared to control (OT), significantly larger STs (d10 through d30), better tissue core integrity (d5 through d20), higher numbers of undifferentiated spermatogonia (d30), meiotic and post-meiotic germ cells (SYCP3: d20 and 30, CREM: d20) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cell (Ki67/VASA) numbers, Leydig cell (INSL3) numbers and testosterone levels were not significantly different between systems. Conclusions: Using a modified culture system (AGPC), germ cell survival and the efficiency of porcine germ cell differentiation were moderately improved ex vivo. We assume that further optimization can be obtained with concomitant modifications in culture media components.

Fertility preservation for prepubertal boys: are we ready for autologous grafting of cryopreserved immature testicular tissue?

Fertility preservation in prepubertal boys is a matter that has been gaining ground fast during the past 20 years. As the life expectancy for childhood cancer survivors continues to increase (5-year survival rate>80%), and as the risk of permanent infertility in adulthood is still high with current treatment options, offering a testicular biopsy to cryopreserve immature testicular tissue (ITT) containing spermatogonial stem cells is now recommended as an experimental option for fertility preservation. Transplantation of cryopreserved testicular prepubertal tissue fragments back to the cured patient appears to be one of the most feasible and promising techniques for fertility restoration. In 2019, the birth of a healthy female baby rhesus monkey after intracytoplasmic sperm injection using spermatozoa recovered from frozen-thawed ITT autografts fueled the hopes of translating the technique to humans. While gonadal tissue autotransplantation has already been successfully achieved in clinical practice for females, it has never been attempted in males to date. In this work, we review acquired knowledge on ITT transplantation in animal models and share our vision on the remaining challenges to autograft currently banked human ITT within pilot clinical trials in the near future.

Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles.

Avascular transplantation of frozen-thawed testicular tissue fragments represents a potential future technique for fertility restoration in boys with cancer. A significant loss of spermatogonia was observed in xeno-transplants of human tissue most likely due to the hypoxic period before revascularization. To reduce the effect of hypoxia-reoxygenation injuries, several options have already been explored, like encapsulation in alginate hydrogel and supplementation with nanoparticles delivering a necrosis inhibitor (NECINH) or VEGF. While these approaches improved short-term (5 days) vascular surfaces in grafts, neovessels were not maintained up to 21 days; i.e., the time needed for achieving vessel stabilization. To better support tissue grafts, nanoparticles loaded with VEGF, PDGF and NECINH were developed. Testicular tissue fragments from 4-5-week-old mice were encapsulated in calcium-alginate hydrogels, either non-supplemented (control) or supplemented with drug-loaded nanoparticles (VEGF-nanoparticles; VEGF-nanoparticles + PDGF-nanoparticles; NECINH-nanoparticles; VEGF-nanoparticles + NECINH-nanoparticles; and VEGF-nanoparticles + PDGF-nanoparticles + NECINH-nanoparticles) before auto-transplantation. Grafts were recovered after 5 or 21 days for analyses of tissue integrity (hematoxylin-eosin staining), spermatogonial survival (immuno-histo-chemistry for promyelocytic leukemia zinc finger) and vascularization (immuno-histo-chemistry for α-smooth muscle actin and CD-31). Our results showed that a combination of VEGF and PDGF nanoparticles increased vascular maturity and induced a faster maturation of vascular structures in grafts.

Organoids as tools to investigate the molecular mechanisms of male infertility and its treatments.

Organoids are 3D structures characterized by cellular spatial organizations and functions close to the native tissue they mimic. Attempts to create organoids originating from several tissues have now been reported, including the testis. Testicular organoids have the potential to improve our knowledge of the mechanisms that regulate testicular morphogenesis, physiology, and pathophysiology. They could especially prove as useful tools to understand the complex mechanisms involved in the regulation of the germ cell niche in infertility cases as they offer the possibility to control and modify the nature of cell types before self-assembly and thereby opening the perspective for developing innovative methods to restore fertility. To date, there are only few studies targeted at testicular organoids' formation and even less describing the generation of organoids with both testis-specific structure and function. While researchers described interesting applications with regards to testicular tissue morphogenesis and drug toxicity, further research is needed before testicular organoids would eventually lead to the generation of fertilizing spermatozoa. This review will present the conventional systems used to induce in vitro maturation of testicular cells, describe the different approaches that have been used for the development of testicular organoids and discuss the potential applications they could have in the field of male reproductive biology.

Fertility preservation for prepubertal boys: lessons learned from the past and update on remaining challenges towards clinical translation.

BACKGROUND: Childhood cancer incidence and survivorship are both on the rise. However, many lifesaving treatments threaten the prepubertal testis. Cryopreservation of immature testicular tissue (ITT), containing spermatogonial stem cells (SSCs), as a fertility preservation (FP) option for this population is increasingly proposed worldwide. Recent achievements notably the birth of non-human primate (NHP) progeny using sperm developed in frozen-thawed ITT autografts has given proof of principle of the reproductive potential of banked ITT. Outlining the current state of the art on FP for prepubertal boys is crucial as some of the boys who have cryopreserved ITT since the early 2000s are now in their reproductive age and are already seeking answers with regards to their fertility. OBJECTIVE AND RATIONALE: In the light of past decade achievements and observations, this review aims to provide insight into relevant questions for clinicians involved in FP programmes. Have the indications for FP for prepubertal boys changed over time? What is key for patient counselling and ITT sampling based on the latest achievements in animals and research performed with human ITT? How far are we from clinical application of methods to restore reproductive capacity with cryostored ITT? SEARCH METHODS: An extensive search for articles published in English or French since January 2010 to June 2020 using keywords relevant to the topic of FP for prepubertal boys was made in the MEDLINE database through PubMed. Original articles on fertility preservation with emphasis on those involving prepubertal testicular tissue, as well as comprehensive and systematic reviews were included. Papers with redundancy of information or with an absence of a relevant link for future clinical application were excluded. Papers on alternative sources of stem cells besides SSCs were excluded. OUTCOMES: Preliminary follow-up data indicate that around 27% of boys who have undergone testicular sampling as an FP measure have proved azoospermic and must therefore solely rely on their cryostored ITT to ensure biologic parenthood. Auto-transplantation of ITT appears to be the first technique that could enter pilot clinical trials but should be restricted to tissue free of malignant cells. While in vitro spermatogenesis circumvents the risk linked to cancer cell contamination and has led to offspring in mice, complete spermatogenesis has not been achieved with human ITT. However, generation of haploid germ cells paves the way to further studies aimed at completing the final maturation of germ cells and increasing the efficiency of the processes. WIDER IMPLICATIONS: Despite all the research done to date, FP for prepubertal boys remains a relatively young field and is often challenging to healthcare providers, patients and parents. As cryopreservation of ITT is now likely to expand further, it is important not only to acknowledge some of the research questions raised on the topic, e.g. the epigenetic and genetic integrity of gametes derived from strategies to restore fertility with banked ITT but also to provide healthcare professionals worldwide with updated knowledge to launch proper multicollaborative care pathways in the field and address clinical issues that will come-up when aiming for the child's best interest.

Generation of Organized Porcine Testicular Organoids in Solubilized Hydrogels from Decellularized Extracellular Matrix.

Cryopreservation of immature testicular tissue (ITT) prior to chemo/radiotherapy is now ethically accepted and is currently the only way to preserve fertility of prepubertal boys about to undergo cancer therapies. So far, three-dimensional culture of testicular cells isolated from prepubertal human testicular tissue was neither efficient nor reproducible to obtain mature spermatozoa, and ITT transplantation is not a safe option when there is a risk of cancer cell contamination of the testis. Hence, generation of testicular organoids (TOs) after cell selection is a novel strategy aimed at restoring fertility in these patients. Here, we created TOs using hydrogels developed from decellularized porcine ITT and compared cell numbers, organization and function to TOs generated in collagen only hydrogel. Organotypic culture of porcine ITT was used as a control. Rheological and mass spectrometry analyses of both hydrogels highlighted differences in terms of extracellular matrix stiffness and composition, respectively. Sertoli cells (SCs) and germ cells (GCs) assembled into seminiferous tubule-like structures delimited by a basement membrane while Leydig cells (LCs) and peritubular cells localized outside. TOs were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality of SCs and LCs, respectively. In both TOs GC numbers decreased and SC numbers increased. However, LC numbers decreased significantly in the collagen hydrogel TOs (p < 0.05) suggesting a better preservation of growth factors within TOs developed from decellularized ITT and thus a better potential to restore the reproductive capacity.

Cryostorage of testicular tissue and retransplantation of spermatogonial stem cells in the infertile male.

Transplantation of own cryostored spermatogonial stem cells (SSCs) is a promising technique for fertility restoration when the SSC pool has been depleted. In this regard, cryopreservation of pre-pubertal testicular tissue or SSCs suspensions before gonadotoxic therapies is ethically accepted and increasingly proposed. SSC transplantation has also been considered to treat other causes of infertility relying on the possibility of propagating SSCs retrieved in the testes of infertile men before autologous re-transplantation. Although encouraging results were achieved in animals and in preclinical experiments, clinical perspectives are still limited by a number of unresolved technical and safety issues, such as the risk of cancer cell contamination of cells intended for transplantation and the genetic and epigenetic stability of SCCs when cultured before re-transplantation. Moreover, while genome editing techniques raise the hope of modifying the SSCs genome before re-transplantation, their application for reproductive purposes might be a step too far for the moment.