Tracking Immature Testicular Tissue after Vitrification In Vitro and In Vivo for Pre-Pubertal Fertility Preservation: A Translational Transgenic Mouse Model.

Pediatric cancer survivors experiencing gonadotoxic chemoradiation therapy may encounter subfertility or permanent infertility. However, previous studies of cryopreservation of immature testicular tissue (ITT) have mainly been limited to in vitro studies. In this study, we aim to evaluate in vitro and in vivo bioluminescence imaging (BLI) for solid surface-vitrified (SSV) ITT grafts until adulthood. The donors and recipients were transgenic and wild-type mice, respectively, with fresh ITT grafts used as the control group. In our study, the frozen ITT grafts remained intact as shown in the BLI, scanning electron microscopy (SEM) and immunohistochemistry (IHC) analyses. Graft survival was analyzed by BLI on days 1, 2, 5, 7, and 31 after transplantation. The signals decreased by quantum yield between days 2 and 5 in both groups, but gradually increased afterwards until day 31, which were significantly stronger than day 1 after transplantation (p = 0.008). The differences between the two groups were constantly insignificant, suggesting that both fresh and SSV ITT can survive, accompanied by spermatogenesis, until adulthood. The ITT in both groups presented similar BLI intensity and intact cells and ultrastructures for spermatogenesis. This translational model demonstrates the great potential of SSV for ITT in pre-pubertal male fertility preservation.

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.

[Reconstruction of spermatogonial niche for male fertility preservation].

Infertility is one of the late side effects of cancer treatment. Expansion of anti-cancer treatment allow patients to have more life time, however infertility is becoming a matter damaging QOL during the young cancer survivors. The passive strategy such as avoiding the gonad-toxic drug or decreasing the total volume of them and shielding the gonads against cancer therapy has been conducted. To preserve the fertility of young female, ovary tissue cryopreservation is becoming a standard over the world after the success of offspring from cryopreserved ovary tissue autograft was reported. Sperm preservation method is established for the male fertility preservation method, however this is only applicable for sexually matured male patients. For the sake of preserving fertility of sexually immature male patients, many trials using cryopreserved testis tissues or testicular cells have been undergone. Recently, in vitro gametogenesis from stem cell of the human and the mouse to primordial germ cell like cell has been achieved. Here the previous challenges and the latest reports for obtaining functional sperm from immature testis and the reconstruction of spermatogonial niche as a potential approach for preserving fertility procedure are described.

Transplantation of rat frozen-thawed testicular tissues: Does fragment size matter?

Cryopreservation of testicular tissue from pre-pubertal boys before gonadotoxic treatment is an important step in fertility preservation. Yet, this approach remains experimental, and there is still few study measuring the effect of tissue size on the graft after cryopreservation and transplantation. The objective of this study is to detect the effect of varying tissue sizes on the efficacy of rat testicular tissue cryopreservation and transplantation. Varying sizes of rat testicular tissues were frozen-thawed and autografted. At the 30th day after grafting, the grafts were collected for histology assessment and immunohistochemistry assay for MAGE-A4 (germ cell marker) and CD34 (blood vessel marker). The transplant recovery, seminiferous tubule integrity, tubular diameter, spermatogonia number, and microsvessel density in testicular fragments sizing in 3 mm in length, 3 mm wide, and 3 mm in thickness were significantly lower than other groups. Whereas, the absorption rate of graft sizing in 1 mm in length, 1 mm in wide, and 1 mm in thickness was significantly higher than other groups. Testicular fragment sizing in 2-3 mm in length, 2-3 mm in wide, and 2 mm in thickness (8 mm3-18 mm3) is suitable for rat testicular tissue cryopreservation and transplantation.

Robotic-Assisted Testicular Autotransplantation.

Silber and Kelly first described the successful autotransplantation of an intra-abdominal testis in 1976. Subsequent authors incorporated laparoscopy and demonstrated the viability of transplanted testes based on serial postoperative exams. We sought to extend this experience with use of the da Vinci surgical robot, thereby demonstrating a novel robotic technique for the management of cryptorchidism. The procedure was performed for an 18-year-old male with a solitary left intra-abdominal testis. Following establishment of pneumoperitoneum, the robot is docked with four trocars oriented towards the left lower quadrant. Testicular dissection is carried out as shown. The gonadal and inferior epigastric vessels are isolated and mobilized; once adequate length is achieved, the former is clipped and transected, and the testicle and inferior epigastric vessels are delivered out of the body. The robot is then undocked and exchanged for the operating microscope. Arterial and venous anastomoses are completed with interrupted and running 9-0 Nylon, respectively, and satisfactory re-anastomosis is confirmed visually and with Doppler. The transplanted testicle is then fixed inferiorly and laterally within the left hemiscrotum, and all incisions are closed. We note that intraoperative testicular biopsy was not performed, for three reasons: (1) to avoid further risk to an already tenuous, solitary organ, (2) because our primary aim was to preserve testicular endocrine function, and (3) because the presence of ITGCN would neither prompt orchiectomy nor obviate the need for ongoing surveillance via periodic self-examination and ultrasonography. The patient is maintained on bed rest for two days and discharged on postoperative day seven in good condition. Over one year since autotransplantation, his now intra-scrotal testicle remains palpable and stable in size. Serum testosterone is unchanged from preoperative measurements. Robotic-assisted testicular autotransplantation is a feasible and efficacious management option for the solitary intra-abdominal testis.

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.

Sertoli cell replacement in explanted mouse testis tissue supporting host spermatogenesis†.

Spermatogenesis takes place in the seminiferous tubules, starting from the spermatogonial stem cell and maturing into sperm through multiple stages of cell differentiation. Sertoli cells, the main somatic cell constituting the seminiferous tubule, are in close contact with every germ cell and play pivotal roles in the progression of spermatogenesis. In this study, we developed an in vitro Sertoli cell replacement method by combining an organ culture technique and a toxin receptor-mediated cell knockout system. We used Amh-diphtheria toxin receptor transgenic mice, whose Sertoli cells specifically express human diphtheria toxin receptor, which renders them sensitive to diphtheria toxin. An immature Amh-diphtheria toxin receptor testis was transplanted with the donor testis cells followed by culturing in a medium containing diphtheria toxin. This procedure successfully replaced the original Sertoli cells with the transplanted Sertoli cells, and spermatogenesis originating from resident germ cells was confirmed. In addition, Sertoli cells in the mouse testis tissues were replaced by transplanted rat Sertoli cells within culture conditions without requiring immunosuppressive treatments. This method works as a functional assay system, making it possible to evaluate any cells that might function as Sertoli cells. It would also be possible to investigate interactions between Sertoli and germ cells more closely, providing a new platform for the study of spermatogenesis and its impairments.

Islet transplantation ameliorates diabetes-induced testicular interstitial fibrosis and is associated with inhibition of TGF-ß1/Smad2 pathway in a rat model of type 1 diabetes.

Islet transplantation (IT) is considered the most effective endocrine replacement therapy for diabetes mellitus (DM). Studies have demonstrated that IT can repair testicular structural injury caused by inflammatory and oxidative stress in a diabetic rat model. However, highly effective exogenous antioxidant and anti-inflammatory drugs can achieve this effect. Testicular interstitial fibrosis caused by long-term hyperglycemia is however difficult to reverse or recover. Thus far, there are no effective drugs that prevent or relieve testicular interstitial fibrosis. Therefore, it is necessary to explore the potential benefit of IT on testicular interstitial fibrosis induced by DM and its underlying molecular mechanisms. In the present study, Wistar rats were used to establish a DM model by intraperitoneal injection of streptozotocin. The diabetic models then underwent IT or received insulin treatment after 12 weeks. IT was more effective than insulin treatment in ameliorating diabetic-induced testicular interstitial fibrosis, Leydig cells apoptosis, testosterone deficiency and poor sperm motility. IT and insulin treatment both significantly inhibited the upregulation of TGF-ß1 and phosphorylated Smad2 in DM, with IT being more effective than insulin. The present study's findings proved that IT effectively protects diabetic-induced testicular interstitial fibrosis probably by inhibiting the TGF-ß1/Smad2 signaling pathway, which offers hope in male patients with DM complicating with testicular interstitial fibrosis.

Testicular subcutaneous allografting followed by immunosuppressive treatment promotes maintenance of spermatogonial cells in rainbow trout (Oncorhynchus mykiss).

Germ cell transplantation and testis graft represent promising biotechnologies that can be applied for the reproduction of commercial or endangered species. However, mechanisms of rejection from the host immune system might remove the transplanted donor cells/tissues and limit the surrogate production of gametes. In this work, we administered emulsion containing-immunosuppressants to verify whether they are capable to prevent immune rejection and promote survival of testis allografts in rainbow trout. In the first part of this study, we demonstrated in vitro that tacrolimus and cyclosporine were able to affect viability, inhibit leucocyte proliferation, and suppress il2 expression in vitro. In in vivo experiments, both doses of tacrolimus (0.5 and 1.5 mg/kg) and the lower dose of cyclosporine (20 mg/kg) significantly inhibited the expression of il2 in head kidney, three days post-injection. A higher dose of cyclosporine (40 mg/kg) was able to inhibit il2 expression for up to seven days post-injection. In the second part, testis allografts were conducted in fish treated weekly with emulsion containing-tacrolimus. Immunohistochemical, conventional histology, and qRT-PCR (vasa) analysis demonstrated the presence of spermatogonial cells by the fifth week, in animals treated with 0.5 mg/kg of tacrolimus similar as found in autografted group. In the group treated with the highest tacrolimus dose (1.5 mg/kg) and in the non-treated group (without immunosuppressant), no germ cells or their respective markers were detected. il2 expression in head kidney was also suppressed in grafted animals treated with tacrolimus compared to non-treated group. These results suggest that tacrolimus may be a promising immunosuppressant for testis allografts or germ cell transplantation in rainbow trout. Co-administration combining tacrolimus (at lower dose) with other immunosuppressive drugs for inhibiting other activation pathways of the immune system, as performed in human organ transplantation, could be an alternative approach to optimize the immunosuppressive effects in host organisms.

The mouse testis tissue culture could resume spermatogenesis as same as in vivo condition after human spermatogonial stem cells transplantation.

OBJECTIVE: The introduction of alternative systems in vivo is very important for cancer patients who are treated with gonadotoxic treatment. In this study, we examine the progression of the spermatogenesis process after human spermatogonial stem cell (SSCs) transplantation in vivo and in tissue culture conditions. MATERIALS AND METHODS: Human SSCs were obtained from a Testicular Sperm Extractions (TESE) sample, and characterization of these cells was confirmed by detecting the promyelocytic leukemia zinc finger (PLZF) protein. These cells, after being labeled with Di-alkyl Indocarbocyanine (DiI), were transplanted to adult azoospermia mouse testes treated with Busulfan 40mg/kg. The host testicular tissue culture was then considered a test group and in vivo transplant a control group. After 8 weeks, immunohistochemical, morphometric and molecular studies were performed. RESULTS: The results of morphometric studies indicated that the mean number of spermatogonia, spermatocytes, and spermatids in the test groups was significantly lower than in the control group (P<0.05) and most of the cells responded positively to DiI tracing. Immunohistochemical study in both groups revealed expression of PLZF, Synaptonemal complex protein 3 (SCP3) and Acrosin Binding Protein (ACRBP) proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively. Also, PLZF, Transition Protein 1 (TP1) and Tektin-1 (Tekt1) human-specific genes had a significant difference in the between test groups and control groups (P<0.05) in molecular studies. CONCLUSION: These results suggest that the conditions of testicular tissue culture after transplantation of SSCs can support spermatogenesis resumption, as well as in an in vivo condition.

Long-Term Monitoring of Donor Xenogeneic Testis Tissue Grafts and Cell Implants in Recipient Mice Using Ultrasound Biomicroscopy.

Testis tissue xenografting and testis cell aggregate implantation from various donor species into recipient mice are novel models for the study and manipulation of testis formation and function in target species. Thus far, the analysis of such studies has been limited to surgical or post-mortem retrieval of samples. Here we used ultrasound biomicroscopy (UBM) to monitor the development of neonatal porcine testis grafts and implants in host mice for 24 wk, and to correlate UBM and (immuno)histologic changes. This led to long-term visualization of gradual changes in volume, dimension and structure of grafts and implants; detection of a 4 wk developmental gap between grafts and implants; and revelation of differences in implant development depending on the craniocaudal site of implantation on the back of host mice. Our data support the reliability and precision of UBM for longitudinal study of transplants, which eliminates the need for frequent surgical sampling.

Germ cell depletion in recipient testis has adverse effects on spermatogenesis in orthotopically transplanted testis pieces via retinoic acid insufficiency.

Germ cell depletion in recipient testes is indispensable for successful transplantation of spermatogonial stem cells. However, we found that such treatment had an adverse effect on spermatogenesis of orthotopically transplanted donor testis tissues. In the donor tissue, the frequency of stimulated by retinoic acid (RA) 8 (STRA8) expression was reduced in germ cells, suggesting that RA signalling indispensable for spermatogenesis was attenuated in germ cell-depleted recipient testes. In this context, germ cell depletion diminished expression of testicular Aldh1a2, which is responsible for testicular RA synthesis, while Cyp26b1, which is responsible for testicular RA metabolism, was still expressed even after germ cell depletion, suggesting an alteration of the RA synthesis/metabolism ratio. These observations suggested that RA insufficiency was one of the causes of the defective donor spermatogenesis. Indeed, repetitive RA administrations significantly improved donor spermatogenesis to produce fertile offspring without any side effects. These findings may contribute to improving fertility preservation techniques for males, especially to prevent iatrogenic infertility induced by chemotherapy in prepubertal cancer patients.

Suitability of hybrid mackerel (Scomber australasicus × S. japonicus) with germ cell-less sterile gonads as a recipient for transplantation of bluefin tuna germ cells.

We aim to establish a small-bodied surrogate broodstock, such as mackerel, which produces functional bluefin tuna gametes by spermatogonial transplantation. When reproductively fertile fish are used as recipients, endogenous gametogenesis outcompetes donor-derived gametogenesis, and recipient fish predominantly produce their gametes. In this study, we assessed fertility of hybrid mackerel, Scomber australasicus × S. japonicus, and its suitability as a recipient for transplantation of bluefin tuna germ cells. Hybrid mackerel were produced by artificially inseminating S. australasicus eggs with S. japonicus spermatozoa. Cellular DNA content and PCR analyses revealed that F1 offspring were diploid carrying both paternal and maternal genomes. Surprisingly, histological observations found no germ cells in hybrid mackerel gonads at 120 days post-hatch (dph), although they were present in the gonad of 30- and 60-dph hybrid mackerel. The frequency of germ cell-less fish was 100% at 120-dph, 63.1% at 1-year-old, and 81.8% at 2-year-old. We also confirmed a lack of expression of germ cell marker (DEAD-box helicase 4, ddx4) in the germ cell-less gonads of hybrid mackerel. By contrast, expression of Sertoli cell marker (gonadal soma-derived growth factor, gsdf) and of Leydig cell marker (steroid 11-beta-hydroxlase, cyp11b1) were clearly detected in hybrid mackerel gonads. Together these results showed that most of the hybrid gonads were germ cell-less sterile, but still possessed supporting cells and steroidogenic cells, both of which are indispensable for nursing donor-derived germ cells. To determine whether hybrid gonads could attract and incorporate donor bluefin tuna germ cells, testicular cells labeled with PKH26 fluorescent dye were intraperitoneally transplanted. Fluorescence observation of hybrid recipients at 14 days post-transplantation revealed that donor cells had been incorporated into the recipient's gonads. This suggests that hybrid mackerel show significant promise for use as a recipient to produce bluefin tuna gametes.

Cryopreservation and Culture of Testicular Tissues: An Essential Tool for Biodiversity Preservation.

Systematic cryo-banking of reproductive tissues could enhance reproductive management and ensure sustainability of rare mammalian genotypes. Testicular tissues contain a vast number of germ cells, including at early stages (spermatogonia and spermatocytes), that can potentially develop into viable spermatozoa after grafting or culture in vitro, and the resulting sperm cells then can be used for assisted reproductive techniques. The objective of this review was to describe current advances, limitations, and perspectives related to the use of testicular tissue preservation as a strategy for the conservation of male fertility. Testes can be obtained from mature or prepubertal individuals, immediately postmortem or by orchiectomy, but testicular biopsies could also be an alternative to collect samples from living individuals. Testicular fragments can be then cryopreserved by using slow or ultra-rapid freezing, or even vitrification methods. The composition of cryopreservation media can vary according to species-specific characteristics, especially regarding the cryoprotectant type and concentration. Finally, spermatozoa have been usually obtained after xenografting of testicular fragments into severely immunodeficient mice, while this method still has to be optimized after in vitro culture conditions.

Engineered reproductive tissues.

Engineered male and female biomimetic reproductive tissues are being developed as autonomous in vitro units or as integrated multi-organ in vitro systems to support germ cell and embryo function, and to display characteristic endocrine phenotypic patterns, such as the 28-day human ovulatory cycle. In this Review, we summarize how engineered reproductive tissues facilitate research in reproductive biology, and overview strategies for making engineered reproductive tissues that might eventually allow the restoration of reproductive capacity in patients.

Progress in translational reproductive science: testicular tissue transplantation and in vitro spermatogenesis.

Since the birth of the first child conceived via in vitro fertilization 40 years ago, fertility treatments and assisted reproductive technology have allowed many couples to reach their reproductive goals. As of yet, no fertility options are available for men who cannot produce functional sperm, but many experimental therapies have demonstrated promising results in animal models. Both autologous (stem cell transplantation, de novo morphogenesis, and testicular tissue grafting) and outside-the-body (xenografting and in vitro spermatogenesis) approaches exist for restoring sperm production in infertile animals with varying degrees of success. Once safety profiles are established and an ideal patient population is chosen, some of these techniques may be ready for human experimentation in the near future, with likely clinical implementation within the next decade.

Regeneration of testis tissue after ectopic implantation of porcine testis cell aggregates in mice: improved consistency of outcomes and in situ monitoring.

Ectopic implantation of donor testis cell aggregates in recipient mice results in de novo formation or regeneration of testis tissue and, as such, provides a unique invivo model for the study of testis development. However, currently the results are inconsistent and the efficiency of the model remains low. This study was designed to: (1) examine several factors that can potentially improve the consistency and efficiency of this model and (2) explore the use of ultrasound biomicroscopy (UBM) for the non-invasive invivo evaluation of implants. Testis cell aggregates, containing ~40% gonocytes, from 1-week-old donor piglets were implanted under the back skin of immunodeficient mice through skin incisions using gel matrices or through subcutaneous injection without using gel matrices. The addition of gel matrices led to inconsistent tissue development; gelatin had the greatest development, followed by collagen, whereas agarose resulted in poor development. The results also depended on the implanted cell numbers since implants with 100×106 cells were larger than those with 50×106 cells. The injection approach for cell implantation was less invasive and resulted in more consistent and efficient testis tissue development. UBM provided promising results as a means of non-invasive monitoring of implants.

Testicular tissue cryopreservation is the preferred method to preserve spermatogonial stem cells prior to transplantation.

RESEARCH QUESTION: Which cryopreservation method better protects reproductive potential: the cryopreservation of a testicular cell suspension (TCS) or the cryopreservation of testicular tissue (TET)? DESIGN: Two cryopreservation strategies for spermatogonial stem cells (SSCs) were compared in a mouse model: cryopreservation as TET or as TCS. Evaluated outcomes were number of viable cells after thawing, number and length of donor-derived colonies after spermatogonial stem cell transplantation (SSCT), number of litters, litter size and number of donor-derived pups after mating. RESULTS: Compared with cryopreserving TCS, cryopreservation of TET resulted in significantly higher numbers of viable cells after thawing (TET: 13.4 â€¯×  104 ± 7.2 â€¯×  104 versus TCS: 8.2 â€¯×  104 ± 2.7 â€¯×  104; P = 0.0002), more (TET: 47.6 ± 19.2 versus TCS: 18.5 ± 13.0; P = 0.0039) and longer (TET: 5.2 ± 1.0 mm versus TCS: 2.7 ± 1.5 mm; P = 0.0016) donor-derived colonies, and more donor-derived pups per litter (TET: 2.2 ± 0.2 versus TCS: 0.5 ± 0.1; P = 0.0008). CONCLUSIONS: Cryopreservation of TET is the preferred method to cryopreserve SSCs prior to SSCT in a mouse model.

Validation of ultrasound biomicroscopy for the assessment of xenogeneic testis tissue grafts and cell implants in recipient mice.

BACKGROUND: Subcutaneous grafting/implantation of neonatal testis tissue/cells from diverse donor species into recipient mice can be used as an in vivo model to study testis development, spermatogenesis, and steroidogenesis. Ultrasound biomicroscopy (UBM) allows obtaining high definition cross-sectional images of tissues at microscopic resolutions. OBJECTIVES: The present study was designed to (a) validate the use of UBM for non-invasive monitoring of grafts/implants overtime and to (b) correlate UBM findings with the morphological attributes of recovered grafts/implants. MATERIALS AND METHODS: Testis tissue fragments (~14 mm3 , each) and cell aggregates (100 × 106 cells, each) obtained from 1-week-old donor piglets (n = 30) were grafted/implanted under the back skin of immunodeficient mice (n = 6) in eight analogous sites per mouse. Three-dimensional transcutaneous Doppler UBM was performed, and a randomly selected graft and its corresponding implant were recovered at 2, 4, 6, and 8 weeks. RESULTS: Graft/implant weight (P = .04) and physical height (P = .03) increased overtime. The dynamics of physical length and volume increases over time differed between tissue grafts and cell implants (P = .02 and 0.01 for sample type*time interactions, respectively). UBM-estimated volume was correlated with the post-recovery weight and volume of the grafts/implants (r = 0.98 and r = 0.99, respectively; P < .001). Pre- and post-recovery length and height of the grafts/implants were positively and strongly correlated (r = 0.50, P = .01; r = 0.70, P = .001) and so were the areas covered by cordal, non-cordal, or fluid-filled cavities between UBM and histology (r = 0.87, P < .001). DISCUSSION AND CONCLUSION: UBM findings correlated with physical attributes of the grafts/implants, validating its use as a non-invasive high-fidelity tool to quantify the developmental changes in ectopic testis tissue grafts and cell implants, potentially leading to a reduction in the number of recipient mice needed for similar experiments.

Significant Benefits of Nanoparticles Containing a Necrosis Inhibitor on Mice Testicular Tissue Autografts Outcomes.

Fertility preservation for prepubertal boys relies exclusively on cryopreservation of immature testicular tissue (ITT) containing spermatogonia as the only cells with reproductive potential. Preclinical studies that used a nude mice model to evaluate the development of human transplanted ITT were characterized by important spermatogonial loss. We hypothesized that the encapsulation of testicular tissue in an alginate matrix supplemented with nanoparticles containing a necrosis inhibitor (NECINH-NPS) would improve tissue integrity and germ cells' survival in grafts. We performed orthotopic autotransplantation of 1 mm³ testicular tissue fragments recovered form mice (aged 4-5 weeks). Fragments were either non-encapsulated, encapsulated in an alginate matrix, or encapsulated in an alginate matrix containing NECINH-NPs. Grafts were recovered 5- and 21-days post-transplantation. We evaluated tissue integrity (hematoxylin-eosin staining), germ cells survival (immunohistochemistry for promyelocytic leukemia zinc-finger, VASA, and protein-boule-like), apoptosis (immunohistochemistry for active-caspase 3), and lipid peroxidation (immunohistochemistry for malondialdehyde). NECINH-NPs significantly improved testicular tissue integrity and germ cells' survival after 21 days. Oxidative stress was reduced after 5 days, regardless of nanoparticle incorporation. No effect on caspase-dependent apoptosis was observed. In conclusion, NECINH-NPs in an alginate matrix significantly improved tissue integrity and germ cells' survival in grafts with the perspective of higher reproductive outcomes.