Results from the first autologous grafting of adult human testis tissue: a case report.

Fertility restoration using autologous testicular tissue transplantation is relevant for infertile men surviving from childhood cancer and, possibly, in men with absent or incomplete spermatogenesis resulting in the lack of spermatozoa in the ejaculate (non-obstructive azoospermia, NOA). Currently, testicular tissue from pre-pubertal boys extracted before treatment with gonadotoxic cancer therapy can be cryopreserved with good survival of spermatogonial stem cells. However, strategies for fertility restoration, after successful cancer treatment, are still experimental and no clinical methods have yet been developed. Similarly, no clinically available treatments can help men with NOA to become biological fathers after failed attempts of testicular surgical sperm retrieval. We present a case of a 31-year-old man with NOA who had three pieces of testis tissue (each ∼2 × 4 × 2 mm3) extracted and cryopreserved in relation to performing microdissection testicular sperm extraction (mTESE). Approximately 2 years after mTESE, the thawed tissue pieces were engrafted in surgically created pockets bilaterally under the scrotal skin. Follow-up was performed after 2, 4, and 6 months with assessment of reproductive hormones and ultrasound of the scrotum. After 6 months, all engrafted tissue was extracted and microscopically analyzed for the presence of spermatozoa. Furthermore, parts of the extracted tissue were analyzed histologically and by immunohistochemical analysis. Active blood flow in the engrafted tissue was demonstrated by doppler ultrasound after 6 months. No spermatozoa were found in the extracted tissue. Histological and immunohistochemical analysis demonstrated graft survival with intact clear tubules and normal cell organization. Sertoli cells and spermatocytes with normal morphology were located near the basement membrane. MAGE-A and VASA positive spermatogonia/spermatocytes were detected together with SOX9 positive Sertoli cells. Spermatocytes and/or Sertoli cells positive for γH2AX was also detected. In summary, following autologous grafting of frozen-thawed testis tissue under the scrotal skin in a man with NOA, we demonstrated graft survival after 6 months. No mature spermatozoa were detected; however, this is likely due to the pre-existing spermatogenic failure.

Ex-Vivo and In-Vivo Expansion of Spermatogonial Stem Cells Using Cell-Seeded Microfluidic Testis Scaffolds and Animal Model.

AIMS: This study was designed to provide both ex-vivo and in-vivo methods for the extraction and expansion of spermatogonial stem cells (SSCs). METHODS: For in-vivo experiments, azoospermic mouse model was performed with Busulfan. Isolation, culture, and characterization of neonate mouse SSC were also achieved. We performed an in-vivo injection of labeled SSCs to the testes with azoospermia. In ex-vivo experiments, extracted SSCs were seeded on the fabricated scaffold consisting of hyaluronic acid (HA) and decellularized testis tissues (DTT). Immunofluorescence staining with PLZF, TP1, and Tekt 1 was performed for SSCs differentiation and proliferation. RESULTS: Several studies demonstrated efficient spermatogenic arrest in seminiferous tubules and proved the absence of spermatogenesis. Transplanted SSCs moved and settled in the basement covering the seminiferous tubules. Most of the cells were positive for Dil, after 4 weeks. An epithelium containing spermatogonia-like cells with Sertoli-like, and Leydig cells were evident in the seminiferous tubules of biopsies, and the IHC staining was significantly positive, 4 weeks after injection of SSCs. The results of the ex-vivo experiments showed positive staining for all markers, which was significantly enhanced in scaffolds of ex-vivo experiments compared with in-vitro seeded scaffolds. CONCLUSION: Ex-vivo SSC differentiation and proliferation using cell-seeded microfluidic testis scaffolds maybe effective for treatment of the azoospermia.

[Nursing cooperation in surgical collection of testis tissues from prepubertal male patients for cryopreservation: A study of 4 cases].

OBJECTIVE: To explore nursing cooperation in surgical collection of the testis tissue from prepubertal male patients for cryopreservation. METHODS: We retrospectively analyzed the methods and effects of perioperative nursing in surgical collection of the testis tissue from 4 prepubertal male patients for cryopreservation in our Center of Reproductive Medicine. Before, during and after operation, we took strict measures in making sterilized ice containers, intraoperative nursing cooperation, protection of the isolated testis tissues and transferring of the samples. RESULTS: Testis tissues were successfully collected from all the 4 prepubertal males, 31, 31, 20 and 34 samples from each case respectively, well protected and subjected to slow cryopreservation after standard processing in the embryo laboratory. CONCLUSION: In surgical collection of the testis tissue for cryopreservation, preparation of sterilized ice containers, intraoperative nursing cooperation and protection and transferring of the samples are essential for standard processing and cryopreservation of the testis tissue in the embryo laboratory.

Engineered Immature Testicular Tissue by Electrospun Mats for Prepubertal Fertility Preservation in a Bioluminescence Imaging Transgenic Mouse Model.

Prepubertal boys with cancer may suffer from reduced fertility and maturity following gonadotoxic chemoradiotherapy. Thus, a viable method of immature testicular tissue (ITT) preservation is required in this cohort. In this study, we used poly-L-lactic acid electrospun scaffolds with two levels of fineness to support the development of ITT transplanted from transgenic donors to wild-type recipient mice. The purpose of this study was to evaluate the potential of ITT transplantation and spermatogenesis after using the two scaffolds, employing bioluminescence imaging for evaluation. The results suggest that ITT from 4-week-old mice possessed the most potential in spermatogenesis on the 70th day, together with the fine electrospun scaffolds. Moreover, bioluminescent imaging intensity was observed in recipient mice for up to 107 days, approximately six times more than the coarse electrospun scaffold and the control group. This occurs since the fine scaffold is more akin to the microenvironment of native testicular tissue as it reduces stiffness resulting from micronization and body fluid infiltration. The thermal analysis also exhibited recrystallization during the biodegradation process, which can lead to a more stable microenvironment. Overall, these findings present the prospect of fertility preservation in prepubertal males and could serve as a framework for future applications.

Immature Testicular Tissue Engineered from Weaned Mice to Adults for Prepubertal Fertility Preservation-An In Vivo Translational Study.

Male pediatric survivors of cancers and bone marrow transplantation often require adjuvant chemoradiation therapy that may be gonadotoxic. The optimal methods to preserve fertility in these prepubertal males are still under investigation. This manuscript presents an in vivo experiment which involved transplantation of immature testicular tissues (ITT) from transgenic donor, to wild-type recipient mice. Donors and recipients were age-mismatched (from 20-week-old donors to 3-week-old recipients, and vice versa) and the transplantation sites involved the abdomen, skin of the head, back muscle, and scrotum. The application of poly-l-lactic acid (PLLA) scaffold was also evaluated in age-matched donors and recipients (both 3-weeks-old). To quantitively evaluate the process of spermatogenesis after ITT transplantation and scaffold application, bioluminescence imaging (BLI) was employed. Our result showed that ITT from 3-week-old mice had the best potential for spermatogenesis, and the optimal transplantation site was in the scrotum. Spermatogenesis was observed in recipient mice up to 51 days after transplantation, and up to the 85th day if scaffold was used. The peak of spermatogenesis occurred between the 42nd and 55th days in the scaffold group. This animal model may serve as a framework for further studies in prepubertal male fertility preservation.

The study and manipulation of spermatogonial stem cells using animal models.

Spermatogonial stem cells (SSCs) are a rare group of cells in the testis that undergo self-renewal and complex sequences of differentiation to initiate and sustain spermatogenesis, to ensure the continuity of sperm production throughout adulthood. The difficulty of unequivocal identification of SSCs and complexity of replicating their differentiation properties in vitro have prompted the introduction of novel in vivo models such as germ cell transplantation (GCT), testis tissue xenografting (TTX), and testis cell aggregate implantation (TCAI). Owing to these unique animal models, our ability to study and manipulate SSCs has dramatically increased, which complements the availability of other advanced assisted reproductive technologies and various genome editing tools. These animal models can advance our knowledge of SSCs, testis tissue morphogenesis and development, germ-somatic cell interactions, and mechanisms that control spermatogenesis. Equally important, these animal models can have a wide range of experimental and potential clinical applications in fertility preservation of prepubertal cancer patients, and genetic conservation of endangered species. Moreover, these models allow experimentations that are otherwise difficult or impossible to be performed directly in the target species. Examples include proof-of-principle manipulation of germ cells for correction of genetic disorders or investigation of potential toxicants or new drugs on human testis formation or function. The primary focus of this review is to highlight the importance, methodology, current and potential future applications, as well as limitations of using these novel animal models in the study and manipulation of male germline stem cells.

Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate.

STUDY QUESTION: Can full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously? SUMMARY ANSWER: Intratesticular xenotransplantation supported the differentiation of immature germ cells from marmoset (Callithrix jacchus) into spermatids and spermatozoa at 4 and 9 months post-transplantation, while in subcutaneous transplants, spermatogenic arrest was observed at 4 months and none of the transplants survived at 9 months. WHAT IS KNOWN ALREADY: Auto-transplantation of cryopreserved immature testis tissue (ITT) could be a potential fertility restoration strategy for patients with complete loss of germ cells due to chemo- and/or radiotherapy at a young age. Before ITT transplantation can be used for clinical application, it is a prerequisite to demonstrate the feasibility of the technique and identify the conditions required for establishing spermatogenesis in primate ITT transplants. Although xenotransplantation of ITT from several species has resulted in complete spermatogenesis, in human and marmoset, ITT has not been successful. STUDY DESIGN, SIZE, DURATION: In this study, we used marmoset as a pre-clinical animal model. ITT was obtained from two 6-month-old co-twin marmosets. A total of 147 testis tissue pieces (~0.8-1.0 mm3 each) were transplanted into the testicular parenchyma (intratesticular; n = 40) or under the dorsal skin (ectopic; n = 107) of 4-week-old immunodeficient Swiss Nu/Nu mice (n = 20). Each mouse received one single marmoset testis tissue piece in each testis and 4-6 pieces subcutaneously. Xenotransplants were retrieved at 4 and 9 months post-transplantation and evaluations were performed with regards to transplant survival, spermatogonial quantity and germ cell differentiation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Transplant survival was histologically evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Spermatogonia were identified by MAGE-A4 via immunohistochemistry. Germ cell differentiation was assessed by morphological identification of different germ cell types on H/PAS stained sections. Meiotically active germ cells were identified by BOLL expression. CREM immunohistochemistry was performed to confirm the presence of post-meiotic germ cells and ACROSIN was used to determine the presence of round, elongating and elongated spermatids. MAIN RESULTS AND THE ROLE OF CHANCE: Four months post-transplantation, 50% of the intratesticular transplants and 21% of the ectopic transplants were recovered (P = 0.019). The number of spermatogonia per tubule did not show any variation. In 33% of the recovered intratesticular transplants, complete spermatogenesis was established. Overall, 78% of the intratesticular transplants showed post-meiotic differentiation (round spermatids, elongating/elongated spermatids and spermatozoa). However, during the same period, spermatocytes (early meiotic germ cells) were the most advanced germ cell type present in the ectopic transplants. Nine months post-transplantation, 50% of the intratesticular transplants survived, whilst none of the ectopic transplants was recovered (P < 0.0001). Transplants contained more spermatogonia per tubule (P = 0.018) than at 4 months. Complete spermatogenesis was observed in all recovered transplants (100%), indicating a progressive spermatogenic development in intratesticular transplants between the two time-points. Nine months post-transplantation, transplants contained more seminiferous tubules with post-meiotic germ cells (37 vs. 5%; P < 0.001) and fewer tubules without germ cells (2 vs. 8%; P = 0.014) compared to 4 months post-transplantation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although xenotransplantation of marmoset ITT was successful, it does not fully reflect all aspects of a future clinical setting. Furthermore, due to ethical restrictions, we were not able to prove the functionality of the spermatozoa produced in the marmoset transplants. WIDER IMPLICATIONS OF THE FINDINGS: In this pre-clinical study, we demonstrated that testicular parenchyma provides the required microenvironment for germ cell differentiation and long-term survival of immature marmoset testis tissue, likely due to the favourable temperature regulation, growth factors and hormonal support. These results encourage the design of new experiments on human ITT xenotransplantation and show that intratesticular transplantation is likely to be superior to ectopic transplantation for fertility restoration following gonadotoxic treatment in childhood. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by the ITN Marie Curie Programme 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568) and the scientific Fund Willy Gepts from the UZ Brussel (ADSI677). D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

Effect of recombinant human vascular endothelial growth factor on testis tissue xenotransplants from prepubertal boys: a three-case study.

RESEARCH QUESTION: Does recombinant human vascular endothelial growth factor (VEGF-165) improve the efficiency of human immature testis tissue (ITT) xenotransplantation? DESIGN: ITT fragments from three prepubertal boys were cultured for 5 days with VEGF-165 or without (control) before xenotransplantation into the testes of immunodeficient mice. Xenotransplants were recovered at 4 and 9 months post-transplantation and vascularization, seminiferous tubule integrity, number of spermatogonia and germ cell differentiation were evaluated by histology and immunohistochemistry. RESULTS: Transplants from donor 1 and donor 2 treated with VEGF demonstrated higher vascular surface (P = 0.004) and vessel density (P = 0.011) overall and contained more intact seminiferous tubules (P = 0.039) with time, compared with controls. The number of spermatogonia was increased over time (P < 0.001) irrespective of treatment and donor, whereas, for the VEGF-treated transplants, the increase was even higher over time (P = 0.020). At 9 months, spermatocytes were present in the xenotransplants, irrespective of treatment. No transplants could be recovered from donor 3, who had already received treatment with cyclosporine for aplastic anaemia before biopsy. CONCLUSIONS: In-vitro pre-treatment of human prepubertal testis tissue with VEGF improved transplant vascularization in two out of three cases, resulting in improved seminiferous tubule integrity and spermatogonial survival during xenotransplantation. Although further studies are warranted, we suggest VEGF to be considered as a factor for improving the efficiency of immature testis tissue transplantation in the future.

Effect of newborn bovine serum on cryopreservation of adult bovine testicular tissue.

Bovine serum is widely used for cryopreservation of various cells and tissues. However, its cryoprotective effects on the cells and tissues are ambiguous and controversial. To test the effects of newborn calf serum (NCS) on cryopreservation of bovine testis tissue, NCS of 0%, 5%, 10% and 20% (v/v) was added into minimum essential medium + 10% dimethyl sulphoxide (DMSO)-based medium according to our previous report. Interestingly, the testicular cell viabilities and spermatogonia percentages from four groups were very close. The results indicated that an increase in the concentration of NCS in freezing medium to 20% has no significant effect on survival of both testicular cells and spermatogonia, and 10% DMSO-based freezing medium can maintain the testicular cell viability and spermatogonia percentage at a relatively high level (83.4 ± 0.7 and 56.5 ± 2.2 respectively). Taken together, NCS is dispensable for cryopreservation of adult bovine testis tissue. Our results provide an evidence for cutting down the costs in cryopreservation research of bovine testis tissue by reducing or giving up the use of serum.

Molecular phenotyping of domestic cat (Felis catus) testicular cells across postnatal development - A model for wild felids.

Molecular characterisation of testicular cells is a pivotal step towards a profound understanding of spermatogenesis and developing assisted reproductive techniques (ARTs) based on germline preservation. To enable the identification of testicular somatic and spermatogenic cell types in felids, we investigated the expression of five molecular markers at the protein level in testes from domestic cats (Felis catus) at different developmental phases (prepubertal, pubertal I and II, postpubertal I and II) classified by single-cell ploidy analysis. Our findings indicate a prominent co-labelling for two spermatogonial markers, UCHL1 and FOXO1, throughout postnatal testis development. Smaller subsets of UCHL1 or FOXO1 single-positive spermatogonia were also evident, with the FOXO1 single-positive spermatogonia predominantly observed in prepubertal testes. As expected, DDX4+ germ cells increased in numbers beginning in puberty, reaching a maximum at adulthood (post-pubertal phase), corresponding to the sequential appearance of labelled spermatogonia, spermatocytes and spermatids. Furthermore, we identified SOX9+ Sertoli cells and CYP17A1+ Leydig cells in all of the developmental groups. Importantly, testes of African lion (Panthera leo), Sumatran tiger (Panthera tigris sumatrae), Chinese leopard (Panthera pardus japonesis) and Sudan cheetah (Acinonyx jubatus soemmeringii) exhibited conserved labelling for UCHL1, FOXO1, DDX4, SOX9 and CYP17A1. The present study provides fundamental information about the identity of spermatogenic and somatic testicular cell types across felid development that will be useful for developing ART approaches to support endangered felid conservation.

Protective effects of chlorogenic acid against ionizing radiation-induced testicular toxicity.

Background: Testicular tissues could damage by ionizing radiation (IR) during the treatment of pelvic cancers. The aim of this study was to investigate both the protective and therapeutic effects of chlorogenic acid (CGA) on IR-induced mouse testis tissue damage. Methods: In this experimental study, 70 mice were divided into 3 groups, including group 1 (normal saline), group 2 (IR + normal saline), and group 3 (IR + 5, 10, 20, 40, and 80 mg/kg) CGA via I.P injection. Animals in groups 2 and 3 received a dose of 2.0 Gy total-body irradiation in a single fraction. At two determined time points (16 h and 35 days after exposure), the testis and caudal part of both epididymis were isolated and underwent subsequent analyses. Results: The results showed that irradiation of mice caused massive damage to spermatogenesis, seminiferous tubules, basal lamina, Leydig cells, and sperm parameters. Further biochemical assessment of the data demonstrated that 40 mg/kg CGA almost restored MDA to a normal level. In addition, the level of SOD, TAC, and GSH were significantly increased in the 40 mg/kg CGA treated group. Molecular evidence confirmed the protective effects of CGA and also revealed that the ratio of Bax/Bcl-2 in the presence of 40 mg/kg CGA was significantly decreased compared to IR and some treated groups. Conclusion: The protective and therapeutic effects of CGA on testis were found to be positively correlated with the dose level.

Transcriptomic Study of Spermatogenesis in the Testis of Hu Sheep and Tibetan Sheep.

Numerous genes involved in male reproduction regulate testis development and spermatogenesis. In this study, the testis tissue transcriptome was used to identify candidate genes and key pathways associated with fecundity in sheep. Histological analysis of testis tissue using hematoxylin-eosin (HE) routine staining was performed for two sheep breeds. Overall, 466 differentially expressed genes (DEGs) were identified between Hu sheep (HS) and Tibetan sheep (TS) through RNA sequencing technology (RNA-Seq), including 226 upregulated and 240 downregulated genes. Functional analysis showed that several terms and pathways, such as "protein digestion and absorption", "cAMP signaling pathway", "focal adhesion", and "p53 signaling pathway" were closely related to testis development and spermatogenesis. Several genes (including COL1A1, COL1A2, COL3A1, SOX9, BCL2, HDC, and GGT5) were significantly enriched in these terms and pathways and might affect the reproduction of sheep by regulating the migration of spermatogenic cells, apoptosis of spermatogenic cells, and secretion of sterol hormones via testicular interstitial cells. Our results provide a theoretical basis for better understanding the molecular mechanisms of reproduction in sheep.

The ameliorative effect of carvacrol on oxidative stress and germ cell apoptosis in testicular tissue of adult diabetic rats.

BACKGROUND: Diabetes is one of the most chronic and widespread diseases causing the damages to the male reproductive system. Nowadays, several studies have been performed to show the role of phenolic compounds in reducing the complications of diabetes. Carvacrol is a phenolic monoterpene which has been shown to have much therapeutic efficacy in various diseases. METHODS: Thirty-two adult male Wistar rats (n = 8 in each group) were used in this experimental study. The induction of diabetes was performed using a single intraperitoneal (IP) injection of STZ (50 mg/kg). Rats were assigned into the following groups: control group, diabetic group, diabetic group daily fed with carvacrol at a dose of 75 mg/kg for 8 weeks, and the control group daily fed with carvacrol at a dose of 75 mg/kg for 8 weeks. RESULTS: Treatment with carvacrol significantly improved the histological morphology of the testis, reduced the tissue activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymes, and diminished the elevated levels of tissue malondialdehyde (MDA) (p < 0.05). Moreover, our results showed that carvacrol significantly decreased Bax and increased Bcl-2 at the levels of gene and protein expression. It also significantly (p < 0.05) reduced the rate of germ cell apoptosis. CONCLUSION: It seems that the treatment with carvacrol mitigates testicular tissue damage in diabetic rats possibly through its antioxidant properties.

Comparative RNA-Seq analysis of differentially expressed genes in the testis and ovary of Takifugu rubripes.

Takifugu rubripes is a classical model organism for studying the role of gonad organogenesis in such physiological processes as fertilization, sex determination, and sexual development. To explicitly investigate the mechanism associated with gonad organogenesis in T. rubripes, we obtained 44.3 million and 55.2 million raw reads from the testis and ovary, respectively, by RNA-seq and from these, 18,523 genes were identified. A total of 680 differentially expressed genes were obtained from comparison transcriptome analysis between the testis and ovary, and of these, 556 genes were up-regulated in the testis, whereas only 124 genes were upregulated in the ovary. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that many of these genes encode proteins involved in signal transduction and gonad development. We mainly focused on the differentially expressed genes that have the potential to develop into the gonad. The generation of large scale transcriptomic data presented in this work would enrich the genetic resources of T. rubripes, which should be valuable to the comparative and evolutionary studies of teleosts.

Advances in cryopreservation of spermatogonial stem cells and restoration of male fertility.

Spermatogenesis is a highly complicated process which initiated by spermatogonial stem cells (SSCs). SSCs are the only cell type that can restore fertility in infertile recipient after SSCs transplantation. SSCs damage during cancer diagnosis and therapy and their depletion may be cause of male infertility in cancer survivors. In this review, used experimental methods regarding SSCs and testis tissue cryopreservation have been reviewed with a special focus on animal models and human which have generated the majority of data about SSCs and the cryopreservation process.

[Androprotect and prospects for fertility treatment]. / Androprotect und Perspektiven der Fertilitätstherapie.

BACKGROUND: Fertility protection is essential for men undergoing potentially gonadotoxic treatment. It is usually offered to adolescents and men in reproductive age by semen cryopreservation. In case of azoospermia, testicular sperm cryopreservation is an additional option. In prepubertal boys no sperm cryopreservation is possible. A purely experimental option is cryopreservation of spermatogonial stem cells in immature testis tissue. METHOD: Transplantation of either immature testis tissue or testicular stem cells or spermatogonia generated in vitro from stem cells are possible options for fertility preservation in boys. OBJECTIVES: In this article, the rationale for cryopreservation of gonadal stem cells and the experimental methods for refertilization are summarized. The current research, national and international clinical and research activities and possible perspectives of further development of fertility preservation are explained.