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

2020 ◽  
Author(s):  
Christine Wyns ◽  
Marc Kanbar ◽  
Maria Grazia Giudice ◽  
Jonathan Poels
Keyword(s):  
Stem Cells ◽  
Fertility Preservation ◽  
Clinical Application ◽  
Germ Cells ◽  
Testicular Tissue ◽  
Reproductive Age ◽  
Reproductive Capacity ◽  
Patient Counselling ◽  
Final Maturation ◽  
Prepubertal Boys

Abstract 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.

192 IDENTIFICATION AND CHARACTERIZATION OF Oct4-EGFP EXPRESSING CELLS IN TRANSGENIC PIG TESTIS

2014 ◽  
Vol 26 (1) ◽  
pp. 210
Author(s):  
M. Nowak-Imialek ◽  
N. Lachmann ◽  
D. Herrmann ◽  
F. Jacob ◽  
H. Niemann
Keyword(s):  
Stem Cells ◽  
Cell Population ◽  
Germ Cells ◽  
Germ Line ◽  
Cell Mass ◽  
Testicular Tissue ◽  
Egfp Expression ◽  
Cell Populations ◽  
Marker Genes ◽  
Transgenic Pigs

We have produced germ line transgenic pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct; Nowak-Imialek et al., 2011 Stem Cells Dev.). Expression of the EGFP reporter construct is confined to germ line cells, the inner cell mass, and trophectoderm of blastocysts, and testicular germ cells, including putative spermatogonial stem cells (SSC). SSC are unique among stem cells because they can both self-renew and differentiate into spermatozoa. In-depth knowledge on porcine SSC has been hampered by the inability to isolate these cells from the complex cell population of the testis. In the Oct4-EGFP transgenic mouse, SSC are the only adult stem cells that express Oct4. Fluorescence microscopy of testicular tissue isolated from transgenic piglets revealed minimum numbers of EGFP-positive cells, whereas testicular tissue isolated from adult transgenic boars contained a high amount of EGFP fluorescent cells. Northern blot analysis confirmed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. Time course and the signal intensity of EGFP expression in Oct4-EGFP testis paralleled mRNA expression of the endogenous Oct4 gene. Here, we used adult Oct4-EGFP transgenic pigs as a model for fluorescence-activated cell sorting (FACS)-based isolation of EGFP-expressing cells from testes. To obtain a single-cell suspension, the testes were enzymatically dissociated using two digestion steps. Thereafter, FACS based on EGFP expression was successfully used to purify specific testicular cell populations. Two cell populations, i.e. EGFP+ (14%) and EGFP– (45%) could be isolated. Subsequently, qualitative PCR analyses were performed on EGFP+, EGFP–, and unsorted cell populations using marker genes specific for pluripotency and undifferentiated germ cells (OCT4, FGFR3, UTF1, PGP9.5, GFRα1, CD90, SALL4), differentiating germ cells (c-KIT), meiosis (BOLL), spermatids (PRM2), and somatic cells (VIM, LHCGR). All of the genes, including OCT4, UTF1, FGFR3, PGP9.5, CD90, SALL4, and GFRα1 were expressed at least 3-fold and up to 12-fold greater in the EGFP-positive population. Vimentin, which is mainly expressed in Sertoli cells and LHCGR, which is mainly expressed in Leydig cells, were expressed in unsorted and EGFP– cell populations and at very low level in EGFP+ cells. Moreover, expression of the c-KIT and PRM2 markers were detected also in EGFP+ cell population, indicating that these cells contain also differentiating spermatogonia. To explore the characteristics of the Oct4-EGFP expressing cells in greater detail, localization in the porcine testis sections and analysis of co-expression with germ cell markers using immunohistochemistry is currently underway.

Current State of Fertility Preservation in Cancer Patients—What is Established and What is Still Controversial?

2016 ◽  
Vol 12 (01) ◽  
pp. 33
Author(s):  
Kutluk Oktay ◽  
Giuliano Bedoschi ◽  
◽  
◽  
◽  
...  
Keyword(s):  
Cancer Patients ◽  
Fertility Preservation ◽  
Cancer Care ◽  
Testicular Tissue ◽  
Semen Cryopreservation ◽  
Young Females ◽  
Current State ◽  
Preservation Method ◽  
Prepubertal Boys ◽  
Straightforward Approach

Fertility Preservation is an essential part of cancer care when treating young females and men. While semen cryopreservation is a straightforward approach for postpubertal men and there is the option of experimental testicular tissue freezing for prepubertal boys, the options for females are more tumultuous. The last 17 years brought us established approaches such the embryo and oocyte cryopreservation and the ovarian cryopreservation is ready to join the list. However, there still is no proven medical fertility preservation method and the controversy around the utility of GnRHa continues.

scholarly journals Cryopreservation of testicular tissue: an alternative to maintain the reproductive capacity in different animal species

2017 ◽  
Vol 47 (11) ◽  
Author(s):  
David Baruc Cruvinel Lima ◽  
Lúcia Daniel Machado da Silva
Keyword(s):  
Clinical Practice ◽  
Germ Cells ◽  
Animal Species ◽  
Testicular Tissue ◽  
Reproductive Capacity ◽  
Significant Progress ◽  
Breeding Programs ◽  
Artificial Breeding ◽  
Made In

ABSTRACT: Cryopreservation of testicular tissue enables the maintenance of reproductive capacity in different animal species, and contributes to the formation of gene banks for endangered species. The spermatogonia present in the testes can be grown in vitro and the sperm obtained can be used in artificial breeding programs. This review aimed to describe the main techniques of testicular cryopreservation, the main cryoprotectants used, as well as the progress made in different animal species thus far. In the last decade, significant progress has been made in obtaining viable and functional germ cells from testicular tissue. However, more research is needed to better establish protocols that can be used in clinical practice with various species.

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

2019 ◽  
Vol 20 (23) ◽  
pp. 5833 ◽  
Author(s):  
Federico Del Vento ◽  
Maxime Vermeulen ◽  
Bernard Ucakar ◽  
Jonathan Poels ◽  
Anne des Rieux ◽  
...  
Keyword(s):  
Germ Cells ◽  
Reproductive Potential ◽  
Testicular Tissue ◽  
Mice Model ◽  
Post Transplantation ◽  
Tissue Integrity ◽  
Prepubertal Boys ◽  
Hematoxylin Eosin ◽  
Alginate Matrix ◽  
Active Caspase

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.

P–125 Evaluation of N-acetylcysteine (NAC) effect on in vitro culture of immature mouse testis following vitrification

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
P Nikoosokhan ◽  
B Ebrahimi ◽  
A Alizadeh ◽  
S Hajiaghalou
Keyword(s):  
Stem Cells ◽  
Cell Survival ◽  
Culture Medium ◽  
Caspase 3 ◽  
Testicular Tissue ◽  
Culture Conditions ◽  
Seminiferous Tubules ◽  
Immature Mouse ◽  
Prepubertal Boys

Abstract Study question Can the Culture of cryopreserved immature mouse testicular tissue in the presence of NAC improves the developmental process and prevent apoptosis induction during the culture? Summary answer An appropriate dosage of NAC in the culture medium of immature mouse testicular tissue was associated with increased cell survival and spermatogonia stem cell regeneration. What is known already Spermatogonial stem cells (SSCs) are the most advanced type of stem cells in the testes of prepubertal boys which are the main targets of oncological treatments. Therefore, the only possible alternative to maintain fertility in prepubertal boys is to preserve SSCs before their depletion by cryopreserving the testicular tissue. Despite the possibility of obtaining viable spermatozoa using cryopreserved testicular tissue cultivated in vitro,cryopreservation methods and damages caused by the culture procedure would be obstacles for maintaining the testicular tissueand it seems that optimizing the culture condition is vital. Study design, size, duration Testis tissues were harvested from 6-days-old immature NMRI male mice (n = 100) after cervical dislocation and vitrified. After 3 days testicular biopsies were warmed and distributed into control, culture Ӏ (not supplemented with NAC) and culture ӀӀ (supplemented with NAC) groups. To determine the appropriate NAC concentration 8 different dosages of NAC were evaluated in terms of cell survival and the best dose, a culture medium containing 125mmol/L NAC was selected to continue the study. Participants/materials, setting, methods Vitrified-warmed fragments (2mm3) obtaining from immature NMRI mice were cultured in vitro for 7 days on agar gel. The effects of culture conditions were assessed by Morphological evaluation of seminiferous tubules (using Hematoxylin-eosin staining). Cell viability, protein expression (caspase–3), and gene expression (Bax, Bcl2, Caspase–3, plzf) were evaluated by flow cytometry, immunofluorescence staining, and real time polymerase chain reaction respectively. Additionally, Malondialdehyde (MDA) concentration in the culture medium was measured by MAD Assay Kit. Main results and the role of chance Significant (p < 0.01) increase in cell viability was observed in the culture ӀӀ group after 7 days of culture compared to the culture Ӏ. Bax/Bcl2 ratio was significantly (p < 0.01) lower in the culture ӀӀ group compared to the control and culture Ӏ group. The expression of caspase–3 showed a significant (p < 0.001) increase in the culture ӀӀ group while immunofluorescence analysis showed low expression of it in all groups. These results were consistent with the high level of Bcl2expression that inhibited Caspase–3 expression and consequently the inhibition of apoptosis, and on the other hand, the presence of NAC showed that plzf expressions significantly (p < 0.001) increased in culture ӀӀ group compared to the control and culture Ӏ group. Although the presence of NAC did not inhibit all the deleterious effects of culture medium on tissue morphology, NAC was able to maintain better integrity of tissue and seminiferous tubules within central regions compared to the group without NAC. The decrease in MDA level in the presence of NAC (culture ӀӀ) was also a good indicator to confirm the desired results obtained from the presence of NAC in the culture medium. Limitations, reasons for caution Although the findings of the study were satisfactory in mice tissue after 1 week of culture, it is essential to replicate the experiments using human tissue and evaluate the quality and reproductive potential of surviving spermatogonia after long-term storage to become clinically applicable. Wider implications of the findings: This study highlights the necessity for further experiments to improve the testicular tissue culture conditions for better spermatogonial survival and differentiation to sperm, as the prepubertal fertility restoration methods are promising to be implemented in the clinic in the near future. Trial registration number Not applicable

scholarly journals Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys

2018 ◽  
Vol 9 ◽  
Author(s):  
Francesca de Michele ◽  
Jonathan Poels ◽  
Maxime Vermeulen ◽  
Jérôme Ambroise ◽  
Damien Gruson ◽  
...  
Keyword(s):  
Germ Cells ◽  
Organotypic Culture ◽  
Testicular Tissue ◽  
Prepubertal Boys

Cryopreservation of mouse testicular tissue: prospect for harvesting spermatogonial stem cells for fertility preservation

2011 ◽  
Vol 95 (7) ◽  
pp. 2399-2403 ◽  
Author(s):  
Sok Siam Gouk ◽  
Yu Feng Jason Loh ◽  
Srinivasan D. Kumar ◽  
Paul F. Watson ◽  
Lilia L. Kuleshova
Keyword(s):  
Stem Cells ◽  
Fertility Preservation ◽  
Testicular Tissue ◽  
Spermatogonial Stem Cells

189 Isolation and purification of rhinoceros and horse spermatogonial stem cells

2019 ◽  
Vol 31 (1) ◽  
pp. 219
Author(s):  
C. Yu-Su ◽  
T. Jensen ◽  
B. Durrant ◽  
M. C. Gómez
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Reproductive Age ◽  
Culture Methods ◽  
Isolation And Purification ◽  
Ceratotherium Simum ◽  
White Rhinoceros

The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is critically endangered. Testicular tissue (TT) of an NWR that died at 46 years was cryopreserved and stored. Spermatogonial stem cells (SSC) have been isolated from TT and, following transplantation or in vitro culture, differentiated into mature spermatozoa. These SSC have been phenotypically characterised by the expression of markers specific for SSC, and in vitro culture methods optimized to isolate, purify, and enrich populations of SSC from mixed germ cells. The reproductive age may affect the percent of SSC in TT, affecting recovery from older animals. We previously identified in TT of NWR the surface markers GFRa1 and GPR125, both of which were expressed at various stages of spermatogenesis. However, characterisation, isolation, and purification of rhinoceros SSC have not been studied. Because of the limited availability of rhinoceros TT, we used the horse as a model for rhinoceros (both species are in the same order: Perissodactyla). In this study, we (1) identified and compared the expression of markers specific for SSC (GFRa1, GPR125, PLZF) and pluripotent markers (SSEA-1, SSEA-4, OCT-4) in rhinoceros and horse mixed germ cells; (2) evaluated whether rhinoceros and horse SSC could be purified and enriched by sequential culture with collagen (2 days) and laminin (3 weeks); and (3) determined whether age may affect the percentages of SSC by comparing the expression of SSC and pluripotent markers in horse mixed germ cells at different reproductive stages: pubertal (PU=1-1.5 years) v. post-pubertal (PP=2-3 years) v. adult (AD=>5 years). Mixed germ cells were isolated from testes of one male of each of 3 rhinoceros species: NWR, southern white rhinoceros (SWR; Ceratotherium simum simum, 49 years), and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis, 5 years), and horses at different reproductive stages (PU=2, PP=2, AD=7). The SWR and GOHR testes were refrigerated overnight, whereas NWR mixed germ cells were isolated from frozen-thawed TT. Flow cytometry analysis showed expression of SSC and pluripotent markers but not for transcription factor PLZF. The marker expression was similarly distributed between the 3 species of rhinoceros, with a larger portion of cells positive for SSEA-4 (mean%±s.e.m.: 6.3±1.1%) and smaller proportion for GDNFa1 (0.4±0.2%), GRP125 (0.1±0.1%), OCT-4 (0.4±0.2%), and SSEA-1 (0.4±0.2%; P<0.05). Similarly, horse expressed the markers GDNFa1 (0.9±0.3%), GRP125 (0.9±0.4%), OCT-4 (1.7±0.5%), and SSEA-1 (0.12±0.1%), but the abundance of SSEA-4 (1.8±0.8%) was less than that of rhinoceros (P<0.05). In rhinoceros, sequential culture enhanced the numbers of cells expressing all markers compared with that before culture, whereas in horses, the increase was observed only for cells expressing GDNFa1, GPR125, and SSEA-1. Reproductive stage did not affect the percentages of horse cells expressing germ cell and pluripotent markers. Overall, these results showed that rhinoceros SSC can be isolated from TT and expressed the same SSC markers as horses, and that differential culture enriched a population of SSC.

scholarly journals Stem Cell Therapy for Male Infertility: A Review

2016 ◽  
Vol 5 (2) ◽  
Author(s):  
Nathan Isaac Dibal ◽  
Musa Samaila Chiroma ◽  
Martha Orendu Attah
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Continuous Production ◽  
Genetic Material ◽  
Spermatogonial Stem Cells ◽  
Rete Testis ◽  
Reproductive Age ◽  
Seminiferous Tubules ◽  
Male Factor ◽  
Induced Pluripotent

Infertility affects 15% of couples in reproductive age world-wide and male factor is solely responsible in about 50% of the cases and contributory in 30-40% of cases. Spermatogonial stem cells (SSCs) are small self-renewing cells found in the basal compartment of seminiferous tubules where they form the foundation of spermatogenesis and are required for the continuous production of sperm. Transplantation of spermatogonial stem cells results in a donor derived sperm production and fertility in rodent and non-rodent species like Goat. Transplantation of cryopreserved spermatogonial stem cells could help oncology patients undergoing radiotherapy or chemotherapy by prior isolation of the SSCs and transplantation after treatment. Induced pluripotent stem cells also have the ability to differentiate into late stage germ cells. The efficacy and safety of SSCs transplantation showed that offspring produced did not show any morphological differences or alteration of genetic material but is most effective through assisted reproduction and better on young/immature Animals. The transfer of germ cells through micro-injection of seminiferous tubules and cannulation of efferent ducts is most effective on rodent testis while injection into the rete testis guided by ultrasound was reported to be the best technique in higher Animals (Bulls and Apes). Researches are still ongoing to get a safe and successful method of SSCs transplantation with no or less side effects on humans.Keywords: Cryopreserved, Pluripotent, self-renewing, Spermatogonia, and Spermatogenesis

scholarly journals Fertility preservation in boys: recent developments and new insights †

2020 ◽  
Vol 2020 (3) ◽  
Author(s):  
E Goossens ◽  
K Jahnukainen ◽  
RT Mitchell ◽  
AMM van Pelt ◽  
G Pennings ◽  
...  
Keyword(s):  
High Risk ◽  
Fertility Preservation ◽  
Patient Management ◽  
Experimental Methods ◽  
Testicular Tissue ◽  
Human Testis ◽  
Clinical Practices ◽  
Future Fertility ◽  
Prepubertal Boys ◽  
Tissue Cryopreservation

Abstract BACKGROUND Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS, REASONS FOR CAUTION The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTEREST(S) The work was funded by ESHRE. None of the authors has a conflict of interest.

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