Jan-bernd Stukenborg - Academia.edu (original) (raw)

Papers by Jan-bernd Stukenborg

Human Reproduction, Feb 14, 2018

Can a systematic scoring procedure provide crucial information on the status of highly heterogene... more Can a systematic scoring procedure provide crucial information on the status of highly heterogeneous immature human testicular tissues in the context of cryopreservation for fertility preservation? SUMMARY ANSWER: We developed a systematic histological score as a novel diagnostic tool which differentiates the patient cohort according to the status of germ cell differentiation and number of spermatogonia (normal, diminished and absent), and which could be relevant in the fertility clinic. WHAT IS KNOWN ALREADY: Cryopreservation of testicular tissue of immature boys is currently considered the option for future fertility restoration. However, experimental techniques for the derivation of sperm as well as valid diagnostic scoring of these immature testis tissues are not yet reported. STUDY DESIGN, SIZE, DURATION: Testicular tissues of 39 patients (aged 2-20 years) who attended our clinic for cryopreservation between 2010 and 2015 were analyzed to determine the variability of testicular tissue composition, germ cell numbers and differentiation status. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human testicular tissue samples were divided into three groups. Group NT included patients suffering from diseases which do not directly affect the testes (n = 6; aged 6-14 years), group AT included patients suffering from diseases that directly affect the testes (n = 14; 2-17 years), and group KS (Klinefelter patients, n = 19; 12-20 years). Based on immunohistochemical stainings for MAGEA4, the differentiation status as well as the numbers of gonocytes, spermatogonia and spermatocytes were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Testicular tissue samples from the NT group contained a mean of 100.3 spermatogonia/mm 3 (×10 3). Highly heterogeneous and significantly lower mean numbers of spermatogonia were scored in testes from boys after cytotoxic exposures or with pre-existing disease (AT group: 35.7 spermatogonia/mm 3 (×10 3); KS group: 1.8 spermatogonia/mm 3 (×10 3)). In addition, the germ cell differentiation status was determined and revealed tissues with either spermatogonia and gonocytes, only spermatogonia, spermatogonia and spermatocytes, or all three germ cell types were present. Based on spermatogonial numbers and differentiation status, we developed a germ cell score which we applied to each individual patient sample. LIMITATIONS REASONS FOR CAUTION: Normal human testicular tissue samples are difficult to obtain for ethical reasons and the sample numbers were small. However, six such samples provide a valid baseline for the normal situation.

International Journal of Andrology, Sep 28, 2019

Increasing rates of male infertility have led to a greater need for relevant model systems to gai... more Increasing rates of male infertility have led to a greater need for relevant model systems to gain further insight into male fertility and its failings. Spermatogenesis and hormone production occur within distinct regions of the testis. Defined by specialized architecture and a diverse population of cell types, it is no surprise that disruption of this highly organized microenvironment can lead to infertility. To date, no robust in vitro system has facilitated full spermatogenesis resulting in the production of fertilization-competent human spermatozoa. Here, we review a selection of current in vitro systems available for modelling the human testis microenvironment with focus on the progression of spermatogenesis and recapitulation of the testis microenvironment.

Leukemia, Mar 8, 2017

Cancer therapies are known to cause fertility impairment as a long-term treatment side effect. Al... more Cancer therapies are known to cause fertility impairment as a long-term treatment side effect. Although postpubertal male cancer patients have the possibility to cryopreserve semen before gonadotoxic treatment, prepubertal boys do not yet produce spermatozoa. 1 Therefore, proposed fertility-preservation strategies for these boys are cryopreservation of testicular tissue followed by autotransplantation of spermatogonial stem cells, tissue grafting or in vitro maturation. 1-3 Numerous animal studies, including those of non-human primates, have proven the successful accelerated recovery of spermatogenesis following the application of fertility-restoration techniques. Hence, at this moment, testicular tissue cryopreservation is offered to patients as a part of clinical research programs, while the fertility-restoration strategy remains experimental. 3 Male patients with leukaemia who are at a high risk of infertility include those receiving allogenic haematopoietic stem cell transplantation, while conventional chemotherapy is associated with a low risk of infertility. 4,5 Therefore, the majority of patients with haematological malignancies do not meet the criteria for testicular tissue cryopreservation at the time of the original diagnosis. However, disease response or relapse may result in patients undergoing potentially sterilizing therapy regimens. This means that many patients with leukaemia have already received chemotherapy before testicular tissue cryopreservation is offered. 1 The potential effect of previous chemotherapy must be taken into account when testis biopsy and storage are considered for these patients. In order to optimize the cryobanking of prepubertal testicular tissue, more information on the effects of cancer therapies on spermatogonial quantities is required. Recently, reference values concerning spermatogonial quantity in human testes throughout healthy prepuberty were established. 6 In the present study, these reference values were used to evaluate the effects of leukaemia treatment on spermatogonial quantity and hence spermatogenic recovery in prepubertal boys with acute lymphoblastic leukaemia. The study material consisted of testis samples from prepubertal boys with acute lymphoblastic leukaemia who had undergone routine bilateral/unilateral testicular biopsy to examine possible testicular leukaemia at the cessation of antileukaemia therapy at University Central Hospitals in Helsinki and Turku between 1979 and 1995. The Research Ethics Committees of Helsinki University Hospital (No 192/13/03/03/2013) and Turku University Hospital (DNR 1905-32/300/05) approved the study. Altogether, 37 boys were identified through hospital records and enrolled in the study. Eighteen of these boys had earlier been included in a study to evaluate the expression of spermatogonial markers during leukaemia therapy, as previously described. 7 Testicular biopsies were performed at the end of the treatment, during the last days of the per oral maintenance therapy. Four of the 37 boys were subjected to two biopsies-one at the end of induction therapy (50 days after diagnosis) and a second biopsy at the end of the treatment (Figure 1). Antileukaemia therapy was carried out as described earlier. 8,9 Briefly, the therapy involved the use of antimetabolites, vinca-alkaloids and anthracyclines. Of the 37

55th ESPE Meeting, Aug 19, 2016

Conclusions Different genes involved in fundamental processes within the testis were affected by ... more Conclusions Different genes involved in fundamental processes within the testis were affected by fetal hypoxia up to pubertal age, suggesting that long term alterations occur as a consequence of IUGR. Moreover, testosterone production was increased in the prepubertal rats, as putative catch-up growth mechanism. Further analyses are needed to elucidate later consequences of IUGR on testis function. Figure 2-TaqMan Low-Density Array analysis of gene expression in IUGR and sham animals at 5, 20 and 40 dpp. (A) Specific differences in gene expression between animal groups, with higher expression in IUGR vs sham animals, and (B) lower expression in IUGR vs sham animals.

Biology of Reproduction, Jul 1, 2009

Human Reproduction, Nov 1, 2019

Defects in neuronal migration cause brain malformations, which are associated with intellectual d... more Defects in neuronal migration cause brain malformations, which are associated with intellectual disability (ID) and epilepsy. Using exome sequencing, we identified compound heterozygous variants (p.Arg71His and p. Leu729ThrfsTer6) in TMTC3, encoding transmembrane and tetratricopeptide repeat containing 3, in four siblings with nocturnal seizures and ID. Three of the four siblings have periventricular nodular heterotopia (PVNH), a common brain malformation caused by failure of neurons to migrate from the ventricular zone to the cortex. Expression analysis using patient-derived cells confirmed reduced TMTC3 transcript levels and loss of the TMTC3 protein compared to parental and control cells. As TMTC3 function is currently unexplored in the brain, we gathered support for a neurobiological role for TMTC3 by generating flies with post-mitotic neuron-specific knockdown of the highly conserved Drosophila melanogaster TMTC3 ortholog, CG4050/tmtc3. Neuron-specific knockdown of tmtc3 in flies resulted in increased susceptibility to induced seizures. Importantly, this phenotype was rescued by neuron-specific

Human Reproduction, Jun 1, 2023

categories (Class I to V) according to their symptoms, ultrasound, HSG and hysteroscopy findings.... more categories (Class I to V) according to their symptoms, ultrasound, HSG and hysteroscopy findings. Participants/materials, setting, methods: Clinical data and operative findings were reviewed from patient files and video recordings. The number of hysteroscopic interventions needed to restore the cavity and the reproductive outcome in women who were desirous for pregnancy were recorded. Predictive power of the model was assessed using the live birth rate as the primary and rate of cavity restoration and number of interventions as the second outcome parameters. Groups were compared using ANOVA, ROC and regression analyses. Main results and the role of chance: Adhesions were classified as class I in 43 (15.3%), class II in 72 (25.6%), class III in 57 (20.3%), class IV in 82 (29.2%) and class V in 27 (9.6%) patients. They were due to previous curettages of pregnancies (79,7%) or retained products of gestation (3.9%), prior uterine surgery (6.8%), prior hysteroscopy of inappropriate technique (6.8%) and tuberculosis (2.8%). The cavity was septate in 12 and unicornuate in 2 patients. The mean age of the study group was 29.8 § 3.7 (20-40). Age was not related with the severity of adhesions (p ¼ 0.335). While the majority of patients with curettage-related adhesions were classified as Class II, uterine surgery, iatrogenic and tuberculosis related adhesions were higher in severity. The number of hysteroscopic adhesiolysis procedures (from 1.0 § 02. to 2.3 § 0.5) needed for optimal restoration of the cavity was directly related and the rate of full restoration (from 100%-18.5%) was indirectly related with the severity of adhesions according to the proposed classification (p ¼ 0.0001 for both). The live birth rates were 54.3%, 45%, 31.7%, 21% and 12.5% for patients in Class I to V, respectively (p ¼ 0.0001). The proposed classification was fairly predictive (AUC: 0.654, 95%CI: 0.582-0.727) for live birth. Limitations, reasons for caution: This is a retrospective analysis of consecutive patients with intrauterine adhesions in routine practice. The followup for reproductive outcome is limited. The study is hospital-based and single-center. Thus, the predictive value of the proposed classification needs to be validated in an external data set preferably in a prospective series. Wider implications of the findings: In patients with intrauterine adhesions, a classification system based on patient symptoms, imaging findings and hysteroscopic appearance of the uterine cavity reliably predicts the postoperative outcome in terms of the extent of anatomical restoration of the uterine cavity and pregnancy and live birth rates. Trial registration number: Not applicable

Frontier Media SA eBooks, 2022

Annals of Oncology, Dec 1, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Fertility and Sterility, Sep 1, 2021

Objective: To normalize age-dependent effects on standardized measures of spermatogonial quantity... more Objective: To normalize age-dependent effects on standardized measures of spermatogonial quantity such as the number of spermatogonia per tubular cross-section (S/T) or fertility index. Design: Published quantitative histologic data on human spermatogonial numbers were used to create Z-scores for reference means and tested on archived testicular tissue samples. Setting: Retrospective cohort study. Patient(s): The sample cohort comprised testicular samples from 24 boys with cancer diagnosis and 10 with Klinefelter syndrome, as part of the fertility preservation programs NORDFERTIL and Androprotect, as well as archived histologic samples from 35 prepubertal boys with acute lymphoblastic leukemia and 20 testicular biobank samples. Intervention(s): None. Main Outcome Measure(s): Z-score values for S/T and fertility index on the basis of morphology and germ cell-specific markers (MAGEA4 and/or DDX4) were calculated, and the impact of cancer therapy exposure and genetic disorders on Z-score values was evaluated. Result(s): The Z-scores for S/T values in the nontreated samples (À2.08 AE 2.20, n ¼ 28) and samples treated with nonalkylating agents (À1.90 AE 2.60, n ¼ 25) were comparable within AE3 standard deviations of the reference mean value but differed significantly from samples exposed to alkylating agents (À12.14 AE 9.20, n ¼ 22) and from patients with Klinefelter syndrome (À11.56 AE 4.89, n ¼ 8). The Z-scores for S/T were correlated with increasing cumulative exposure to alkylating agents (r ¼ À0.7020). Conclusion(s): The Z-score values for S/T allow for the quantification of genetic and cancer treatment-related effects on testicular tissue stored for fertility preservation, facilitating their use for patient counseling. (Fertil Steril Ò 2021;116:713-20. Ó2021 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo.

Haematologica, Dec 9, 2021

Disclosures: no conflicts of interest to disclose. Contributions: KMBF collected and analyzed dat... more Disclosures: no conflicts of interest to disclose. Contributions: KMBF collected and analyzed data, prepared figures, drafted the manuscript; NN and JBS prepared samples, set up the experimental design, collected, analyzed and interpreted data, and drafted the manuscript; AJ set up the experimental design and collected data; AKL, CL, HAMBO, JMDP, MS and VN collected data; CK and AS collected data and were responsible for clinical patient care, SK supervised clinical patient care, clinical and laboratory testing and documentation in the University Hospital of Munster; KJ set up the experimental design, collected and interpreted data, and drafted the manuscript. All authors had significant intellectual contribution in reviewing the manuscript and approved the final article.

Frontiers in Toxicology, 2022

Background: Retrospective studies in adult survivors of childhood cancer show long-term impacts o... more Background: Retrospective studies in adult survivors of childhood cancer show long-term impacts of exposure to alkylating chemotherapy on future fertility. We recently demonstrated germ cell loss in immature human testicular tissues following exposure to platinum-based chemotherapeutic drugs. This study investigated the effects of platinum-based chemotherapy exposure on the somatic Sertoli cell population in human fetal and pre-pubertal testicular tissues.Methods: Human fetal (n = 23; 14–22 gestational weeks) testicular tissue pieces were exposed to cisplatin (0.5 or 1.0 μg/ml) or vehicle for 24 h in vitro and analysed 24–240 h post-exposure or 12 weeks after xenografting. Human pre-pubertal (n = 10; 1–12 years) testicular tissue pieces were exposed to cisplatin (0.5 μg/ml), carboplatin (5 μg/ml) or vehicle for 24 h in vitro and analysed 24–240 h post-exposure; exposure to carboplatin at 10-times the concentration of cisplatin reflects the relative clinical doses given to patients. ...

Molecular Human Reproduction, 2020

Successful in vitro spermatogenesis was reported using immature mouse testicular tissues in a fra... more Successful in vitro spermatogenesis was reported using immature mouse testicular tissues in a fragment culture approach, raising hopes that this method could also be applied for fertility preservation in humans. Although maintaining immature human testicular tissue fragments in culture is feasible for an extended period, it remains unknown whether germ cell survival and the somatic cell response depend on the differentiation status of tissue. Employing the marmoset monkey (Callithrix jacchus), we aimed to assess whether the maturation status of prepubertal and peri-/pubertal testicular tissues influence the outcome of testis fragment culture. Testicular tissue fragments from 4- and 8-month-old (n = 3, each) marmosets were cultured and evaluated after 0, 7, 14, 28 and 42 days. Immunohistochemistry was performed for identification and quantification of germ cells (melanoma-associated antigen 4) and Sertoli cell maturation status (anti-Müllerian hormone: AMH). During testis fragment cu...

MHR: Basic science of reproductive medicine, 2017

Can enzymatically dispersed testicular cells from adult men reassemble into seminiferous cord-lik... more Can enzymatically dispersed testicular cells from adult men reassemble into seminiferous cord-like structures in vitro? SUMMARY ANSWER: Adult human testicular somatic cells reassembled into testicular cord-like structures via dynamic interactions of Sertoli and peritubular cells. WHAT IS KNOWN ALREADY: In vitro approaches using dispersed single cell suspensions of human testes to generate seminiferous tubule structures and to initiate their functionality have as yet shown only limited success. STUDY DESIGN, SIZE, DURATION: Testes from 15 adult gender dysphoria patients (mean ± standard deviation age 35 ± 9.3 years) showing spermatogonial arrest became available for this study after sex-reassignment surgery. In vitro primary testicular somatic cell cultures were generated to explore the self-organizing ability of testicular somatic cells to form testis cords over a 2-week period. Morphological phenotype, protein marker expression and temporal dynamics of cell reassembly were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cell suspensions obtained by two-step enzymatic digestion were plated onto glass coverslips in 24-well plates. To obtain adherent somatic cells, the supernatant was discarded on Day 2. The culture of the attached cell population was continued. Reassembly into cord-like structures was analyzed daily by microscopic observations. Endpoints were qualitative changes in morphology. Cell types were characterized by phase-contrast microscopy and immunohistochemistry. Dynamics of cord formation were recorded by time-lapse microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: Primary adult human testicular cells underwent sequential morphological changes including compaction and reaggregation resulting in round or elongated cord-like structures. Time-lapse video recordings within the first 4 days of culture revealed highly dynamic processes of migration and coalescence of reaggregated cells. The cellular movements were mediated by peritubular cells. Immunohistochemical analysis showed that both SRY-related high mobility box 9-positive Sertoli and α-smooth muscle actin-positive peritubular myoid cells interacted and contributed to cord-like structure formation. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Owing to scarcity of normal human testicular tissue, testes from gender dysphoria patients were used in the study. The regressed status might influence the experimental responses of primary cells. We observed basic morphological features resembling in vivo testicular cords, however, the proof of functionality (e.g. support of germ cells) will need further studies. WIDER IMPLICATIONS OF THE FINDINGS: The proposed in vitro culture system may open opportunities for examination of testicular cell interactions during testicular tubulogenesis. Further refinement of our approach may enable initiation of ex vivo spermatogenesis.

L'Endocrinologo, 2013

C. Kollin, J.B. Stukenborg, M. Nurmio, E. Sundqvist, T. Gustafsson, O. Söder, J. Toppari, A. Nord... more C. Kollin, J.B. Stukenborg, M. Nurmio, E. Sundqvist, T. Gustafsson, O. Söder, J. Toppari, A. Nordenskjöld, E. M. Ritzén J Clin Endocrinol Metab 2012; 97: 4588-4595 L’incompleta discesa dei testicoli è un’anomalia molto comune nei neonati. La prevalenza nei nati a termine varia dal 2 al 9%, riducendosi all’1-3% a 3 mesi di età per la discesa spontanea. A un anno di vita il criptorchidismo monolaterale costituisce circa l’85% di tutti i casi. Questa condizione non dovrebbe essere trascurata, in quanto associata ad aumentato rischio di sviluppo di tumore testicolare e infertilità. Tuttavia, la tempistica del trattamento chirurgico è stata controversa per molto tempo. Sulla base di studi istopatologici è stato suggerito che l’intervento chirurgico dovrebbe essere effettuato entro 1-2 anni di vita, poiché dopo tale età il numero di spermatogoni si riduce significativamente nei testicoli criptorchidi. Questo è uno studio randomizzato controllato svolto confrontando i risultati dell’intervento chirurgico per criptorchidismo congenito effettuato a 9 mesi o 3 anni di età. Scopo dello studio è stato quello di valutare se l’esecuzione dell’intervento chirurgico a 9 mesi di vita sia più vantaggiosa rispetto all’intervento a 3 anni di età e di identificare marcatori ormonali precoci importanti per lo sviluppo testicolare. Sono state eseguite 213 biopsie durante l’orchidopessi e sono stati analizzati il numero di cellule germinali e cellule di Sertoli per 100 sezioni trasversali di tubuli seminiferi e l’area della superficie tubulare e di tessuto interstiziale. Sono stati determinati i livelli di inibina B, FSH, LH e testosterone. Il volume testicolare è stato valutato ecograficamente e per mezzo di un righello prima della biopsia. Il numero di cellule germinali e di Sertoli e il volume testicolare a 9 mesi di vita erano significativamente maggiori che a 3 anni di età. I testicoli intra-addominali mostravano la maggiore deplezione di cellule di Sertoli a 3 anni. La deplezione cellulare nei bambini criptorchidi è risultata maggiore rispetto a quella fisiologica in soggetti di pari età. A entrambe le età il volume testicolare correlava con il numero di cellule germinali e di Sertoli. Nessuno degli ormoni valutati durante i primi 6 mesi di vita (LH, FSH, testosterone e inibina B) era in grado di predire il numero di cellule germinali e di Sertoli a 9 o a 36 mesi di età né di predire la discesa testicolare spontanea. In conclusione, i dati morfometrici e volumetrici mostrano che l’orchidopessi a 9 mesi di vita è più vantaggiosa per il successivo sviluppo testicolare rispetto all’intervento eseguito a 3 anni di età. I risultati istologici hanno mostrato che a 9 mesi di vita i testicoli ritenuti hanno un numero significativamente maggiore di cellule germinali e di Sertoli, un maggiore diametro dei tubuli seminiferi e un maggior rapporto tubuli/tessuto interstiziale rispetto ai 3 anni di età. Il volume testicolare, inoltre, rifletteva il numero di cellule germinali nella prima infanzia, mentre i parametri ormonali non erano in grado di predire la struttura cellulare del testicolo o una discesa spontanea. Questo studio evidenzia per la prima volta come anche nella prima infanzia – come già noto nell’adulto – vi sia una correlazione tra volume testicolare e attività spermatogenetica. Il follow-up mediante esame del liquido seminale dei ragazzi precedentemente criptorchidi potrà fornire importanti dati circa il potenziale di fertilità.

Human Reproduction Open, 2020

Goossens et al. are being evaluated in mouse and primate models. However, important practical, me... more Goossens et al. 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 coordinating 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 multidisciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss.

Reproduction and Fertility

Testicular samples obtained for fertility preservation often need to be transported between clini... more Testicular samples obtained for fertility preservation often need to be transported between clinics. This study aimed to mimic this short-term hypothermic storage (4–8°C) and explore the impact of these conditions and the transport medium composition on prepubertal rat testicular tissue samples. Testicular tissue samples obtained from 7 days post-partum rats were transferred to six compositionally different basal culture media and a balanced salt solution, which had been kept at 4–8°C prior to transfer. The samples were preserved for either 12 or 24 h in these hypothermic conditions. The potential effects of the short-term storage were evaluated by assessing the morphology, measuring the testosterone levels by radioimmunoassay and analysing 96 genes with TaqMan Low-Density Arrays. Levels of gene expression related to energy, apoptosis, and angiogenesis pathways were altered after hypothermic storage for 12 and especially 24 h. We observed only minor differences in gene expression pr...

Frontiers in Endocrinology

Editorial on the Research Topic: Research advances in male fertility: New horizons for investigat... more Editorial on the Research Topic: Research advances in male fertility: New horizons for investigating human testicular function and development of clinical fertility preservation approaches The topic for this special issue on Research Advance in Male Fertility was defined and initiated by the three authors in 2021 inviting submissions to address a broad range of original research papers and reviews. Ten submissions involving 64 authors were published after peer review between October 2021 and July 2022. The accepted two reviews and eight original papers present a wide array of themes and topics describing new and exciting strategies from basic science to clinical applications (Figure 1). As of mid-August 2022, the submissions attracted already 12.000 views and 4.000 downloads of the open access submissions. The early response reveals that male fertility preservation is currently a hot topic in the field of reproductive medicine. This is because poor male reproductive health leads to nearly half of failed pregnancy attempts. Declining semen quality over the past 50 years indicates the role of changing lifestyle and exposure to environmental and toxic components as potential contributing factors. Sperm retrieval is feasible in adult infertile men by using surgical procedures like TESE (Testicular sperm extraction) and can be combined with cryopreserving semen samples for future Assisted Reproductive Technologies (ART) treatments. Prepubertal patients undergoing gonadotoxic treatment to cure malignant or non-malignant diseases or patients with specific genetic Frontiers in Endocrinology frontiersin.org 01