A 20-year overview of fertility preservation in boys: new insights gained through a comprehensive international survey.

STUDY QUESTION: Twenty years after the inception of the first fertility preservation programme for pre-pubertal boys, what are the current international practices with regard to cryopreservation of immature testicular tissue? SUMMARY ANSWER: Worldwide, testicular tissue has been cryopreserved from over 3000 boys under the age of 18 years for a variety of malignant and non-malignant indications; there is variability in practices related to eligibility, clinical assessment, storage, and funding. WHAT IS KNOWN ALREADY: For male patients receiving gonadotoxic treatment prior to puberty, testicular tissue cryopreservation may provide a method of fertility preservation. While this technique remains experimental, an increasing number of centres worldwide are cryopreserving immature testicular tissue and are approaching clinical application of methods to use this stored tissue to restore fertility. As such, standards for quality assurance and clinical care in preserving immature testicular tissue should be established. STUDY DESIGN SIZE DURATION: A detailed survey was sent to 17 centres within the recently established ORCHID-NET consortium, which offer testicular tissue cryopreservation to patients under the age of 18 years. The study encompassed 60 questions and remained open from 1 July to 1 November 2022. PARTICIPANTS/MATERIALS SETTING METHODS: Of the 17 invited centres, 16 completed the survey, with representation from Europe, Australia, and the USA. Collectively, these centres have cryopreserved testicular tissue from patients under the age of 18 years. Data are presented using descriptive analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Since the establishment of the first formal fertility preservation programme for pre-pubertal males in 2002, these 16 centres have cryopreserved tissue from 3118 patients under the age of 18 years, with both malignant (60.4%) and non-malignant (39.6%) diagnoses. All centres perform unilateral biopsies, while 6/16 sometimes perform bilateral biopsies. When cryopreserving tissue, 9/16 centres preserve fragments sized ≤5 mm3 with the remainder preserving fragments sized 6-20 mm3. Dimethylsulphoxide is commonly used as a cryoprotectant, with medium supplements varying across centres. There are variations in funding source, storage duration, and follow-up practice. Research, with consent, is conducted on stored tissue in 13/16 centres. LIMITATIONS REASONS FOR CAUTION: While this is a multi-national study, it will not encompass every centre worldwide that is cryopreserving testicular tissue from males under 18 years of age. As such, it is likely that the actual number of patients is even higher than we report. Whilst the study is likely to reflect global practice overall, it will not provide a complete picture of practices in every centre. WIDER IMPLICATIONS OF THE FINDINGS: Given the research advances, it is reasonable to suggest that cryopreserved immature testicular tissue will in the future be used clinically to restore fertility. The growing number of patients undergoing this procedure necessitates collaboration between centres to better harmonize clinical and research protocols evaluating tissue function and clinical outcomes in these patients. STUDY FUNDING/COMPETING INTERESTS: K.D. is supported by a CRUK grant (C157/A25193). R.T.M. is supported by an UK Research and Innovation (UKRI) Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health at the University of Edinburgh is supported by MRC (MR/N022556/1). C.L.M. is funded by Kika86 and ZonMW TAS 116003002. A.M.M.v.P. is supported by ZonMW TAS 116003002. E.G. was supported by the Research Program of the Research Foundation-Flanders (G.0109.18N), Kom op tegen Kanker, the Strategic Research Program (VUB_SRP89), and the Scientific Fund Willy Gepts. J.-B.S. is supported by the Swedish Childhood Cancer Foundation (TJ2020-0026). The work of NORDFERTIL is supported by the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115), the Swedish Research Council (2018-03094; 2021-02107), and the Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2021-00073; 2022-00317; 2023-00353). C.E is supported by the Health Department of the Basque Government (Grants 2019111068 and 2022111067) and Inocente Inocente Foundation (FII22/001). M.P.R. is funded by a Medical Research Council Centre for Reproductive Health Grant No: MR/N022556/1. A.F. and N.R. received support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. K.E.O. is funded by the University of Pittsburgh Medical Center and the US National Institutes of Health HD100197. V.B-L is supported by the French National Institute of Cancer (Grant Seq21-026). Y.J. is supported by the Royal Children's Hospital Foundation and a Medical Research Future Fund MRFAR000308. E.G., N.N., S.S., C.L.M., A.M.M.v.P., C.E., R.T.M., K.D., M.P.R. are members of COST Action CA20119 (ANDRONET) supported by COST (European Cooperation in Science and Technology). The Danish Child Cancer Foundation is also thanked for financial support (C.Y.A.). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Gonadal development and function after immature testicular tissue banking as part of high-risk gonadotoxic treatment.

BACKGROUND: Experimental fertility preservation programs have been started to safeguard the future fertility of prepubertal and pubertal males requiring high-risk gonadotoxic treatment protocols. However, long-term follow-up studies evaluating the effects on their gonadal development and function related to the testicular biopsy procedure are rather limited. DESIGN: This two-center follow-up study (between 2002 and 2020) evaluated the gonadal development and function of a cohort of 59 prepubertal and pubertal males who have been offered immature testicular tissue banking (TTB) prior to conventional high-risk chemo- and/or radiotherapy (HR-C/R) or conditioning therapy before hematopoietic stem cell transplantation (CT-HSCT). The aim is to investigate the long-term impact of the testicular biopsy procedure and the high-risk gonadotoxic treatment. Testicular growth and the reproductive hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and inhibin B (INHB) were analyzed after treatment completion, and compared between males accepting TTB and those refusing TTB (control) as well as between HR-C/R and CT-HSCT treatment protocols. RESULTS: Of the 59 prepubertal and pubertal males included, 25 were treated by HR-C/R and 34 required CT-HSCT. TTB was accepted for 39 males and refused for 20 males. Most patients were prepubertal at diagnosis (85%), at TTB (79%), and at treatment completion (76%), and pubertal or postpubertal at their last follow-up visit (66%). After 5.0 (1.0-13.0) years post treatment, most patients show normal testicular volumes (83%) and normal LH (89%), FSH (87%), T (87%), and INHB (79%) serum levels. The testicular biopsy procedure did not have an effect on testicular growth, LH, FSH, T, and INHB. Significantly more small postpubertal testicular volumes (p = .0278) and low INHB serum levels (p = .0130) were recorded after CT-HSCT, especially after myeloablative conditioning. CONCLUSION: The clinical follow-up data demonstrate no effect related to the biopsy procedure, but a substantial risk for impaired gonadal development after high-risk gonadotoxic treatment, in particular myeloablative CT-HSCT. Longer follow-up studies with a larger study population are needed to confirm these preliminary findings.

Testicular Tissue Banking for Fertility Preservation in Young Boys: Which Patients Should Be Included?

Due to the growing number of young patients at risk of germ cell loss, there is a need to preserve spermatogonial stem cells for patients who are not able to bank spermatozoa. Worldwide, more and more clinics are implementing testicular tissue (TT) banking programs, making it a novel, yet indispensable, discipline in the field of fertility preservation. Previously, TT cryopreservation was predominantly offered to young cancer patients before starting gonadotoxic chemo- or radiotherapy. Nowadays, most centers also bank TT from patients with non-malignant conditions who need gonadotoxic conditioning therapy prior to hematopoietic stem cell (HSCT) or bone marrow transplantation (BMT). Additionally, some centers include patients who suffer from genetic or developmental disorders associated with prepubertal germ cell loss or patients who already had a previous round of chemo- or radiotherapy. It is important to note that the surgical removal of TT is an invasive procedure. Moreover, TT cryopreservation is still considered experimental as restoration methods are not yet clinically available. For this reason, TT banking should preferably only be offered to patients who are at significant risk of becoming infertile. In our view, TT cryopreservation is recommended for young cancer patients in need of high-risk chemo- and/or radiotherapy, regardless of previous low-risk treatment. Likewise, TT banking is advised for patients with non-malignant disorders such as sickle cell disease, beta-thalassemia, and bone marrow failure, who need high-risk conditioning therapy before HSCT/BMT. TT retrieval during orchidopexy is also proposed for patients with bilateral cryptorchidism. Since patients with a medium- to low-risk treatment generally maintain their fertility, TT banking is not advised for this group. Also for Klinefelter patients, TT banking is not recommended as it does not give better outcomes than a testicular sperm extraction later in life.

Testicular biopsy for fertility preservation in early-diagnosed Klinefelter patients: patient characteristics and long-term follow-up.

RESEARCH QUESTION: Which early-diagnosed Klinefelter syndrome patients have been offered cryopreservation of testicular tissue as part of fertility preservation before spermatogonial stem cell (SSC) loss? Do these Klinefelter syndrome patients present with behavioural, cognitive and/or psychological problems? Does a testicular biopsy procedure have long-term effects on the gonadal development of Klinefelter syndrome patients? DESIGN: Early-diagnosed Klinefelter syndrome patients followed between 2009 and 2020 and offered testicular tissue banking in an experimental context at the Universitair Ziekenhuis Brussel were included. The prevalence of behavioural, cognitive and/or psychological problems was determined. Changes in testicular volume and in gonadal function (LH, FSH, testosterone and inhibin B [INHB]) were studied. RESULTS: Of the 48 Klinefelter syndrome patients included, 22 had testicular tissue removed (biopsy group) and 26 had no surgical intervention (control group). The need for specialized education was significantly higher in prenatally (P = 0.0159) and prepubertally (P = 0.0002) diagnosed Klinefelter syndrome patients. Psychological problems were significantly more prevalent in Klinefelter syndrome patients who did not opt for fertility preservation (P = 0.0447). In the first 4.2 (1.9-9.1) years after testicular biopsy, no difference in testicular volume was observed between the biopsied and the contralateral non-biopsied testes (P > 0.9999). After pubertal onset, no differences in LH, FSH, testosterone and INHB were found between the biopsy and the control groups (P = 0.1324 for LH, P > 0.9999 for FSH, P = 0.5433 for testosterone, P > 0.9999 for INHB). CONCLUSION: Early-diagnosed Klinefelter syndrome patients presented with behavioural, cognitive and/or psychological problems. Only psychological problems seemed to influence the decision towards fertility preservation. Follow-up data confirm that harvesting testicular tissue does not have a long-term impact on the gonadal development of Klinefelter syndrome patients.

Setting Up a Cryopreservation Programme for Immature Testicular Tissue: Lessons Learned After More Than 15 Years of Experience.

Young boys undergoing gonadotoxic treatments are at high risk of spermatogonial stem cell (SSC) loss and fertility problems later in life. Stem cell loss can also occur in specific genetic conditions, eg, Klinefelter syndrome (KS). Before puberty, these boys do not yet produce sperm. Hence, they cannot benefit from sperm banking. An emerging alternative is the freezing of testicular tissue aiming to preserve the SSCs for eventual autologous transplantation or in vitro maturation at adult age. Many fertility preservation programmes include cryopreservation of immature testicular tissue, although the restoration procedures are still under development. Until the end of 2018, the Universitair Ziekenhuis Brussel has frozen testicular tissues of 112 patients between 8 months and 18 years of age. Testicular tissue was removed in view of gonadotoxic cancer treatment (35%), gonadotoxic conditioning therapy for bone marrow transplantation (35%) or in boys diagnosed with KS (30%). So far, none of these boys had their testicular tissue transplanted back. This article summarizes our experience with cryopreservation of immature testicular tissue over the past 16 years (2002-2018) and describes the key issues for setting up a cryopreservation programme for immature testicular tissue as a means to safeguard the future fertility of boys at high risk of SSC loss.

Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics.

OBJECTIVE: To assess whether testicular cell dynamics are altered during long-term culture after testicular tissue cryopreservation. DESIGN: Experimental basic science study. SETTING: Reproductive biology laboratory. PATIENT(S): Testicular tissue with normal spermatogenesis was obtained from six donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Detection and comparison of testicular cells from fresh and frozen tissues during long-term culture. RESULT(S): Human testicular cells derived from fresh (n = 3) and cryopreserved (n = 3) tissues were cultured for 2 months and analyzed with quantitative reverse-transcription polymerase chain reaction and immunofluorescence. Spermatogonia including spermatogonial stem cells (SSCs) were reliably detected by combining VASA, a germ cell marker, with UCHL1, a marker expressed by spermatogonia. The established markers STAR, ACTA2, and SOX9 were used to analyze the presence of Leydig cells, peritubular myoid cells, and Sertoli cells, respectively. No obvious differences were found between the cultures initiated from fresh or cryopreserved tissues. Single or small groups of SSCs (VASA(+)/UCHL1(+)) were detected in considerable amounts up to 1 month of culture, but infrequently after 2 months. SSCs were found attached to the feeder monolayer, which expressed markers for Sertoli cells, Leydig cells, and peritubular myoid cells. In addition, VASA(-)/UCHL1(+) cells, most likely originating from the interstitium, also contributed to this monolayer. Apart from Sertoli cells, all somatic cell types could be detected throughout the culture period. CONCLUSION(S): Testicular tissue can be cryopreserved before long-term culture without modifying its outcome, which encourages implementation of testicular tissue banking for fertility preservation. However, because of the limited numbers of SSCs available after 2 months, further exploration and optimization of the culture system is needed.