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Cancer affected prepubertal boys will undergo testicular biopsy for testicular fragments cryopreservation. The biopsy will be performed by an attending urologist, before any gonadotoxic therapy is initiated. Testicular biopsy specimens will be divided immediately on the operating table. Two thirds of the specimen will be cryopreserved for potential use by the patient at a later date and will have the patient's details and will stay at the sperm bank of Hadassah Medical Center. The other third of specimen will be used for research purposes in an effort to advance the science of isolating and culturing human SSC's for fertility research.

An open testicular biopsy will be performed by an attending urologist under general anesthesia.

Testicular Biopsy

Doron Kabiri, MD

Due to remarkable advances in cancer treatments, the investigators are witnessing a growing population of long-term survivors of childhood malignancies. However, fertility in adult life may be severely impaired by gonadotoxic therapies. Since prepubertal boys cannot produce spermatozoa, banking of testicular tissue prior to gonadotoxic treatment is a crucial step towards fertility preservation for this population. Several centers around the world are now cryopreserving testicular tissue for prepubertal boys in anticipation that future technologies will allow the utilization of the banked samples for fertility restoration.

Testicular tissue cryopreservation has now emerged as the leading strategy for fertility preservation in prepubertal boys before gonadotoxic treatments. Fertility restoration can theoretically be obtained after allo-transplantation of testicular tissue fragments that preserve both the SSCs and the supporting microenvironment (spermatogonial stem-cell niche).

As treatment regimens for pediatric oncologic malignancies have improved, more and more survivors are entering their reproductive years. Since gonadal damage is a relatively common and unfortunate consequence of the treatments used to cure pediatric cancer as well as certain hematological conditions and immunodeficiencies, maintenance of fertility is extremely important with regard to long-term quality of life for these survivors. Consideration must be given to whether a child's fertility is likely to be impacted by his or her treatment. Ideally, this should occur before the start of therapy, when a window of opportunity may exist to preserve the patient's future reproductive potential. Developments in the area of sperm banking and reproductive technologies have made it possible to offer viable options to preserve fertility to pubertal males undergoing cancer therapy. Pubertal males can produce a semen sample prior to starting gonadotoxic therapy and cryopreserve the sperm for future use. Because current methods of oocyte fertilization can utilize as few as one motile sperm, this method has proven to be successful even when the number of sperm, which are frozen, is small. Unfortunately, prepubertal males pose a particular challenge for fertility preservation. These boys cannot produce semen for cryopreservation by masturbation. They also do not have mature spermatozoa. Although the prepubertal testis does not produce mature spermatozoa, it does contain the diploid stem-germ cells from which haploid spermatozoa will be derived. For these at risk prepubertal boys, as well as for pubertal boys who may not be able to sperm bank, a recent and encouraging approach is the use of cryopreserved testicular tissue. While important strides have been made in animal research in this area, the use of testicular tissue cryopreservation in humans remains experimental. If prepubertal testicular tissue could be acquired and banked before starting gonadotoxic therapy, this tissue could then be thawed and the stored germ cells reimplanted into the patient's own testes, a procedure known as germ-cell transplantation. Alternatively, the stored cells could be matured in vitro until they can achieve fertilization by use of intracytoplasmic sperm injection (ICSI).

Research with animal models has demonstrated that there are several methods to use germ cells to obtain mature spermatozoa for fertilization including autotransplantation, and xenotransplantation. Autotransplantation is considered more acceptable than xenotransplantation, although both have been successful in mouse models. In rodents, autotransplantation has resulted in restored spermatogenesis and mice have reproduced in vivo. Investigators have demonstrated that microinjecting a crude suspension of germ cells into mice rendered sterile can restore spermatogenesis and fertility. Similar success has been reported in rats and larger mammals. Restoration of spermatogenesis is also possible with cryopreserved cells. It has been estimated that 70% of spermatogonial cells can survive after freezing and thawing. Malignant contamination remains one of the main concerns surrounding transplantation, though the few studies that address this issue are controversial in their findings. Progress in addressing this question has been made in mouse models, particularly with the use of fluorescence-activated cell sorting to negatively sort malignant cells from cell suspensions.

Because cryopreservation and transplantation of SSCs is currently successful in animals, application in humans seems likely in the near future. The possibility of infertility is an issue many families need to grapple with as they agree to initiate chemotherapy on their sons. Since this method requires testicular biopsies, both parental desire and the acceptability of SSC collection are important to assess. Recently, researchers interviewed 318 parents regarding their acceptance of such an idea. They asked parents to think hypothetically about the following scenario, "If there was an experimental procedure available at diagnosis, would you allow your sons to undergo a testicular biopsy in an attempt to collect SSCs?" At diagnosis, SSC collection by means of testicular biopsy was theoretically approved by 61% of these parents. They also observed that the desire to cryopreserve SSCs was not related to potential harmfulness of the oncological treatment, indicating that even a minor chance of infertility was considered as a major burden in respect to the ultimate quality of life. These data indicate that the translation of current animal experiments on SSC collection and transplantation into clinical care is desired by parents from prepubertal boys with cancer.

This is a novel, experimental protocol that requires collaborations between clinicians and basic scientists. There are several key clinical and translational points that have to be considered, including the beliefs and concerns of parents and prepubertal boys with cancer, the ability to perform a testicular biopsy adequately without negative sequelae, as well as tissue storage for fertility and research purposes, notably the viability of biopsy samples for downstream in vitro and functional genomics studies.

The goal of the present proposal is to develop clinical, translational, and basic science initiatives which will allow for an extensive, dedicated program for the acquisition and storage of testicular tissue from prepubertal males seen at Hadassah Medical center as well as at collaborating institutions in Israel and across the world. Developing the means for tissue distribution and handling as well as long-term tissue storage are critical components, especially since testicular tissue might be decades in storage prior to use in a fertility setting. Most importantly, a cryopreservation protocol for human testicular tissue has to be developed de novo for this initiative to ultimately become a standard option for fertility preservation for prepubertal males with cancer as well as for those with blood diseases or immunodeficiency who will be undergoing stem cell transplant.

An open testicular biopsy will be performed by an attending urologist (effort will be made to perform the procedure concurrently during a procedure when the patient is under general anesthesia for another purpose, i.e. central line placement, bone marrow aspirates/biopsies). This procedure will occur before any gonadotoxic therapy is initiated. Nevertheless, in cases of emergent chemotherapy treatment, the procedure may be done after chemotherapy since the toxicity for testicular stem cells is not known. A 10 mm incision is made in the superior pole of the testis and an approximately 5% portion of the extruded seminiferous tubules is excised (about 2 mm x 4 mm x 10 mm; approximately 1 gram of tissue). This is the size of a normal testis biopsy done for clinical reasons such as evaluation for cancer. In infants or very young boys the size of the incision and the biopsy is under the discretion of the urologist.

Testicular biopsy specimens will be divided immediately on the operating table. Two thirds of the specimen (termed Specimen #1) will be cryopreserved in Sperm Wash Media for potential use by the patient at a later date. A freezing protocol (slow freezing) currently in place for adult human testicular tissue cryopreservation will be implemented for the current proposal, and has been published in the literature for use with prepubertal testicular tissue. Specifically, 0.7 M dimethyl sulfoxide (DMSO) is used as a cryoprotective agent. Specimen #1 of the biopsy will have the patient's name, date of birth, current date, and medical record number attached. A separate sheet will also be sent with the specimen who will include phone number and current address. These specimens will stay at the sperm bank of Hadassah Medical Center.

As stated above, the other third of specimen will be used for research purposes in an effort to advance the science of isolating and culturing human SSCs for fertility research. These portions will be stripped of all personal health identifiers at the time of division and a unique identifying code will be assigned.

The risks of this study pertain to the testicular biopsy. The side effects from this procedure are mild. Pain related to the procedure is expected and is easily controlled with mild to moderate analgesic medications.

The benefit is that the patient may be able to use the frozen testicular biopsy tissue and/or cultured SSC's derived from the biopsy to preserve their fertility in the future. At this time, however, it is imperative to note that this procedure is purely experimental. The time frame for this study is defined from the date of recruitment until the date of clinical utilization of the cryopreserved tissue for fertility restoration up to forty years.

We will collect any adverse outcomes, which occur during (intra-operatively) or after the testicular biopsy including pain, bleeding and/or infection. Events will be tracked and recorded for one week after the procedure, a time frame well beyond the 2-3 day recovery time that is associated with this type of biopsy. Events clearly related to the subject's cancer or cancer treatment and not specifically to the testicular biopsy will not be collected/reported.

Fertility Preservation in Prepubertal Boys: An Experimental Approach

The investigators will report the number of cancer affected prepubertal boys that will undergo testicular cryopreservation for future fertility restoration.

Hadassah Medical Organization

Patients meeting eligibility criteria are offered experimental procedure of testicular biopsy for clinical storage and use in laboratory research. The intervention does not involve a drug or device.

Testicular biopsy for clinical storage and laboratory research.

Rod T Mitchell, MBChB

Mark Brougham, MBChB

Prepubertal boys treated for cancer are at risk of infertility as a result of their treatment. At present there are no options to preserve fertility in these boys. With informed consent, the investigators aim to cryopreserve (freeze) testis tissue biopsies taken from boys undergoing cancer therapy with a high risk of infertility. A portion of cryopreserved tissue will be securely stored, which in the future may be used to restore fertility in the patients. A second portion of the tissue will be used for laboratory work aimed at understanding the conditions required for development of the germ cells.

Patients will be recruited from the Oncology Department at the Royal Hospital for Children and Young People (RHCYP) in Edinburgh. Tissue will be obtained from prepubertal patients with cancer who are about to undergo treatment with a high risk (\>80%) of infertility. Informed consent will be obtained from the patients legal guardian (and patient if applicable) for the procedure and subsequent use of tissue. A testis biopsy from one testis will be performed and if possible will coincide with a planned routine procedure requiring general anaesthetic in order to avoid the need for additional anaesthesia.

The procedure will take place at RHCYP during the initial assessment and prior to the commencement of cancer treatment and will not result in delay of treatment. Tissue will be assessed for the presence of sperm for storage using existing methods. In addition, tissue will be cryopreserved by Tissue and Cells Directorate, SNBTS in Edinburgh. The tissue will be stored for up to 55 years in accordance with current legislation (HFEA Act as amended 2005). Research will be carried out at the MRC Centre for Reproductive Health in accordance with Human Tissue (Scotland) Act 2006.

Objectives

1. Primary Objective

   a) Cryopreservation of testicular tissue from pre- and peri-pubertal patients undergoing potentially sterilising therapy for future therapeutic use to preserve fertility.

   i. Development/refinement of inclusion and exclusion criteria ii. Exploration of patients and parents attitudes to this approach to fertility preservation iii. Development of the required multidisciplinary group to provide this
2. Secondary Objectives

   1. Follow-up patients after testis tissue cryopreservation
   2. Laboratory research - testicular germ cell development for fertility preservation i. Development of optimal freezing methods for testicular tissue ii. In-vitro culture methods for germ cell maturation iii. Development of xenografting approaches for maturation of germ cells

Design Prospective cohort study of pre- and peri-pubertal boys

Study Population Pre- and peri-pubertal boys due to undergo therapy with high risk of subsequent infertility

Main Study Endpoint Successful long-term cryostorage of testicular tissue for potential future use in fertility preservation, and follow-up of patients for effects of testicular biopsy on subsequent endocrine function

Establishment of laboratory techniques for the development of methods to support long term storage of tissue and methods for in vivo/in vitro spermatogenesis

Methods

Patient Recruitment Patients will be recruited from the Oncology Department at the RHCYP in Edinburgh. Testicular cryopreservation will be offered to eligible patients providing they meet the inclusion criteria. Age appropriate patient information sheets will be provided to the patients.

Patient Consent Consent will be for testis tissue cryopreservation and storage. Patients will also have the option of allowing tissue to be used for research purposes, however declining this aspect will not prevent them from consenting to cryopreservation of tissue. Due to the young age and immaturity of the study patients, written informed consent for a testicular biopsy under general anaesthetic will be obtained from the parents/guardian of the patient, unless the patients are considered able to give fully informed consent themselves. Patient assent will be sought from those boys not considered competent to provide written informed consent due to their young age.

Surgical Procedure The testicular biopsy will take place in operating theatres at the RHCYP before cancer gonadotoxic treatment begins. This will, whenever possible, coincide with a planned routine procedure requiring general anaesthetic in order to avoid the need for additional anaesthesia, hence this will not result in delay to commencing treatment. A blood sample will be taken for viral screening which is a requirement for tissue storage. The blood sample will be stored to allow further testing for infectious diseases as required over time by Tissues Services and the HTA.

Testicular volume will be assessed by visual inspection and manual palpation at surgery. The amount of tissue removed will typically be approximately 0.6-0.8mls from one testis, and never in excess of 50% of the total volume of a single testis. The procedure will involve a unilateral open testicular biopsy through a scrotal incision.

Since treatment with cytotoxic therapy will not have started prior to the biopsy, the risk of infection is minimal. In patients with solid tumours bleeding is unlikely to be a major problem since platelet numbers and function are generally normal in these patients. In patients with leukaemia thrombocytopenia is likely to be present at diagnosis, but haemostasis is routinely secured by platelet transfusion for operative procedures in these patients.

The biopsy will be taken for testis tissue cryopreservation, with a portion of the sample going for laboratory research (if consent has been obtained for this purpose). In pubertal patients, the tissue for cryopreservation will be immediately analysed by an experienced embryologist for the presence of sperm using standard methods. If sperm are present then a portion of the sample will be used for Testicular Sperm Extraction (TESE) and storage, whilst remaining tissue will be cryopreserved using methods described below.

Cryopreservation of testicular tissue Testis tissue will be stored under the licence terms of the HTA. Samples are collected into sterile Hank's balanced saline solution (HBSS; 14175-129; Life Technologies, Merelbeke, Belgium), and transported to Tissue Services for cryopreservation and storage.

1. Cryopreservation of Immature Testicular Tissue Cryopreservation of ITT is performed by placing two tissue pieces in each cryovial containing 0.7 M DMSO (D2650; Sigma-Aldrich, Bornem, Belgium), with 0.1 M sucrose (+S; 10274-5c; VWR, Leuven, Belgium) and 10 mg/ ml HSA as previously described (Keros, Hultenby et al. 2007) (Wyns, Curaba et al. 2007). Equilibration is performed at +4°C for 30 min. Using a programmable freezer, the vials are cooled at 1°C/min with holding at 0°C for 5 min, followed by cooling at 0.5°C/min until 20°C. At this temperature, the program is put on hold for 10 min to allow manual seeding. The program continues at a rate of 0.5°C/min until 240°C, held for 10 min, and continued to 270°C at 7°C/min, with subsequent plunging into liquid nitrogen.
2. Cryopreservation of Mature Testicular Tissue Each biopsy is minced carefully with two fine needles in a Petri dish with 2 mL HBSS or Human Tubule Fluid, supplemented with 2.5% HSA (adapted from Verheyen, De Croo et al. 1995). After centrifugation of the tissue/cell suspension at 500g for 10 min and removal of the supernatant, the pellet is resuspended in 200 µL HBSS or Human Tubule Fluid, supplemented with 2.5% HSA. SpermFreeze (Vitrolife) (is added slowly at a ratio of 1:1 (v/v) to the suspension. After 20 min of equilibration at 37°C, the sperm suspension is aspirated into a 500-µL cryostraw (IMV, l'Aigle Cedex, France). Cooling and freezing are carried out under computer control using a standard programme for ejaculated spermatozoa (Verheyen, Pletincx et al. 1993). The straws are placed into the chamber of a programmable biological freezer and cooled according to the following programme: (i) cooling rate of -1°C/min from room temperature to 4°C (ii) hold for 1 min (iii) freezing rate of -5°C/min from 4°C to -80°C (iv) plunge into liquid nitrogen (-196 °C).

Laboratory research The primary aim of this application is to store testis tissue from patients that may be used in the future for fertility preservation if required. However, consent will also be sought for use of an extra portion of tissue for laboratory research. In addition to obtaining tissue as described above, the investigators will also use tissues obtained from an alternative source of tissue obtained from a collaborating centre (John Radcliffe Hospital, Oxford, UK). This second source of human prepubertal testis material will be stored under a HTA licence (reference: 11106) in which consent has previously been obtained for the use of the tissue in approved research, including animal research.

This testis tissue will be used for three main aspects of laboratory research

1. Optimal freezing methods for pre-pubertal testis tissue and cells

   Freezing conditions need to be optimized and improved with xeno-free clinical grade components before clinical application can be considered. The investigators will assess the use of various cryoprotectants on survival and developmental potential of germ cells within the portion of pre-pubertal tissue taken for research. Post-thaw viability and functionality will be assessed by immunohistochemistry, flow cytometry and RT-PCR. To evaluate the functional properties of the tissue after freezing, the tissue will be xenografted or used for in-vitro culture.
2. In-vitro culture of pre-pubertal testis tissue

   Testis tissue organ culture using a soft agar system has resulted in full spermatogenesis using neonatal mouse testis (Sato, Katagiri et al. 2011). This method will be adapted for use with human pre-pubertal testis tissue by investigating the effects of different culture media, serum and the addition of gonadotrophins/other growth factors to stimulate germ cell differentiation. This will be evaluated by histological analysis and expression of markers for spermatogonia (e.g. MAGE-A4, Thy-1), spermatocytes (e.g. SCP-3, Prohibitin) and spermatids (e.g. Protamine-2, SP-10) which will be studied with different methodologies, including qPCR, flow cytometry and protein expression. Modifications of the culture conditions by adding the following substances will be considered: (1) Knock-out Serum Replacement (KO-SR) as it induced complete spermatogenesis in neonatal mouse testicular tissue culture (Sato, Katagiri et al. 2011); (2) Retinoic acid as it has a known role in meiosis and spermatogenic differentiation; (3) Testosterone as high concentrations of testosterone are essential for spermatogenesis in vivo.
3. Xenografting

Testis xenografting represents a model system to investigate the factors required to establish the spermatogonial stem cell (SSC) niche (Mitchell, Saunders et al. 2010). Testis tissue will be xenografted in mice, ectopically (dorsal skin, scrotal, kidney capsule) or directly into the testis in order to establish the SSC pool. The investigators will examine which factors are responsible for the maintenance of the SSC niche using established advanced immunohistochemical co-localisation studies. The investigators will determine protein expression in the SSCs and the surrounding niche, including somatic cells, basal lamina and vasculature. This will include proteins that have been shown to be important in SSC self-renewal/differentiation in rodents, such as c-kit/KITL and GDNF/Gfrα1. Grafts will also be maintained for longer periods in order to investigate subsequent differentiation of the germ cells beyond the spermatogonial stage by investigating morphology and expression of meiotic markers such as described for in-vitro work. The effects on the establishment of spermatogenesis by exposure of xenografts to exogenous gonadotrophins (LH/hCG +/- FSH) will also be determined. Moreover, the xenografting approach will be used to obtain SSCs which can then be propagated and/or transplanted into seminiferous tubules to achieve colonization.

Follow up of Patients Patients will be followed up in the Oncology clinic as normal until they are no longer required to have regular surveillance imaging when they will be transferred to the joint Oncology/Endocrinology 'Late Effects' clinic at the RHCYP in Edinburgh. Patients will be reviewed every 3 months through childhood and puberty to monitor growth and pubertal status. Subsequent follow up will be between 3 monthly and annually. Puberty will be assessed by clinical examination using Tanner Staging and also by measuring blood levels of gonadotrophins, testosterone and Inhibin B. Testicular volumes will be measured using a Prader orchidometer and an annual ultrasound of the testes will be performed to assess testicular growth and monitor for scarring or damage as a result of the biopsy. A key outcome is the prevalence of gonadal failure (divided into spermatogenic and endocrine) in boys who have had testicular biopsies in comparison with those with similar and other diagnoses who have not. This will ensure that in the unlikely event that there are adverse effects of the biopsy this will be detected and it will also allow modification of the selection criteria for biopsy and testis tissue cryopreservation.

The same protocol applies to all subjects.

Project is on male fertility and therefore by definition is only applicable to males. Similar programmes are already established for females.

Cryopreservation of Prepubertal Testicular Tissue for Preservation of Fertility in Young Boys With Cancer

Cryopreservation of Prepubertal Testicular Tissue for Preservation

Measurement of reproductive hormones in blood

Most advanced germ cell stage identified in tissue after culture or xenotransplant.

University of Oxford

University of Edinburgh

Immune System Diseases

All Conditions

Urinary Tract, Sexual Organs, and Pregnancy Conditions

Autoimmune Diseases

Infertility

Testicular biopsy is performed to obtain testicular tissue for cryopreservation

Testicular biopsy

Rachel Neelley

Kyle Orwig, PhD

Testicular tissue cryopreservation is an experimental procedure where a young boy's testicular tissue is retrieved and frozen. This technique is reserved for young male patients who are not yet producing mature sperm, with the ultimate goal that their tissue may be used in the future to restore fertility when experimental techniques emerge from the research pipeline.

Fertility status has an important impact on the post-treatment quality of life for cancer survivors and other patients that receive gonadotoxic therapies (e.g., prior to bone marrow transplantation). Semen cryopreservation is an established fertility preserving therapy, but is not accessible or appropriate for all men. Currently there are no therapies to preserve the future fertility of preadolescent boys. However, new reproductive therapies are under development and may one day offer "fertile hope" to those survivors that do not currently have access to fertility preserving therapies. Clinical management of fertility threatening diseases and treatments must have foresight of the gonadotoxic side effects and the potential for infertility. When no established fertility sparing options are available, it is reasonable to offer harvesting and cryopreservation of testicular tissue as a possible means of fertility preservation.

This study will harvest testicular tissue from eligible patients. Separate portions of the harvested tissue and/or cells will be 1) designated for research and 2) cryopreserved and maintained for participating patients as a resource for future elective procedures to attempt fertility restoration.

Testicular Tissue Cryopreservation for Fertility Preservation

Testicular Tissue Cryopreservation for Fertility Preservation in Patients Facing Infertility-causing Diseases or Treatment Regimens

The investigators will publish individual participant data. The investigators will also report each individual participants data back to them. Participants will be identified with unique ID numbers. No identifiable information will be shared.

A segment of testicular specimen (\~5%) will be removed and assessed for presence of spermatogonial stem cells by immunofluorescence and hematoxylin staining

Study title	NCT ID	Conditions	Interventions	Eligibility	Organization
Fertility Preservation in Prepubertal Boys: An Experimental Approach	NCT02529826	Oncology [See A...	Testicular Biop...	3 Months - 18 Years	Hadassah Medical Organization
Cryopreservation of Prepubertal Testicular Tissue for Preservation of Fertility in Young Boys With Cancer	NCT06063551	Fertility Disor... Childhood Cance...	Testicular Biop...	1 Day - 18 Years	University of Edinburgh
Testicular Tissue Cryopreservation for Fertility Preservation	NCT02972801	Cancer Autoimmune Diso...	Testicular biop...	-	University of Pittsburgh
Fertility Preservation in Prepubertal Boys: An Experimental Approach	NCT02529826	Oncology [See A...	Testicular Biop...	3 Months - 18 Years	Hadassah Medical Organization
Testicular Tissue Cryopreservation for Fertility Preservation	NCT02972801	Cancer Autoimmune Diso...	Testicular biop...	-	University of Pittsburgh

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