Trial Title:
Cryopreservation of Prepubertal Testicular Tissue for Preservation of Fertility in Young Boys With Cancer
NCT ID:
NCT06063551
Condition:
Fertility Disorders
Childhood Cancer
Study type:
Interventional
Study phase:
N/A
Overall status:
Recruiting
Study design:
Allocation:
N/A
Intervention model:
Single Group Assignment
Intervention model description:
The same protocol applies to all subjects.
Primary purpose:
Supportive Care
Masking:
None (Open Label)
Intervention:
Intervention type:
Procedure
Intervention name:
Testicular Biopsy
Description:
Testicular biopsy for clinical storage and laboratory research.
Arm group label:
Testicular Biopsy
Summary:
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.
Detailed description:
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.
Criteria for eligibility:
Criteria:
Inclusion Criteria:
- Pre- and peripubertal boys
- Due to receive cancer treatment that is predicted to have a high risk (>80%) of
infertility
- Consent to removal of testicular tissue for storage and research
- Unable to produce a semen sample by masturbation
Exclusion Criteria:
- Participation in study would delay treatment
- Too unwell to undergo the procedure
- Pre-existing testicular pathology/previous testicular surgery (eg orchidopexy)
- Able to produce a semen sample for cryopreservation
Gender:
Male
Gender based:
Yes
Gender description:
Project is on male fertility and therefore by definition is only applicable to males.
Similar programmes are already established for females.
Minimum age:
1 Day
Maximum age:
18 Years
Healthy volunteers:
No
Locations:
Facility:
Name:
University of Edinburgh
Address:
City:
Edinburgh
Zip:
EH164TJ
Country:
United Kingdom
Status:
Recruiting
Contact:
Last name:
Rod T Mitchell, MBChB
Phone:
01316518307
Email:
rod.mitchell@ed.ac.uk
Contact backup:
Last name:
Mark Brougham, MBChB
Phone:
01315360000
Email:
mark.brougham@nhslothian.scot.nhs.uk
Start date:
April 1, 2016
Completion date:
April 2061
Lead sponsor:
Agency:
University of Edinburgh
Agency class:
Other
Collaborator:
Agency:
University of Oxford
Agency class:
Other
Source:
University of Edinburgh
Record processing date:
ClinicalTrials.gov processed this data on November 12, 2024
Source: ClinicalTrials.gov page:
https://clinicaltrials.gov/ct2/show/NCT06063551
