Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT06063551 |
| Other study ID # |
140175 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
April 1, 2016 |
| Est. completion date |
April 2061 |
Study information
| Verified date |
September 2023 |
| Source |
University of Edinburgh |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial 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.
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.
