Testicular tissue cryopreservation in boys. Ethical and legal issues: Case report

G. Bahadur1,3, R. Chatterjee1 and D. Ralph2

1 University College London and UCLH Trust, Fertility and Reproductive Medicine Laboratories, Department of Obstetrics and Gynaecology, 86–96 Chenies Mews, London WC1E 6HX and 2 The Institute of Urology and Nephrology, 48 Riding House Street, London W1P 7PN, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Case 1
 Case 2
 Materials and methods
 Results
 Discussion
 References
 
Sperm preservation prior to chemotherapy and radiotherapy is common practice in adult males. Spermatozoa are usually retrieved from an ejaculated sample although there are occasions when testicular tissue is used as the source. These techniques of sperm preservation present minimal ethical objections as the patients give their informed consent. Sperm preservation in children presents practical and ethical dilemmas in that the children cannot always give their informed consent, there are no regulatory guidelines and there is no guarantee that spermatogenesis is occurring. With the rapid advances in reproductive technology and the possible future use of immature germ cells by in-vitro maturation or transplantation, the demand for immature testicular tissue preservation is likely to increase. More information for the parents and oncologists with regard to this subject is needed to allow informed decisions to be made on behalf of the children. These issues are discussed using two cases of children having testicular tissue preservation.

Key words: boys/counselling/cryopreservation/ethics/testicular tissue


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Case 1
 Case 2
 Materials and methods
 Results
 Discussion
 References
 
Antimitotic chemotherapy or radiation therapy can induce failure of spermatogenesis and in adults sperm cryopreservation is a practical and effective option prior to treatment (Steele et al., 1995Go; Padron et al., 1997Go). Improvement in the prognosis of cancer and increased long-term survival mean that preservation of spermatozoa should be considered before initiating therapy.

With the pace of advances in assisted reproductive technologies primarily with the successful application of intracytoplasmic sperm injection (ICSI) procedures (Rosenlund et al., 1998Go) and gamete maturation techniques, focus has been turning towards preserving spermatozoa of almost any quality. This has been extended to preserving testicular and ovarian tissue (Nayudu, 1994Go; Bahadur and Steele, 1996Go; Newton et al., 1996Go; Allan and Cotman, 1997Go; Silber et al., 1997Go) in the hope that future advances in technology will allow viable gametes to be derived from these frozen materials. The most recent successes include a pregnancy after ICSI of a cryopreserved human oocyte (Nawroth and Kissing, 1998Go) and the birth of a boy after the use of a secondary spermatocyte (Sofikitis et al., 1998Go). In the UK, spermatid injection (Fishel et al., 1997Go; Tesarik, 1997Go) is not allowed on the grounds of health and safety of offspring born from such precursor cells. The pace of advance means that parents and oncologists come under pressure for information or a decision (Hewitt et al., 1998Go). The logical extension of preserving gonadal tissue is now being applied to children about to embark on cytotoxic therapy for cancers.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Case 1
 Case 2
 Materials and methods
 Results
 Discussion
 References
 
Two cases are presented of boys that had testicular tissue cryopreserved prior to chemotherapy for malignant disease. In both cases the parents (and the older child) were told that chemotherapy could lead to sterility with failure of sperm production. They were also told that immature spermatozoa, such as those which were likely to be present in the boys' testicles, cannot currently be used to fertilize oocytes even with the use of ICSI procedures. However, it was suggested that advances in reproductive biology (e.g. in-vitro sperm maturation) might eventually allow germ cells from an immature testis to be used for fertilization. Parental counselling and consent was significant in both cases.


    Case 1
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 Abstract
 Introduction
 Materials and methods
 Case 1
 Case 2
 Materials and methods
 Results
 Discussion
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An 8 year old boy with Ewing's sarcoma who had already had one dose of chemotherapy presented for surgical testicular tissue storage. The mother decided that her son's testicular tissue should be stored and this decision was strongly influenced by another patient having a similar procedure at another hospital. This decision was made 24 h prior to the next dose of chemotherapy and she was counselled. Despite the initial offer to the parent for counselling of the patient, this did not take place. An in-house consent given by the mother for freezing and possible future use of the tissue was already in place before surgery. Local Ethics Committee permission for the procedure was also obtained.


    Case 2
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 Introduction
 Materials and methods
 Case 1
 Case 2
 Materials and methods
 Results
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A 13 year old boy with a newly diagnosed Hodgkin's lymphoma presented for testicular tissue preservation prior to the commencement of chemotherapy. The patient was prepubertal, had never ejaculated or had nocturnal emissions. Three early morning urine samples for spermatouria (Kulin et al., 1989Go; Schaefer et al., 1990Go; Jorgensen et al., 1991Go) over a period of 1 week were negative. The boy was accompanied by his mother and was judged to be able to give informed consent which was witnessed.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Case 1
 Case 2
 Materials and methods
 Results
 Discussion
 References
 
Both boys had a general anaesthetic and an open testicular biopsy performed on one testicle only. A small segment of the biopsy was sent in Bouin's solution for histological analysis and the larger part frozen for storage. Both patients made an unremarkable recovery and commenced their chemotherapy the following day.

Freezing technique
Three freezing procedures were used: (i) an egg-yolk based medium containing 10% glycerol, normally used for freezing spermatozoa; (ii) IVF medium (Medicult, Redhill, Surrey, UK) supplemented with 10% glycerol and 10% heat-inactivated serum; and (iii) phosphate-buffered saline (PBS) containing propan-1,2-diol (1.5 mol/l) and sucrose (0.1 mol/l) (Peek et al., 1982Go; Pilikian et al., 1982Go; Phillip et al., 1983Go; Mahadevan and Trounson, 1983Go; Gosden et al., 1994Go). There was an insufficient amount of tissue sample to freeze by the variety of methods that we had anticipated and none was available to be frozen without any buffer or cryoprotectant to serve as a control.

The tissue was collected immediately and placed in ice-cold PBS (0.5 ml) to wash off traces of blood and then transferred immediately to fresh ice cold PBS (0.5 ml) buffer, where two cuts were made to give three tissue fragments. The tissue was incubated for 15–20 min at room temperature in three separate sterile Petri dishes containing 0.5 ml of each of the above media. The tissue was punctured twice with a sterile syringe needle in the main body of the tissue to assist in the flow of buffer to the inside of the tissue (Bahadur and Steele, 1996Go; Newton et al., 1998Go). After the incubation period, the tissue in the first two media was placed in the cryovials together with their incubation medium for a further 30 min on ice. They were then placed in nitrogen vapour phase (in a tanker 45.7 cm above the liquid nitrogen layer) for 1.5 h, cooling rate ~10°C per min, before being plunged into liquid nitrogen for storage. The third tissue sample was incubated for a further 10 min with fresh PBS/propan,1,2-diol supplemented with sucrose. Both the tissue and the new buffer (0.5 ml) was then placed in ice for 30 min and frozen in nitrogen vapour as above. Each Nunc cryovial (Life Technologies, Paisley, UK) was labelled with the boy's name, the date of freezing and a brief marking of the type of cryoprotection method to enable future differentiation of vials. Each vial was sealed with Nescofilm (Bahadur and Tedder, 1997Go).

Consent
Consent for testicular tissue cryopreservation was obtained from the 13 year old boy who was considered mature enough to understand the process and this consent was witnessed by his mother. Consent for the 8 year old boy was given by his mother. The consent covered the possible use of the material, the duration of storage and the procedure in dealing with the tissue in the event of mental incapacitation or death. In view of the unique circumstances, special emphasis was placed on the current non-existence of technologies for the use of the frozen tissue, the non-established freezing procedures and lack of proof for the viability of frozen tissue. These points were covered in the written consent. Should the necessary technologies become available in the future, the relevant issues for the patient were: the genetic safety of any `intense sperm maturation' procedure, the prevailing attitude of society, the future policy of the Human Fertilisation and Embryology Authority (HFEA), and the legal position at the time. Genetic predisposition towards cancers was also mentioned generally.

Consideration was given to possible future litigation between son and parents which is not unknown in the modern age. One open area could be that of assault charges in a situation which could not constitute the `best interest of the child': namely, that intervention was on `social' rather than `medical' grounds. Parental control over the stored gametic material was restricted to storage only, thus allowing the patient greater control over his own genetic material. Transition of absolute control was discussed and it was anticipated that this would normally occur at the point of that child's maturity. We are presently interpreting this along the line of Gillick (1996) competence although the latter model may be inappropriate. We made clear that, should legal and regulatory controls be introduced in the future, then these would take precedence over the present discussions. Failure to emphasize this point could lead to unnecessary complications in the future between the patient and clinic.


    Results
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 Abstract
 Introduction
 Materials and methods
 Case 1
 Case 2
 Materials and methods
 Results
 Discussion
 References
 
Case 1
Due to the size of the testicle, only small quantities of tissue were retrieved. The histological assessment of 100 seminiferous tubules revealed the presence of immature Sertoli cells and spermatogonia only. No spermatocytes, spermatids or spermatozoa were identified in the sample.

Case 2
This testicle was of a larger size and therefore larger biopsies were taken. Spermatogenesis at the level of spermatids was in abundance and occasional spermatozoa were found in less than 5% of the tubules. A lymphomatous infiltrate was not apparent in the tissue.

Both children made an uneventful recovery and were discharged back to the Oncology Service the same day.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Case 1
 Case 2
 Materials and methods
 Results
 Discussion
 References
 
From our experience, the main impetus and decision-making for freezing children's gonadal tissue is from parents who themselves may be under peer pressure. It might be useful because of the speed of events in cancer treatment, to distinguish consent obtained under emotional, peer-pressured conditions and rational, fully informed consideration. There is a clear need to develop parental counselling and, in the absence of guidance, to stimulate debate and to begin formulating guidelines for tissue storage which can be helpful to clinicians.

Scientific and biological background
The rationale for freezing children's gonadal tissue comes from the hope that future advances in reproductive technology may enable them to be used for fertility treatment and that any social and legal objections to such treatment would be eroded by scientific advances. It is therefore essential to understand the current knowledge gained from immature germ cell use in order that decisions are kept in perspective.

The classification of spermatids (de Kretser and Kerr, 1994Go) is as follows: Sa, round; Sb1, round with flagellum; Sb2, elongating; Sc, elongating, nucleus fully elongated; Sd1, elongated, head still not separated from mid-piece; Sd2, mature, large cytoplasmic sheath in mid-piece. One apparently normal pregnancy and live birth was recorded after Sa round spermatid injection attempts in six patients (Barak et al., 1998Go). However, in the same report no pregnancies were recorded after Sb or Sc elongated spermatids were injected for seven patients.

To utilize germ cells there are two clear strategies to follow: the in-vitro maturation of the gametes or germ cell or tissue transplantation. The latter would involve surgically grafting the pieces of tissue onto an existing testicle or injecting preparations of the germ cells (crude or purified) into the rete testes. The infusion can be monitored and optimized with ultrasonography, needle placement and dye flow. The advantage of injection into the rete testes is that a much higher volume of infusion can be expected. By contrast, microinjection into the seminiferous tubules as well as the efferent ducts is not the most favoured approach for germ cell transfer into the testes, where microinjections are performed using glass needles and the help of a mechanical micromanipulator (Schlatt et al., 1999Go).

Cases 1 and 2 are different in that case 1 will most likely need in-vitro maturation or germ cell transplantation in the future, whereas case 2, where abundant spermatids and occasional spermatozoa occurred, could be treated by ICSI with frozen–thawed testicular spermatozoa. The use of round spermatids in conjunction with ICSI has so far been disappointing with probably no more than 10 live births worldwide, and these were from patients who occasionally had mature spermatozoa in their ejaculates or testicular biopsies (Gianaroli et al, 1999Go). It is interesting to note that there would seem to be a higher frequency of apoptotic DNA fragmentation in round spermatids from patients with a complete lack of spermatogenesis compared with those with incomplete spermatogenic disorders (Tesarik et al., 1998aGo). Despite the important implications of this work, caution is needed. The ploidy of spermatids to be injected cannot be determined. The rapid 24 h maturation in vitro compared with the 72 days taken for spermatogenesis and 22 days for spermiogenesis in vivo raises the question of overriding the natural endogenous control mechanisms with whatever effect that may have on the DNA, involving rapid division and condensation. Short-term in-vitro culture of spermatogenic cells did not improve the incidence of fertilization but overall the more mature the spermatids, the higher the incidence of fertilization that was noted (Aslam and Fishel, 1999Go). There was a higher incidence of a single nucleus formation in zygotes derived from round spermatid injection compared with elongated spermatid injection (Barak et al., 1998Go). It is interesting to note that when injecting secondary spermatocytes into mouse oocytes, electroactivation was needed to create live and apparently healthy offspring (Kimura and Yanagimachi, 1995Go).

From preliminary human and animal data, much still remains unknown about the mechanism of fertilization with these immature sperm precursor cells. Processes such as genomic imprinting, changes in the nuclear proteins (Tesarik and Kopecny, 1989Go), oocyte activation (Sousa et al., 1996Go) and cell-cycle synchronization of the gametes need to be studied to elucidate the mechanism. Whether spermatocytes and spermatids from boys follow the same pattern as those in adults is unknown: certainly in immature human oocytes the Ca2+ signalling is deficient (Herbert et al., 1997Go).

Furthermore, there are concerns regarding the possible risks to health of children born from the use of spermatids with ICSI (Tesarik et al., 1998bGo). In limited animal models, germ cell transplantation has been encouraging but extrapolation to human application will require extreme caution (Brinster and Zimmermann, 1994Go; Jiang and Short, 1995Go; Russell et al., 1996Go). On the other hand, the use of frozen stored tissue grafts is problematical. Trials using a mouse lymphoma model established that lymphoma was transmitted by grafts of both fresh and frozen ovarian tissue (Shaw et al., 1996Go; Gosden et al., 1997Go). The normal healthy recipient mice died 9–43 days after receiving ovarian tissue from the donor with lymphoma. Viruses such as human immunodeficiency virus (HIV) (Nicholson and Johnson, 1994Go), hepatitis (Pereira et al., 1995) and cancers (Weissmann et al., 1995Go) can be transmitted by grafts. It is therefore possible that blood-borne cancers such as leukaemia, systemic cancers such as lymphoma and metastasizing cancers could be transmitted by grafts of gonadal tissue. Equally, it is important that patients are screened against HIV and hepatitis, and that gonadal micrometastases are rigorously excluded prior to tissue use. The pregnancy outcome after childhood cancer does not support the level of germ cell mutations which could result in increased congenital malformation or neonatal mortality. However, much larger patient numbers would be needed to rule out with confidence any of these associations (Bahadur and Ralph, 1999Go).

Regulatory and legal background
In the UK the storage of children's gonadal tissue does not presently come under regulatory control. The HFEA (1990) and HFEA Code of Practice (1995) state that gametes must be stored on licensed premises and a consent to storage must be given by the person who provided the gametes. The HFEA defines a `gamete' to be `a reproductive cell, such as an ovum or spermatozoon, which has a haploid set of chromosomes and which is able to take part in fertilization with another of the opposite sex to form a zygote' (Deech, 1998Go).

Using this definition in conjunction with the six Tanner grades of puberty (Tanner, 1989Go), if a boy is pre-pubertal (pre-Tanner Grade 2) then his testicular tissue is unlikely to contain gametes as defined by HFEA and thus can be stored on unlicensed premises. If, however, such material were subsequently to be developed in vitro in some way as to create `gametes' as defined above, the storage and use of that material would require a licence (Deech, 1998Go).

Storage of testicular tissue from boys who have reached at least Tanner Grade 2 stage will require a licence and the patient can give effective consent in accordance with the requirements set out in the HFEA (1990). Substituted consent is not possible under the 1990 Act. Thus consent to storage cannot be given on behalf of any child who has reached Tanner Grade 2 and whose testicular tissue is to be stored. However, a child under the age of 16 years can give an effective consent in accordance with the 1990 Act's requirement if he is capable of understanding the proposed course of action.

Consent to the removal of testicular tissue from any male of whatever age is covered by statute (Tissues Act, 1961Go). For children, parental consent will be required in accordance with Children's Act, 1989. In taking any decision, the best interest of the child needs to be taken into account in conjunction with the opinion of the child's clinician. The present guideline on `best interest' relates to saving life, or to ensuring improvement or prevention of deterioration in physical or mental health, making true justification for taking gametes problematic.

The procedure of testicular biopsy is inevitably intrusive and may involve pain or possibly future side-effects of the trauma to the testicle. It can only therefore only be justified on the assumption that it will lead to an improvement in future quality of life due to advances in reproductive technology and changes in the regulatory framework. Hence, it is essential that consideration be given to adopting counselling and safety procedures that maximize the possibility of future regulatory support. Furthermore at some stage, responsibility for storage and use of the tissue would have to pass from the parents to the children and it makes sense to address the issues arising from this in the original consent form. For example, our in-house consent form limits parental responsibility to safe-keeping of the tissue and excludes the use of the gamete should the boy die or become mentally incapable of informed consent. Therefore there is no direct possibility for the parents to create grandchildren from their son's gamete.

With regard to the actual procedures, numerous methods already exist for treating adult testicular tissue (Verheyen et al., 1995Go; Hovatta et al., 1996Go; Salzbrunn et al., 1996Go; Khalifeh et al., 1997Go; Oates et al., 1997Go). An approach with minimal manoeuvre was favoured since so little is known about children's testes and the possible necessity of proximal sperm support cells in future maturation processes. Purified germ cells appear to be giving poorer results in transplantation (Avarbock et al., 1996Go; Soder, 1997Go).

In conclusion, there appears to be a need to preserve young boys' gonadal tissue prior to chemo- and radiotherapy for future restorative fertility application. This need is being driven by the pace of technological advances and by parents keen to preserve the quality of life for their children following cytotoxic procedures. As storage of some children's testicular tissue is possible at unlicensed UK centres, it is important that the same high standards operational at UK licensed centres are applied. In the main these are: safe areas for storage, the need for verbal and written information, protocols and established lines of communication for the long-term maintenance of frozen tissue, openness to patient information and counselling provision. As far as possible, a valid and effective parental or next of kin's consent should be in place for the taking and storing of tissue and one which would allow a mechanism of total control to pass in time to the person from whom the genetic material is derived. The two cases reported here also highlight the contrasting potential work-up in the utilization of frozen tissue samples.


    Notes
 
3 To whom correspondence should be addressed at: University College London and UCLH Trust, Fertility and Reproductive Medicine Laboratories, Department of Obstetrics and Gynaecology, 86–96 Chenies Mews, London WC1E 6HX, UK. Back


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 Abstract
 Introduction
 Materials and methods
 Case 1
 Case 2
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 24, 1999; accepted on March 10, 2000.