Gonadal tissue cryopreservation in boys with paediatric cancers

G. Bahadur1,3 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 Middlesex Hospital, Institute of Urology and Nephrology (St Peter's Hospital), London, UK

Abstract

In considering gonadal tissue cryopreservation in children about to undergo chemotherapy or radiotherapy for cancer, numerous issues need to be confronted. This relates firstly to a dearth of information available on the subject of children's gonads coupled to ill-defined fertility preservation procedures, technologies and perhaps less well-publicized regulatory, ethical and legal controls. There may be benefits in considering gonadal tissue preservation for children prior to chemotherapy or radiotherapy, in the hope that future technologies can utilize their immature gametes. Whether or not freezing of gonadal tissue is encouraged prior to cancer therapy, there is a growing demand from parents and clinicians for more information to be made available. This paper reviews our current knowledge of children's gonads in terms of physiology and of fertility experience gained after childhood cancer treatment. It further examines the various issues and concerns regarding children's gonadal tissue storage, its potential use, the present law and the demands and pressures under which clinicians find themselves with patients and parents. This information is important for both the counselling process and decision-making when presented with potential fertility issues in paediatric oncology patients.

Key words: cancer/chemotherapy/children/cryopreservation/fertility

Introduction

With advances in intracytoplasmic sperm injection (ICSI) and interest in gamete extraction and maturation (Hovatta et al., 1996Go; Abir et al., 1997Go; Allan and Cotman, 1997Go; Antinori et al., 1997Go; Oates et al., 1997Go; Porcu et al., 1997Go; Schlegel et al., 1997; Tesarik, 1997Go), focus has turned towards preserving gonadal tissue for future use (Karrow and Crister, 1997Go).

Children provide a double-edged sword when considering such issues of gamete preservation. These partly relate to obtaining proper informed consent, similarly obtained under `Gillick competence', for taking, storing and using gonadal tissue (Elliston, 1996Go; McLean, 1997Go). Also, our scientific investigations and knowledge of children's gonadal tissue is lagging behind that of adults'. We review firstly the physiology of children's gonads and in particular male gonads, and we report on the published experience gained from treating paediatric oncology patients.

Studies on human male germ cells from birth to puberty are few (Mancini et al., 1960Go; Sasaki, 1968Go; Viler, 1970; Bustos-Obregon et al., 1975Go; Hadziselimovic, 1977Go; Hedinger, 1982Go; Muller and Skakkebaek, 1983Go; Nistal and Paniagua, 1984Go), probably reflecting earlier sensitive ethical considerations. The testes are growing organs not only in puberty but also in the first 10 years of life. During this age span, the testicular median value increases from 1.1 ml (range 0.3–1.9) to 3.0 ml (range 2.8–3.4). Testicular growth in the 0–10 year period is mainly due to the increased tubular length. The median length of the seminiferous tubules per boy was 181 m (range 27–361) in the 0–1 year period and 411 m (range 277–660) in the group 5–10 years. Prepubertal testicular growth which follows includes an increase of both diameter and the length of the seminiferous tubules. The total number of germ cells increases steadily during childhood (0–10 years), which indicates that replication of germ cells takes place before puberty (Muller and Skakkebaek, 1983Go).

A variety of sperm cells have been described in studies of prepubertal boys. The primary spermatocytes largely seem to appear at the beginning of puberty, when spermatogenesis is established shortly before. The presence of primary spermatocytes is evident in some studies, but these appear to undergo degeneration or progress to abnormal spermatids which in turn degenerate. The seminiferous epithelium of normal infant and child testes generally consisted of immature Sertoli cells (light cells and occasionally some dark cells), and different types of spermatogonia. These include only fetal spermatogonia (only during infancy) and the Ap, Ad and B (from age 4 years onwards) spermatogonial types. In addition, some seminiferous tubules of some testes showed primary spermatocytes which could be recognised by the characteristic chromatin distribution during the meiotic prophase. Ultrastructural examination revealed the presence of synaptonemal complexes in these cells as well as many degenerating spermatocytes. Whilst spermatids showing many morphological maturative abnormalities, such as binucleate spermatids, were occasionally seen, spermatozoa were never found (Nistal and Paniagua, 1984Go).

The Sertoli cells maintain their immature appearance in these tubules. As in the tubules devoid of spermatocytes, dark Sertoli cells were encountered in a reduced proportion. No mature, adult Leydig cells were observed in the testicular interstitium. Primary spermatocytes were found only in some boys between the ages of 4–13 years. When they appeared they were found in both testes and in only 5–25% of the seminiferous tubules. The lack of complete spermatogenesis in these tubules might be related to the lack of testosterone and the lack of Sertoli cell maturation. The Sertoli cell is the primary target of follicle stimulating hormone (FSH), and its maturation is regulated by the FSH–luteinizing hormone (LH) balance. However, the presence of meiotic spermatocytes in human testes long before puberty is a remarkable feature, although these cells degenerate and do not progress to spermatozoa. From birth to puberty, the developing testis undergoes a marked germ cell proliferation at the end of infancy (about 3–4 years of age), and at 8–9 years of age. This has been interpreted as two failed premature attempts at spermatogenesis. These are also the ages at which the spermatocyte containing tubules are seen. It has been postulated that the enclosure of the male germ cell by the immature Sertoli cell is to prevent the spermatocyte from entering meiosis (Nistal and Paniagua, 1984Go). Large infantile germ cells; pre-spermatogonia (Gondos, 1975Go), pro-spermatogonia (Hilscher et al., 1974Go), gonocytes (Gondos, 1975Go) and primitive type A spermatogonia (Vilar, 1970Go; Steinberger, 1974Go) have been shown to be present throughout the prepubertal period, while adult type A were observed to be the more common germ cells in childhood.

One in 650 children will develop cancer by the age of 15 years, of whom 50–60% will be cured. Overall, there is a 5–10% second tumour rate in long-term survivors of childhood cancer. By the year 2000, this means that 1 in 1000 young adults will be a survivor of childhood cancer. Girls who have received TBI (total body irradiation) or abdominal irradiation are at risk of primary ovarian failure, i.e. failure to initiate or complete puberty and/or premature menopause (secondary amenorrhoea before 40 years) (Davies, 1993Go).

As most requests currently relate to boys, we need to know the status of the child's testes to assess the reality of embarking on highly sensitive and invasive techniques. Investigations of adult patients have shown that chemotherapy causes gonadal damage (Chatterjee and Goldstone, 1996Go), but much less information is available about the impact of chemotherapy on gonadal function in children with malignant disease. Being prepubertal during therapy is sometimes thought to confer protection against chemotherapy-induced gonadal damage. Twelve prepubertal patients, mean age 10.2 ± 3.0 years, receiving chemotherapy were studied after a mean interval of 6.42 years (range 2.0–16.5 years). All patients were evaluated for testicle size, sperm counts, LH and FSH after gonadotrophin releasing hormone (GnRH) test, and testosterone concentrations. Puberty had progressed normally in all patients. No significant difference in testosterone and basal LH concentrations between patients and adult controls were detected. However, appreciable differences were detected in peak LH concentrations and in basal and peak FSH concentrations. Seven patients had exaggerated LH response to GnRH, indicating Leydig cells dysfunction. Eight out of 12 patients suffered azoospermia and only one patient had a normal semen analysis (Mustieles et al., 1995Go). There was no evidence of gonadal protection in prepubertal males. Therefore there was a high incidence of germinal cell damage. This gains support from the finding that testicular function following treatment of Hodgkin's disease in childhood leads to a high level of germinal epithelium damage with elevated FSH concentrations persisting up to 17 years from the end of chemotherapy (Shafford et al., 1993Go). In paediatric Hodgkin's disease cases, severe germ cell damage was present in all 19 patients studied. However, comparing testicular function of prepubescent versus pubescent state at the time of treatment appears to show a trend for improved outcome in the younger patients (Heikens et al., 1996Go).

In another study (Jaffe et al., 1988Go), the reproductive outcome in 27 males long-term was associated with the chemotherapy doses. However, they concluded that there was a need for further studies for more accurate correlations, and fertility potential could not be predicted by testicle size examination or gonadotrophin and testosterone values. Following cyclophosphamide therapy, spermatogenesis dysfunction occurred in six out of 15 boys before and after puberty. However, therapy after puberty was associated with spermatogenic dysfunction in all four boys in the study group. Interestingly, menstrual dysfunction in girls receiving cyclophosphamide therapy was non-existent and girls are less affected than boys (Nicholson and Byrne, 1993Go). In contrast, testicular function in boys treated for acute lymphoblastic leukaemia (ALL) was compatible to chemotherapy (Blatt et al., 1981Go). Partial support is obtained from another source, where it had been suggested that recent strong chemotherapy for the treatment of ALL might cause severe but not fatal damage to children's testicular tissue (Kobayashi et al., 1996Go).

Testicular failure in boys may be a result of Leydig cell dysfunction or germinal epithelium dysfunction, or both. Direct irradiation of the testes in TBI results in permanent Leydig cell failure and ablation of the germinal epithelium. This results in infertility and a need for lifelong testosterone therapy (Shalet et al., 1985Go; Castillo et al., 1990Go) from around 12–13 years of age. Cytotoxic chemotherapy can cause germinal epithelium damage which could possibly be reversed. Precocious puberty does occur following cranial radiotherapy in boys, but is much less common in girls.

Girls with ovarian failure will require oestrogen replacement therapy. However, some girls with evidence of ovarian failure after completion of their therapy will go on to have regular menses and normal FSH and oestradiol concentrations. This suggests that prospects for fertility are good at least in their early child-bearing years, although they may go on to have a premature menopause (Davies, 1993Go).

It would seem vital that the maturation feasibility of the near full 76 day cycle of these sperm precursor cells in boys be established. This is likely to be precisely timed and major breakthroughs in in-vitro culture processes are needed. It would appear that late stage spermatocytes, once they have undergone a first meiotic division, would become haploid with 23 chromosomes and would have to undergo a second meiotic division, a type of pseudo-mitosis, to give the late stage or secondary spermatocytes which are haploid 23 chromatid cells (Setchell, 1978Go). On preliminary consideration, these cells containing chromosomes or chromatids would have reproductive potential when placed in an oocyte combined with the ever increasing permutations of technologies. However, it is unclear what could be obtained by such a fusion with an oocyte. The result would perhaps be a triploid? The area of human spermatogenesis and somatic cell genetics would need further re-evaluation with the advent of modern day fluorescent in-situ hybridization (FISH) testing (Guttenbach et al., 1997Go; Rademaker et al., 1997Go) in genetics, which still has to find its application to these type of cells.

The impetus of preserving children's gonads follows on the heels of reports of live human births resulting from the transfer of embryos fertilized with round (Tesarik et al., 1995Go; Tesarik and Mendoza, 1996Go) and elongated spermatids (Fishel et al., 1995Go, 1996Go), which encouraged various in-vitro fertilization (IVF) clinics to apply these methods where no mature spermatozoon was found. It is therefore essential to understand the current knowledge gained in immature germ cell use to keep decisions taken on children's gonads in perspective. The classification of spermatids (de Krester 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.

In spite of these 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, oocyte activation, and cell-cycle synchronization of the gametes need to be studied to elucidate the mechanism. One apparently normal pregnancy and live birth was recorded after Sa round spermatid injection attempts in six patients. However, in the same report no pregnancies were recorded after Sb or Sc elongated spermatids for seven patients. One of the most conspicuous features concerning the zygote was the development of only a single nucleus. Such single-nucleated zygotes occurred more with round spermatids rather than elongated spermatids. One obvious question relating to this unusual behaviour of the zygote nuclei might be a sign of abnormal development or whether such zygotes subsequently could give rise to normal embryos for transfer into the mother (Barak et al., 1998Go). The cause of such frequent single-nuclei development following round spermatid and elongated spermatid injection into the oocyte remains unknown but could reflect the differences in the mechanism of fertilization compared to fully mature spermatozoa. It is interesting to note that electroactivation of mouse oocyte was needed upon injecting secondary spermatocytes to create live, apparently healthy offspring (Kimura and Yanagimichi, 1995).

Bernabeu et al. (1998) reported no pregnancies when round spermatids (Sa type) derived from ejaculated semen or testicular biopsy, from six and two patients respectively, although culturing round spermatids appeared to increase the fertilization rate. One live birth was recorded from the use of an Sb2 type spermatid. Vanderzwalmen et al. (1997) subdivided patients into testicular biopsies showing incomplete spermatogenesis (11 cycles) and biopsies showing some foci of complete spermatogenesis (26 cycles). In the former group, although the fertilization rate (2PN zygotes) was 12%, no pregnancy could be established. In the latter group, the fertilization rate (2PN) was 37% and the pregnancy rate was 19% per cycle. In relation to the five recorded pregnancies, the type of spermatids used were one with round Sa type spermatid, three with elongating (two Sb2, one Sc) spermatids and one with elongated Sd2 type spermatid. These results clearly demonstrate that an early germinal block markedly worsens prognosis. Poor prognosis was found (Amer et al., 1997Go) where the two established pregnancies were from elongated testicular spermatids. In boys, although there is no evidence of germinal block as experienced with infertile men, it would be interesting to know how the germ cells might behave. These observations in the adult patient group suggest that a crucial development step may occur at the round spermatid stage. In-vitro culturing may improve fertilization rates for spermatocytes and it might be possible in future to apply transmeiotic in-vitro development of the primary spermatocytes into spermatids by injection into germinal vesicle oocytes, thus allowing both the male and female nucleus to undergo simultaneous reduction divisions within the oocyte. Intense in-vitro maturation of spermatozoa is also fraught with transcription errors, especially since sperm cells alone do not possess the ability to repair the DNA (Ashwood-Smith and Edwards, 1996Go).

The spermatids' transition into spermatozoa is marked by salient changes in the composition of nuclear proteins, histones being progressively removed and replaced by protamines, basic proteins rich in S-S bonds responsible for nuclear condensation, whereas an inverse protamine-histone occurs in the male nucleus after fertilization. Variable amounts of protamine may alter the normal sequence of post-fertilization chromatin functions, including the early transcriptional activity detected in human pronuclear zygotes (Tesarik and Kopecny, 1989Go). Using in-situ hybridization, protamines PRM1, PRM2 and transition protein TNP2 transcripts have been demonstrated in mature human spermatozoa (Wykes et al., 1997). The persistence of such transcripts in mature human spermatozoa appears to suggest that, after spermiogenesis, these transcripts are sequestered in some manner while they await their fate. As yet, there exists no information regarding the fate or potential role of these transcripts during fertilization and the formation of a viable male pronucleus.

Another area of concern is the manner in which various sperm precursor cells might induce activation of the oocyte at fertilization, as shown by the Ca2+ oscillation signals (Sousa et al., 1996Go). Preliminary studies show that the presence of oocyte-activating factor in round spermatids in men with complete spermatogenesis failure is deficient compared with spermatids from men with normal spermatogenesis. Suboptimal oocyte activation may have far reaching developmental consequences extending to poor fertilization, implantation and high early abortion rate (Sousa et al., 1996Go). Whether spermatocytes and spermatids from boys follow the same pattern remains an interesting question. Interestingly, in immature human oocytes the Ca2+ signalling is deficient (Herbert et al., 1997Go). It has become clear that genetic imprinting plays an important role in human embryogenesis and in processes leading to the development of paediatric cancers and other human diseases (Rachmilewitz et al., 1992Go; Hochberg et al., 1993Go). Differences in the methylation pattern of gamete DNA is likely to be involved in genomic imprinting. Little is known of the mechanism that regulates de-novo methylation and demethylation during gametogenesis. Mouse round and elongating spermatids contained DNA methyltransferase (Jue et al., 1995Go). The purpose of DNA methyltransferase expression in haploid, post-replicative and postmeiotic germ cells is unknown although it might be involved in further de-novo methylation events. Any risk of genetic imprinting abnormality can only usefully be unravelled by keeping accurate records of spermatid morphologies and having available, in future, lymphocytes from resulting children to test the five imprinted human genes (Tesarik et al., 1998Go). However, any approach to germ cell maturation should be one which avoids the need to involve this uncertain area of health and development.

It is important to understand the role of the sperm centriole and how it manifests itself in the human embryo. The spermatids possess a pair of centrioles, proximal and distal, of which the latter disappears in late stage Sd2 spermatids. The mature spermatozoon then has one centriole, associated with a micro-tubule organizing centre which is responsible for the organization of the zygote microtubules including those of the mitotic spindles employed in the forthcoming cleavage divisions (Sathananthan et al., 1991Go). The human egg has an inactive non-functional centrosome. The prominent centriole (proximal) associated with pericentriolar material which is transmitted to the embryo at fertilization persists during sperm incorporation. Centriolar duplication occurs at the pronuclear stage (interphase) and the centrosome initially organizes a sperm aster when male and female pronuclei break down (prometaphase). The astral centrosome containing diplosomes (two typical centrioles) splits and relocates at opposite poles of a bipolar spindle to establish bipolarization, a prerequisite to normal cell division. Single or double centrioles occupy pivotal positions on spindle poles and paternal and maternal chromosomes organize on the equator at the metaphase spindle, at syngamy. Bipolarization occurs in all monospermic and in most dispermic ova. Dispermic embryos occasionally form two sperm asters initially and produce tripolar spindles (tripolarization). Anaphase and telophase follows producing two or three cells respectively, completing the first cycle. Descendants of the sperm centriole were found at every stage of preimplantation embryo development and were traced from fertilization and cleavage (first four cell cycles) to the morula and hatching blastocyst stage. Centrioles were associated with nuclei at interphase, when they were often replicating and occupied pivotal positions on spindle poles during mitosis. Sperm remnants were associated with centrioles and were found at most stages of cleavage. Centrioles were found in trophoblast, embryoblast and endoderm cells in hatching blastocysts (Sathananthan, 1997Go). This continued influence of sperm centriole not only provides a possible clue for the poor pregnancy and even worse live birth outcome with spermatids, but also highlights the importance of sperm quality.

The highly variable and unpredictable outcomes of human spermatid conception attempts, in terms of fertilization, implantation, fetal development and pregnancy loss, remain a major problem. The completeness of the meiotic reduction in spermatids does not mean all epigenetic, nuclear and cytoplasmic modifications have attained a behaviour level compatible with the fully mature cell state. This has led to some concerns on the possible risks to health of children born from the use of spermatids with ICSI (Tesarik et al., 1998Go). The genetic association of the level of spermatogenesis in adults means counselling of patients becomes imperative.

In limited animal models, germ cell transplantation has been encouraging but extrapolation to human application will require extreme caution. Following germ cell transplantation into the seminiferous tubular lumen of another mouse some spermatogenesis could be established (Russell et al., 1996Go). Under light and electron microscopy donor mouse cells formed normal associations as viewed in cross-sectional tubules. Spermatogonia were found exclusively in the basal compartment, indicating that they were translocated from the tubule lumen through the Sertoli cell junctions, eventually to reside on the basal lamina. Some tubules looked entirely normal whilst others showed impairment. In some tubules a generation of cells was missing from a cell association. A variety of degenerating cells and structural abnormalities were responsible for this impairment. However, the most common abnormalities were seen during the elongation phase of spermatogenesis. Elongation abnormalities and subsequent degeneration of the cells led to the presence of fewer than expected elongated spermatids (Russell et al., 1996Go). A second report involved primordial germ cell and gonocyte transplantation from rat fetuses and neonates (Jiang and Short, 1995Go) via the rete testis into the lumen of the seminiferous tubules of adult rats. The donor cells apparently differentiated into mini-tubules or irregular segments of seminiferous epithelium within the lumen of the host seminiferous tubules, and exhibited qualitatively normal spermatogenesis in 10 out of 16 recipients. With the synchronization of spermatogenesis between host and donor seminiferous epithelia this seemed a reasonable transplantation success. In the final example (Brinster and Zimmermann, 1994Go), the stem cells isolated from the testes of donor male mice repopulated the sterile adult recipient testes when injected into the seminiferous tubules. Donor cell spermatogenesis in recipient testes showed normal morphological characteristics and produced mature spermatozoa. In the adult male, a population of the diploid stem-cell spermatogonia continuously undergo self-renewal and produce progeny cells, which initiate the complex process of cellular differentiation that results in mature spermatozoa. In each of the three examples, healthy adult recipients were used and it would be interesting to have results of self implantation. In the immediate future, these animal models provide useful tools in the study of spermatogenesis mechanism.

Any in-vitro maturation of germ cells or in-vivo grafting of tissue would at least have to be evaluated for genetic safety. Ethical arguments against transplanting tumour-free tissue back to the patient should not arise provided the procedures are well established in model systems. Immunological responses against the tissue should be minimal as the tissue is `self'. Grafting of tissue on the existing testes would be a better option but the many questions pertaining to the safety of the process in an already damaged testis need to be addressed. Another possibility is to graft the whole testes onto a father's testes for the duration of about 6 months' treatment, then regraft them back to the child. This would be an interesting approach but there is no guarantee that the father's hormonal stature would not induce full spermatogenesis in the child's testes and, besides, the approach involves more surgery.

At puberty, however, spermaturia appears to give a reasonable indicator of the status of spermatogenesis (Nielsen et al., 1986Go; Kulin et al., 1989Go; Schaefer et al., 1990Go; Jorgensen et al., 1991Go; Pederson et al., 1993) and the likely success of sperm storage. These collective reports, however, cover a large age range, 10–18 years, and together with the level of patient drop-out, lead us to acknowledge its qualitative usefulness only. It is interesting to note also that spermaturia is a more common and regular event during early and mid-puberty rather than in more mature subjects, as graded by the Tanner stage pubic hair distribution (Nielsen et al., 1986Go). In particular, spermarche may occur when little or no pubic hair has developed and the testes have grown only slightly. This indicates that the mechanism of spermaturia in early and late puberty could be different. It had been suggested that spermaturia in non-virilized boys could be a result of a spontaneous, continuous flow of spermatozoa to the urethra in contrast with the peristaltic flow during ejaculation occurring at a later stage of puberty. In 274 semen assays of 134 pubertal boys, normal figures for semen volume, sperm concentration, morphology and motility were observed 12–14 months after the first ejaculation (Janczewski and Bablok, 1985aGo,bGo). The percentage of normally motile spermatozoa became standard after 21–23 months of first ejaculation. There were changes in semen characteristics from azoospermia through cryptozoospermia, oligozoospermia and asthenozoospermia to normospermia. Azoospermia dominates until the fifth month after the first ejaculation, oligozoospermia from the sixth to 11th month, asthenozoospermia from the 12th to the 20th month, and normospermia from the 21st month. With advances in ICSI, cryopreservation of motile spermatozoa should be undertaken irrespective of the quality or quantity, but only after giving the patient proper information and obtaining a valid consent. If spermatozoa are dead then the balance of decision may be against sperm storage. However, this needs to be carefully discussed in the light of even newer technologies that have come about with the advent of cloning sheep (Wilmut et al., 1997Go). The use of an electrical burst of energy after the introduction by ICSI of a dead spermatozoon in an oocyte would be a possible way towards inducing fertilization; advances in natural sperm factors promoting such fertilization may have possible applications in future. However, safety issues under present consideration are likely to weigh heavily against the use of dead gametes and request for regulatory permission for future human use is highly unlikely unless overall safety in human procreation can somehow be demonstrated.

Against this background we need to find a balance and need to know of the trauma involved in the invasive process of taking testicular biopsy. The pain and discomfort of having a biopsy needs to be considered, especially when the patient is unwell. Secondly, there is the issue of biopsy trauma induced testicular cancer (Swerdlow et al., 1997Go). In a study of 1075 boys with cryptorchidism, to determine the risk of testicular cancer in relation to undescended testes, biopsy appeared to be a stronger risk factor for testicular cancer, the commonest malignancy in men aged 15–34 years, than any other factor previously identified. Association between tubal sterilization and endometrial cancer has been considered but nothing significant has yet emerged (Rosenblatt and Thomas, 1997Go).

The pregnancy outcome and offspring born after childhood cancer do not provide evidence of germ cell mutagenesis, which can manifest in increased congenital malformations, neonatal mortality or cancers in offspring. However, much larger patient numbers would be needed to rule any of these associations out with confidence (Li et al., 1979Go; Otake et al., 1990Go; Yoshimoto et al., 1990Go; Hawkins, 1991Go, 1994Go; Dodds et al., 1993Go).

In the UK, the HFEA Act states that gametes must be stored on licensed premises and 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), storage of testicular tissue from boys who have reached at least Tanner Grade 2 stage will require a licence. 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. If a boy is prepubertal (pre-Tanner Grade 2) then his testicular tissue can be stored on unlicensed premises. If, however, such material were subsequently 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). At that stage an effective consent would also have to be given in accordance with the HFEA Act 1990. Consent to the removal of testicular tissue from any male of whatever age is covered by common law.

In females, ovarian tissue will not contain gametes according to the HFEA's definition for much of the menstrual cycle. Any gamete or gametes that are present will be localized in certain parts of the ovary. The HFEA requires that clinicians consider on a case by case basis whether ovarian tissue contains any gametes by reference to the stage of the woman's cycle or by appropriate testing or assessment of the tissue itself.

The legal position regarding research on children's germ cells needs to be understood. Future avenues may include, for example, xenotransplantation of germ cells in sterilized animal gonads. Under the HFEA Act (1990) one does not need the consent of the boys concerned to use sterilized mice or hamsters to develop the spermatozoa, but if the intention is to store the spermatozoa or inseminate mouse or hamster eggs then the boys' consent is needed. The consent of the parents will not suffice at this stage. If the boy has since died, it follows that the spermatozoa cannot be stored or used. However, if the boy is still alive and capable of giving informed consent then one may proceed with the experimental assessment of the xenotransplanted matured spermatozoa.

Safe in-vitro maturation and application of immature germ cells is still very distant and the HFEA plans to keep advances under review.

Notes

3 To whom correspondence should be addressed Back

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Submitted on August 8, 1998; accepted on October 7, 1998.