Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system.

STUDY QUESTION: Can complete oocyte development be achieved from human ovarian tissue containing primordial/unilaminar follicles and grown in vitro in a multi-step culture to meiotic maturation demonstrated by the formation of polar bodies and a Metaphase II spindle? SUMMARY ANSWER: Development of human oocytes from primordial/unilaminar stages to resumption of meiosis (Metaphase II) and emission of a polar body was achieved within a serum free multi-step culture system. WHAT IS KNOWN ALREADY: Complete development of oocytes in vitro has been achieved in mouse, where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. Human oocytes have been grown in vitro from the secondary/multi-laminar stage to obtain fully grown oocytes capable of meiotic maturation. However, there are no reports of a culture system supporting complete growth from the earliest stages of human follicle development through to Metaphase II. STUDY DESIGN, SIZE, DURATION: Ovarian cortical biopsies were obtained with informed consent from women undergoing elective caesarean section (mean age: 30.7 ± 1.7; range: 25-39 years, n = 10). PARTICIPANTS/MATERIALS, SETTING, METHODS: Laboratory setting. Ovarian biopsies were dissected into thin strips, and after removal of growing follicles were cultured in serum free medium for 8 days (Step 1). At the end of this period secondary/multi-laminar follicles were dissected from the strips and intact follicles 100-150 µm in diameter were selected for further culture. Isolated follicles were cultured individually in serum free medium in the presence of 100 ng/ml of human recombinant Activin A (Step 2). Individual follicles were monitored and after 8 days, cumulus oocyte complexes (COCs) were retrieved by gentle pressure on the cultured follicles. Complexes with complete cumulus and adherent mural granulosa cells were selected and cultured in the presence of Activin A and FSH on membranes for a further 4 days (Step 3). At the end of Step 3, complexes containing oocytes >100 µm diameter were selected for IVM in SAGE medium (Step 4) then fixed for analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Pieces of human ovarian cortex cultured in serum free medium for 8 days (Step 1) supported early follicle growth and 87 secondary follicles of diameter 120 ± 6 µm (mean ± SEM) could be dissected for further culture. After a further 8 days, 54 of the 87 follicles had reached the antral stage of development. COCs were retrieved by gentle pressure from the cultured follicles and those with adherent mural granulosa cells (n = 48) were selected and cultured for a further 4 days (Step 3). At the end of Step 3, 32 complexes contained oocytes >100 µm diameter were selected for IVM (Step 4). Nine of these complexes contained polar bodies within 24 h and all polar bodies were abnormally large. Confocal immuno-histochemical analysis showed the presence of a Metaphase II spindle confirming that these IVG oocytes had resumed meiosis but their developmental potential is unknown. LIMITATIONS, REASONS FOR CAUTION: This is a small number of samples but provides proof of concept that complete development of human oocytes can occur in vitro. Further optimization with morphological evaluation and fertilization potential of IVG oocytes is required to determine whether they are normal. WIDER IMPLICATIONS OF THE FINDINGS: The ability to develop human oocytes from the earliest follicular stages in vitro through to maturation and fertilization would benefit fertility preservation practice. STUDY FUNDING/COMPETING INTEREST(S): Funded by MRC Grants (G0901839 and MR/L00299X/1). No competing interests.

Non-growing follicle density is increased following adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) chemotherapy in the adult human ovary.

STUDY QUESTION: Do the chemotherapeutic regimens of ABVD (adriamycin, bleomycin, vinblastine and dacarbazine) or OEPA-COPDAC (combined vincristine, etoposide, prednisone, doxorubicin (OEPA) and cyclophosphamide, vincristine, prednisone, dacarbazine (COPDAC)) used to treat Hodgkin lymphoma (HL), affect the density, morphology and in vitro developmental potential of human ovarian follicles? SUMMARY ANSWER: Ovarian tissue from women treated with ABVD contained a higher density of non-growing follicles (NGFs) per cubic millimetre and increased numbers of multiovular follicles but showed reduced in vitro growth compared with patients with lymphoma who had not received chemotherapy, patients treated with OEPA-COPDAC, age-matched healthy women and age-related model-predicted values. WHAT IS KNOWN ALREADY: Chemotherapy regimens can cause a loss of follicles within the ovary, which depends on the drugs given. Early stage HL is commonly treated by ABVD, a non-alkylating regimen that apparently has ovarian sparing qualities; thus it is important to investigate the histological appearance and distribution of follicles within ABVD-treated ovarian tissue. STUDY DESIGN, SIZE, DURATION: Thirteen ovarian biopsies were obtained from HL patients (six adolescents and seven adults) and one biopsy from a non-HL patient. Two HL patients and the non-HL patient had received no treatment prior to biopsy collection. The remaining 11 HL patients received one of two regimens: ABVD or OEPA-COPDAC. Tissue was analysed histologically and compared to biopsies from healthy women, and in a subgroup of patients, tissue was cultured for 6 days in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian biopsies were obtained from patients undergoing ovarian cryopreservation for fertility preservation and from healthy women at the time of Caesarian section ('obstetric tissue'). Follicle number and maturity were evaluated in sections of ovarian cortical tissue, and compared to an age-related model of mean follicle density and to age-matched contemporaneous biopsies. The developmental potential of follicles was investigated after 6 days of tissue culture. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 6877 follicles were analysed. ABVD-treated tissue contained a higher density of NGFs per cubic millimetre (230 ± 17) (mean ± SEM) than untreated (110 ± 54), OEPA-COPDAC-treated (50 ± 27) and obstetric (20 ± 4) tissue (P < 0.01), with follicle density 9-21 SD higher than predicted by an age-related model. Biovular and binucleated NGFs occurred frequently in ABVD-treated and in adolescent-untreated tissue but were not observed in OEPA-COPDAC-treated or obstetric tissue, although OEPA-COPDAC-treated tissue contained a high proportion of morphologically abnormal oocytes (52% versus 23% in untreated, 22% in ABVD-treated and 25% in obstetric tissue; P < 0.001). Activation of follicle growth in vitro occurred in all groups, but in ABVD-treated samples there was very limited development to the secondary stage, whereas in untreated samples from lymphoma patients growth was similar to that observed in obstetric tissue (untreated; P < 0.01 versus ABVD-treated, NS versus obstetric). LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: Although a large number of follicles were analysed, these data were derived from a small number of biopsies. The mechanisms underpinning these observations have yet to be determined and it is unclear how they relate to future fertility. WIDER IMPLICATIONS OF THE FINDINGS: This study confirms that the number of NGFs is not depleted following ABVD treatment, consistent with clinical data that female fertility is preserved. Our findings demonstrate that immature follicle density can increase as well as decrease following at least one chemotherapy treatment. This is the first report of morphological and follicle developmental similarities between ABVD-treated tissue and the immature human ovary. Further experiments will investigate the basis for the marked increase in follicle density in ABVD-treated tissue. STUDY FUNDING/COMPETING INTERESTS: Funded by UK Medical Research Council Grants G0901839 and MR/L00299X/1. The authors have no competing interests.

Re-implantation of cryopreserved ovarian cortex resulting in restoration of ovarian function, natural conception and successful pregnancy after haematopoietic stem cell transplantation for Wilms tumour.

With the improvement of long-term cancer survival rates, growing numbers of female survivors are suffering from treatment-related premature ovarian insufficiency (POI). Although pre-treatment embryo and oocyte storage are effective fertility preservation strategies, they are not possible for pre-pubertal girls or women who cannot delay treatment. In these cases, the only available treatment option is ovarian cortex cryopreservation and subsequent re-implantation. A 32-year-old woman had ovarian cortex cryopreserved 10 years previously before commencing high-dose chemotherapy and undergoing a haematopoietic stem cell transplant for recurrent adult Wilms tumour, which resulted in POI. She underwent laparoscopic orthotopic transplantation of cryopreserved ovarian cortex to the original site of biopsy on the left ovary. She ovulated at 15 and 29 weeks post-re-implantation with AMH detectable, then rising, from 21 weeks, and conceived naturally following the second ovulation. The pregnancy was uncomplicated and a healthy male infant was born by elective Caesarean section at 36+4 weeks gestation. This is the first report of ovarian cortex re-implantation in the UK. Despite the patient receiving low-risk chemotherapy prior to cryopreservation and the prolonged tissue storage duration, the re-implantation resulted in rapid restoration of ovarian function and natural conception with successful pregnancy.

An externally validated age-related model of mean follicle density in the cortex of the human ovary.

PURPOSE: The ability to accurately estimate a woman's ovarian reserve by non-invasive means is the goal of ovarian reserve prediction. It is not known whether a correlation exists between model-predicted estimates of ovarian reserve and data generated by direct histological analysis of ovarian tissue. The aim of this study was to compare mean non-growing follicle density values obtained from analysis of ovarian cortical tissue samples against ovarian volume models. METHODS: Non-growing follicle density values were obtained from 13 ovarian cortical biopsies (16-37 years). A mean non-growing follicle density was calculated for each patient by counting all follicles in a given volume of biopsied ovarian cortex. These values were compared to age-matched model generated densities (adjusted to take into consideration the proportion of ovary that is cortex) and the correlation between data sets tested. RESULTS: Non-growing density values obtained from fresh biopsied ovarian cortical samples closely matched model generated data with low mean difference, tight agreement limits and no proportional error between the observed and predicted results. CONCLUSION: These findings validate the use of the adjusted population and ovarian volume models, to accurately predict mean follicle density in the ovarian cortex of healthy adult women.

The immature human ovary shows loss of abnormal follicles and increasing follicle developmental competence through childhood and adolescence.

STUDY QUESTION: Do the ovarian follicles of children and adolescents differ in their morphology and in vitro growth potential from those of adults? SUMMARY ANSWER: Pre-pubertal ovaries contained a high proportion of morphologically abnormal non-growing follicles, and follicles showed reduced capacity for in vitro growth. WHAT IS KNOWN ALREADY: The pre-pubertal ovary is known to contain follicles at the early growing stages. How this changes over childhood and through puberty is unknown, and there are no previous data on the in vitro growth potential of follicles from pre-pubertal and pubertal girls. STUDY DESIGN, SIZE, DURATION: Ovarian biopsies from five pre-pubertal and seven pubertal girls and 19 adult women were analysed histologically, cultured in vitro for 6 days, with growing follicles then isolated and cultured for a further 6 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian biopsies were obtained from girls undergoing ovarian tissue cryopreservation for fertility preservation, and compared with biopsies from adult women. Follicle stage and morphology were classified. After 6 days in culture, follicle growth initiation was assessed. The growth of isolated secondary follicles was assessed over a further 6 days, including analysis of oocyte growth. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-pubertal ovaries contained a high proportion of abnormal non-growing follicles (19.4 versus 4.85% in pubertal ovaries; 4004 follicles analysed; P = 0.02) characterized by indistinct germinal vesicle membrane and absent nucleolus. Follicles with this abnormal morphology were not seen in the adult ovary. During 6 days culture, follicle growth initiation was observed at all ages; in pre-pubertal samples there was very little development to secondary stages, while pubertal samples showed similar growth activation to that seen in adult tissue (pubertal group: P = 0.02 versus pre-pubertal, ns versus adult). Isolated secondary follicles were cultured for a further 6 days. Those from pre-pubertal ovary showed limited growth (P < 0.05 versus both pubertal and adult follicles) and no change in oocyte diameter over that period. Follicles from pubertal ovaries showed increased growth; this was still reduced compared with follicles from adult women (P < 0.05) but oocyte growth was proportionate to follicle size. LIMITATIONS, REASONS FOR CAUTION: These data derive from only a small number of ovarian biopsies, although large numbers of follicles were analysed. It is unclear whether the differences between groups are related to puberty, or just age. WIDER IMPLICATIONS OF THE FINDINGS: These findings show that follicles from girls of all ages can be induced to grow in vitro, which has important implications for some patients who are at high risk of malignant contamination of their ovarian tissue. The reduced growth of isolated follicles indicates that there are true intrafollicular differences in addition to potential differences in their local environment, and that there are maturational processes occurring in the ovary through childhood and adolescence, which involve the loss of abnormal follicles, and increasing follicle developmental competence. STUDY FUNDING/COMPETING INTEREST(S): Funded by MRC grants G0901839 and G1100357. No competing interests.

Physiological sex steroid replacement in premature ovarian failure: randomized crossover trial of effect on uterine volume, endometrial thickness and blood flow, compared with a standard regimen.

BACKGROUND: Premature ovarian failure (POF) is currently managed by non-physiological sex steroid regimens which are inadequate at optimizing uterine characteristics. Previous short-term studies have demonstrated some benefits of a sex steroid replacement (SSR) regimen devised to replicate the physiological cycle. This study aimed to directly compare the effects of longer-term administration of physiological SSR (pSSR) and standard SSR (sSSR) regimens on the uterine volume, blood flow and endometrial thickness (ET) in women with POF. METHODS: In a controlled crossover trial, 34 women with POF were randomized to receive 12 months of 4-week cycles of transdermal estradiol and vaginal progesterone (pSSR) followed by 12 months of 4-week cycles of oral ethinylestradiol and norethisterone (sSSR), or vice versa. Each treatment period was preceded by a 2-month washout period. At 0, 3, 6 and 12 months of each treatment period, transvaginal ultrasound examined the uterine volume and ET, as primary end-points, and uterine artery resistance (UARI) and pulsatility indices (UAPI), as secondary end-points. Serum estradiol, progesterone and gonadotrophins were also measured. RESULTS: Of the 29 women eligible for the uterine analysis, 17 completed the entire study protocol, but 25 women contributed data to statistical analysis of treatment effect. There was a greater estimated mean ET with the use of pSSR (4.8 mm) compared to that with standard therapy (3.0 mm), with an estimated difference of 1.8 mm [95% confidence interval (CI), 0.7 to 2.8, P=0.002]. The estimated mean uterine volume was also greater during physiological treatment (24.8 cm(3)) than during standard treatment (20.6 cm(3)), but the estimated difference of 4.2 cm(3) (95% CI -0.4 to 8.7) was not statitsically significant, P=0.070. The small differences between the two treatments in the mean UARI and mean UAPI were not statistically significant. The estimated treatment differences were fairly constant across the treatment periods, suggesting that prolonged treatment does not increase response. CONCLUSIONS: pSSR has a greater beneficial effect upon ET in women with POF in comparison with standard therapy. A similar trend was seen for uterine volume. Further studies are required to optimize treatment and to assess pregnancy rate and outcome. Trial Registration www.ClinicalTrials.gov, NCR00732693.

Do phthalates affect steroidogenesis by the human fetal testis? Exposure of human fetal testis xenografts to di-n-butyl phthalate.

CONTEXT: Phthalates are ubiquitous environmental chemicals. Fetal exposure to certain phthalates [e.g. di-n-butyl phthalate (DBP)] causes masculinization disorders in rats, raising concern for similar effects in humans. We investigated whether DBP exposure impairs steroidogenesis by the human fetal testis. OBJECTIVE: The aim of the study was to determine effects of DBP exposure on testosterone production by normally growing human fetal testis xenografts. DESIGN: Human fetal testes (14-20 wk gestation; n=12) were xenografted into castrate male nude mice that were treated for 4-21 d with vehicle, or 500 mg/kg·d DBP, or monobutyl phthalate (active metabolite of DBP); all mice were treated with human chorionic gonadotropin to mimic normal human pregnancy. Rat fetal testis xenografts were exposed for 4 d to DBP as a positive control. MAIN OUTCOME MEASURES: Testosterone production was assessed by measuring host serum testosterone and seminal vesicle (SV) weights at termination, plus testis gene expression (rats). RESULTS: Human fetal testis xenografts showed similar survival (∼80%) and total graft weight (8.6 vs. 10.1 mg) in vehicle and DBP-exposed hosts, respectively. Serum testosterone (0.56 vs. 0.64 ng/ml; P>0.05) and SV weight (67.2 vs. 81.9 mg; P>0.05) also did not differ. Exposure to monobutyl phthalate gave similar results. In contrast, exposure of rat fetal xenografts to DBP significantly reduced SV weight and testis Cyp11a1/StAR mRNA expression and lowered testosterone levels, confirming that DBP exposure can inhibit steroidogenesis in xenografts, further validating the negative findings on testosterone production in the human. CONCLUSIONS: Exposure of human fetal testes to DBP is unlikely to impair testosterone production as it does in rats. This has important safety and regulatory implications.

Survivors of childhood cancer lost to follow-up can be re-engaged into active long-term follow-up by a postal health questionnaire intervention.

UNLABELLED: Lifelong long-term follow-up (LTFU) is recommended for all survivors of childhood cancer. National guidelines recommend risk-stratified levels of follow-up by a multidisciplinary team, in an age-appropriate environment. Many survivors do not participate in long-term follow-up. OBJECTIVE: To re-engage childhood cancer survivors lost to follow-up in late effects programmes by means of postal questionnaire. POPULATION AND METHODS: Retrospective cohort study of all children (<19 years) diagnosed with cancer in a single institution in the UK between 1971 and 2003. All lost to follow-up survivors (not seen in clinic >2 years) were sent a postal health and well-being questionnaire. RESULTS: 831 patients were diagnosed with childhood cancer between 1971 and 2003, with 575 long-term survivors (overall survival rate 69%). Information was available on 550 survivors (males 290 (53%), median age (range) at review 18.8 (5.4-44.2) years and at diagnosis 5.0 (0.0-18.8) years, and disease free survival (range) was 10.8 (1.0-37.4) years. Of the 550 survivors, 256 (46%) were lost to follow-up. 99 (39%) of lost to follow-up survivors returned completed postal questionnaires (58% female). 45% of responders reported at least one late effect, 36% mild-moderate, and 8% severe-life threatening. 19% reported two or more late effects. 74% of all childhood cancer survivors are now in active follow-up. CONCLUSIONS: Almost half (46%) of all long-term survivors of childhood cancer are lost to follow-up, Postal follow-up is an effective means of re-engaging more than one third of survivors of childhood cancer in active long-term follow-up, half of whom had at least one late effect.

Measuring anti-Müllerian hormone for the assessment of ovarian reserve: when and for whom is it indicated?

Our understanding of female reproductive function has been hampered by our inability to directly assess the number of non-growing primordial follicles present in the ovary, the ovarian reserve. Female reproductive hormones (FSH and LH, the inhibins and steroids) reflect the activity of the larger growing follicles and thus are largely informative of peri-ovulatory ovarian activity. In contrast anti-Müllerian hormone (AMH) is a product of the granulosa cells of small growing follicles, whose number (and therefore circulating AMH concentrations) is reflective of the ovarian reserve. AMH declines with age in adult women, and emerging data suggest a relationship with remaining reproductive lifespan and age at the menopause. Early studies demonstrated that AMH concentrations are stable across the menstrual cycle, adding to its clinical utility. The most established role for AMH measurement is in women about to start IVF treatment, where it is predictive of the ovarian response and is of clear value in identifying women at risk of ovarian hyperstimulation syndrome or whose response will be poor and thus their expectations can be tailored. AMH is detectable in childhood, and although relationships to puberty are not yet available, it appears that AMH rises to a peak in the early 20s. Developing indications include in assessment and individualisation of the risk to fertility from chemotherapy, in the diagnosis of PCOS and as a tumour marker in granulosa cell tumours. The increasingly routine use of AMH by IVF clinics heralds much wider adoption in a range of clinical situations across the reproductive lifespan.

Data-driven assessment of the human ovarian reserve.

Human ovarian physiology is still poorly understood, with the factors and mechanisms that control initiation of follicular recruitment and loss remaining particularly unclear. Conventional hypothesis-led studies provide new data, results and insights, but datasets from individual studies are often small, allowing only limited interpretation. Great power is afforded by the aggregation of data from multiple studies into single datasets. In this paper, we describe how modern computational analysis of these datasets provides important new insights into ovarian function and has generated hypotheses that are testable in the laboratory. Specifically, we can hypothesize that age is the most important factor for variations in individual ovarian non-growing follicle (NGF) populations, that anti-Müllerian hormone (AMH) levels generally rise and fall in childhood years before peaking in the mid-twenties, and that there are strong correlations between AMH levels and both NGF populations and rates of recruitment towards maturation, for age ranges before and after peak AMH levels.

Individual ovarian volumes obtained from 2-dimensional and 3-dimensional ultrasound lack precision.

Measuring ovarian volume has been suggested as a possible screening test to assess a woman's ovarian reserve. For such a screening tool to be clinically useful, knowledge of its precision and reproducibility is essential. Recent advances in ultrasound scanning techniques allow the measurement of volumes in three dimensions rather than the traditional estimation from two dimensions. Transvaginal 2-dimensional (2D) and 3-dimensional (3D) ultrasound examinations were performed on 49 women attending a tertiary centre for investigation or treatment for subfertility between January and May 2006. Two observers calculated ovarian volume using both 2D (prolate ellipsoid formula) and 3D techniques [virtual organ computer-aided analysis (VOCAL)] with rotation steps of 30 degrees (3D-30). For the four comparisons (inter- and intra-observer; 2D and 3D-30) intraclass coefficients of 0.97 to 0.98, and standard errors ranging from 17% to 14% (for inter-observer 2D and intra-observer 3D, respectively) were obtained. The corresponding coefficients of repeatability ranged from 33% to 28%. These results suggest that measurement of transvaginal ovarian volumes using both 2D and 3D ultrasound is imprecise for individuals. The imprecision is greater for lower ovarian volumes, which may be important in clinical practice. The average of two or more measurements is likely to be more accurate than a single measurement.

Male fertility and strategies for fertility preservation following childhood cancer treatment.

Infertility in the male is a potential complication of childhood cancer treatment for long-term survivors. The risk is dependent primarily on the treatment used, but also on the underlying disease. Chemotherapy (especially alkylating agents) and radiotherapy, even in low doses, may damage the seminiferous epithelium and impair spermatogenesis in both children and adults. Leydig cell function and testosterone production are generally preserved after chemotherapy and low dose radiotherapy, whilst larger doses of radiotherapy may result in hypogonadism. Patients treated with potentially gonadotoxic treatments require regular multidisciplinary follow-up including assessment of puberty and gonadal function. Currently the only option available for fertility preservation in young males treated for cancer is semen cryopreservation. For pre-pubertal patients, techniques for fertility preservation remain theoretical and as yet unproven. These include hormonal manipulation of the gonadal environment before treatment, germ cell transplantation and testis xenografting, which have all shown promise in a variety of animal studies. Refinement of these techniques requires investigations in relevant animal models. In the present chapter we include data which suggest that the common marmoset (Callithrix jacchus) monkey, a New World primate, exhibits important parallels with human testicular development and may help us to understand why the pre-pubertal testis is vulnerable to effects of cytotoxic therapy on future fertility.

Germ cell differentiation in the marmoset (Callithrix jacchus) during fetal and neonatal life closely parallels that in the human.

BACKGROUND: Testicular germ cell tumours (TGCT) are thought to originate from fetal germ cells that fail to differentiate normally, but no animal model for these events has been described. We evaluated the marmoset (Callithrix jacchus) as a model by comparing perinatal germ cell differentiation with that in humans. METHODS: Immunohistochemical profiling was used to investigate germ cell differentiation (OCT4, NANOG, AP-2gamma, MAGE-A4, VASA, NANOS-1) and proliferation (Ki67) in fetal and neonatal marmoset testes in comparison with the human and, to a lesser extent, the rat. RESULTS: In marmosets and humans, differentiation of gonocytes into spermatogonia is associated with the gradual loss of pluripotency markers such as OCT4 and NANOG, and the expression of germ cell-specific proteins such as VASA. This differentiation occurs asynchronously within individual cords during fetal and early postnatal life. This contrasts with rapid and synchronous germ cell differentiation within and between cords in the rat. Similarly, germ cell proliferation in the marmoset and human occurs throughout perinatal life, in contrast to rats in which proliferation ceases during this period. CONCLUSIONS: The marmoset provides a good model for normal human germ cell differentiation and proliferation. The perinatal marmoset may be a useful model in which to establish factors that lead to failure of normal germ cell differentiation and the origins of TGCT.

Ovarian cryopreservation for fertility preservation: indications and outcomes.

Female fertility preservation provides significantly different challenges to that for the male, with the only established method being cryopreservation of embryos thus necessitating the involvement of a male. Other, experimental, options include oocyte or ovarian tissue cryopreservation. The latter has been regarded as a potential method for more than a decade, but has resulted in the birth of only five babies. It is not possible to be certain how many women have had ovarian tissue cryopreserved. Oocyte cryopreservation also remains experimental, but approximately 100-fold more babies have been born through this technique over the last two decades. Ovarian tissue cryopreservation has the potential advantages of preservation of a large number of oocytes within primordial follicles, it does not require hormonal stimulation when time is short and indeed may be appropriate for the pre-pubertal. Disadvantages include the need for an invasive procedure, and the uncertain risk of ovarian contamination in haematological and other malignancies. We here review this approach in the context of our own experience of 36 women, highlighting issues of patient selection especially in the young, and uncertainties over the effects of cancer treatments on subsequent fertility. Of these 36 women, 11 have died but 5 have had spontaneous pregnancies. So far, none have requested reimplantation of their stored ovarian tissue. Ovarian cryopreservation appears to be a potentially valuable method for fertility preservation, but the indications and approaches best used remain unclear.

Hodgkin's lymphoma in children aged 5 years or less - the United Kingdom experience.

PURPOSE: The aim of this study is to describe the natural history of Hodgkin's Lymphoma (HL) in a large unselected group of children aged 5 years or below at diagnosis, who were treated on a standard treatment programme in the United Kingdom between 1982 and 2000. METHODS: Eighty-one unselected children with HL aged 5 years or under at diagnosis, treated on the United Kingdom Children's Cancer Study Group (UKCCSG) Hodgkin's trials HD1 (1982-1992) and HD2 (1992-2000), were included in the study. RESULTS: Sixty-one patients (81%) presented with early stage disease (n=66). Fifty-three patients (65%) received combination chemotherapy, 28 (34%) received involved field radiotherapy (IF-RT) and 4 patients were treated with combined modality therapy. Eighteen children relapsed after primary therapy. CONCLUSIONS: Children treated with IF-RT had a higher rate of primary treatment failures as well as increased late treatment-related morbidity.

Undetectable salivary testosterone in young women with premature ovarian failure.

BACKGROUND: The extent of androgen deficiency in young women with premature ovarian failure (POF) is unclear. AIM: Cross-sectional study of androgen status in young women with POF. PATIENTS: Twenty women with POF: six had Turner syndrome (group A); eight had iatrogenic POF either secondary to bilateral oophorectomy or treatment of malignancy (group B); and six had idiopathic POF (group C). The median age was 30.5 years (range 19-39); in groups B and C the median duration of ovarian failure was 10.0 years (range 1-35). METHODS: After a 2-month wash-out period without sex steroid replacement (SSR), serum testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEAS), SHBG, salivary testosterone (SalT) and the free androgen index [FAI = (serum T/SHBG) x 100] were measured. RESULTS: Median serum A4 was 4.6 nmol/l (10th, 90th centiles, 3.6, 5.1) and DHEAS was 3.2 micromol/l (10th, 90th centiles, 2.3, 9.3). Although median serum T was relatively low at 1.4 nmol/l (10th, 90th centiles, 1.1, 1.7), median SHBG was also low at 34 nmol/l (10th, 90th centiles 22.2, 67.5) and the median calculated FAI was within the normal range at 3.7 (10th, 90th centiles, 2.3, 7.0). However, SalT was undetectable in almost all subjects in the three groups of POF. CONCLUSIONS: Serum T and SHBG are relatively low in young women with POF and their FAI is therefore within the normal range. However, SalT, which measures free testosterone, is consistently low to undetectable in these young women with POF. The reliability of the FAI as a marker of androgen deficiency remains questionable.

Subfertility in children and young people treated for solid and haematological malignancies.

Children treated for cancer may exhibit impaired fertility in later life. A number of chemotherapeutic agents have been identified as being gonadotoxic, and certain treatment regimens are particularly associated with subsequent infertility. Radiotherapy can also cause gonadal damage, most notably after direct testicular or pelvic irradiation or following total body irradiation. Because of the varied nature of the cytotoxic insult, it can be difficult to predict the likelihood of infertility in later life. Currently, cryopreservation of spermatozoa, oocytes or embryos is the only method of preserving fertility in patients receiving gonadotoxic therapy. This is only applicable to postpubertal patients and can be problematic in the adolescent age group. At present there is no provision for the prepubertal child, although there are a number of experimental methods being investigated. However, in addition to the many scientific and technical issues to be overcome before clinical application of such techniques, a number of ethical and legal issues must also be addressed to ensure a safe and realistic prospect for future fertility in these patients.

A comparison of self-reported satisfaction between adolescents treated in a "teenage" unit with those treated in adult or paediatric units.

BACKGROUND: Despite recommendations that adolescents should have in-patient management amongst their peers, there is little literature to support this. The study aim was to evaluate and contrast patient satisfaction for teenage cancer patients treated in two settings. The first is a split site unit (a paediatric ward and adult cancer centre in different locations within one city) and the second, a dedicated adolescent unit for patients aged 13-20. PROCEDURE: Eligible patients aged 13-20 years received treatment from September 1997 to June 2000 and totalled sixty-five adolescents. The patients were identified at both centres from departmental databases. Postal questionnaires (the Youth Satisfaction Questionnaire) were sent to those eligible. RESULTS: Patients receiving treatment in the teenage cancer unit (TCU) were not significantly more satisfied overall than those receiving treatment in adult or paediatric units. However, significant differences were noted in: recreational and relaxation facilities (P < 0.005, P < 0.0002), studying space (P < 0.004), ward noise (P < 0.02), and company of the same age (P < 0.0001). The Grade Point Average (a score of all specific items) was higher in favour of the TCU (P < 0.03). Patients at both centres were dissatisfied with hospital food and menus offered. CONCLUSIONS: Adolescents with cancer are satisfied with the overall care they receive independent of whether it is a TCU or a split site unit. Teenagers are significantly more satisfied with environmental aspects of care in the TCU. More research is required to establish the correct provision for teenagers with cancer. This is the first study that contrasts satisfaction between different centres and thus adding to an understanding of the needs of teenagers with cancer.