Roger G. Gosden

Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin's lymphoma.

BACKGROUND Infertility is a common late effect of chemotherapy and radiotherapy, and has a substantial effect on the quality of life for young survivors of cancer. For men, semen cryopreservation is a simple way of preserving reproductive potential but for women, storage of mature eggs rarely proves successful, and the alternative-immediate in vitro fertilisation with cryopreservation of embryos-is not always appropriate. Reimplantation of cryopreserved ovarian tissue has been shown to restore natural fertility in animals. We applied this technique in a woman who had received sterilising chemotherapy for lymphoma. METHODS A 36-year-old woman underwent a right oophorectomy with cryopreservation of ovarian cortical strips before receiving high-dose CBV chemotherapy for a third recurrence of Hodgkins lymphoma. 19 months later, when serum sex steroid analysis confimed a postmenopausal state, two ovarian cortical strips were thawed and reimplanted-one onto the left ovary and another at the site of the right ovary. FINDINGS 7 months after reimplantation of ovarian cortical strips, the patient reported resolution of hot flashes and, for the first time, oestradiol was detected in the serum. This finding was associated with a decrease in the concentrations of follicle-stimulating hormone and luteinising hormone, and ultrasonography revealed a 10 mm thick endometrium, a poorly visualised left ovary, and a 2 cm diameter follicular structure to the right of the midline. The patient had one menstrual period, but by 9 months after the implantation, her sex steroid concentrations had returned to those seen with ovarian failure. INTERPRETATION Orthotopic reimplantation of frozen/thawed ovarian cortical strips is a well tolerated technique for restoring ovarian function in women treated with sterilising chemotherapy for cancer.

Rare congenital disorders, imprinted genes, and assisted reproductive technology

CONTEXT During the past two decades, assisted reproductive technologies (ARTs) have revolutionised the treatment of infertility. ARTs now account for between 1% and 3% of annual births in many western countries and in-vitro fertilisation (IVF) services are growing worldwide. In general, the incidence of abnormalities at birth is reassuringly low and children develop normally. Nevertheless, it is important to monitor the safety of ARTs as clinical protocols evolve and new technologies emerge. STARTING POINT Three recent studies all report an unexpectedly high incidence of Beckwith-Wiedemann syndrome (BWS) in children conceived with ARTs. Six of 149 cases were reported from a British BWS registry (J Med Genet 2003; 40: 62-64); the same numbers were recorded in a French registry (Am J Hum Genet 2003; 72: 1338-41), and a further seven children have been reported in the USA (Am J Hum Genet 2003; 72: 156-60). These frequencies are extraordinarily high for such a rare congenital condition and such findings are reminiscent of reports of sporadic cases of the imprinting disorder, Angelman syndrome, which has also been linked with ARTs. WHERE NEXT? Continuing surveillance of children conceived with ARTs is needed, including monitoring birth defects, development, and cancer. Studies will need to be prospective and multicentre, and should include molecular characterisation of epigenetic abnormalities, including the methylation status of imprinting control regions within imprinted gene clusters.

Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue.

OBJECTIVE To develop an efficient isolation technique for human primordial follicles. DESIGN Prospective, experimental study of ovarian biopsies collected from healthy women undergoing elective cesarean section. Ovarian blocks either were fixed for histology and follicle counting or partially disaggregated with type 1A collagenase before or after cryopreservation. After partial disaggregation, follicles were isolated by microdissection. SETTING Leeds General Infirmary. MAIN OUTCOME MEASURE(S) Follicle viability was assessed with live-dead stains using 5-(and 6-) carboxyfluorescein diacetate, succinimidyl ester and propidium iodide, respectively, and using electron microscopy. The numbers recovered were expressed as a percentage of the numbers of primordial follicles in comparable blocks of tissue and the viability of the whole follicle and oocyte were scored separately. RESULT(S) On average, 18.0 +/- 3.8 and 15.9 +/- 2.2 (mean +/- SEM) follicles per block were recovered from fresh and cryopreserved ovarian tissue, respectively, corresponding to recovery rates of 57.9% +/- 8.8% and 56.2% +/- 16.7%. In the fresh group, the percent viability of whole follicles and oocytes were 71.6% +/- 2.4% and 91.3% +/- 2% compared with 71.5% +/- 4.7% and 95% +/- 4.3% in the frozen-thawed group. Electron microscopy confirmed that the majority of the cells lacked ultrastructural signs of damage after isolation and cryopreservation. CONCLUSION(S) Primordial follicles can be isolated from fresh and cryopreserved human ovarian tissue with similar high efficiency and viability rates.

Spindles, mitochondria and redox potential in ageing oocytes

Studies of human oocytes obtained from women of advanced reproductive age revealed that spindles are frequently aberrant, with chromosomes sometimes failing to align properly at the equator during meiosis I and II. Chromosomal analyses of donated and spare human oocytes and cytogenetic and molecular studies on the origin of trisomies collectively suggest that errors in chromosome segregation during oogenesis increase with advancing maternal age and as the menopause approaches. Disturbances in the fidelity of chromosome segregation, especially at anaphase I, leading to aneuploidy are prime causes of reduced developmental competence of embryos in assisted reproduction, as well as being responsible for the genesis of genetic disease. This review provides an overview of spindle formation and chromosome behaviour in mammalian oocytes. Evidence of a link between abnormal mitochondrial function in oocytes and somatic follicular cells, and finally disturbances in chromosome cohesion and segregation, and cell cycle control in aged mammalian oocytes, are also discussed.

Successful pregnancy after microsurgical transplantation of an intact ovary.

This case demonstrates the potential for using whole-ovary transplantation between monozygotic twins who are discordant for premature ovarian failure, in order to restore fertility in the affected ...

Low temperature storage and grafting of human ovarian tissue.

Ovarian tissue storage at low temperatures is a promising new method for protecting young cancer patients from the sterilizing effects of chemotherapy and/or radiotherapy. Tissue can be stored and returned to the body in due course as a thin cortical graft since the primordial follicles are distributed peripherally and are relatively resistant to ischaemia. Slices of tissue donated by healthy patients were trimmed to a uniform size and preserved by slow freezing to liquid nitrogen temperatures for up to 2 months in one of the following cryoprotectants: dimethylsulphoxide, ethylene glycol, glycerol, propylene glycol. Their viability was assessed by counting follicles in histological sections 18 days after grafting under the kidney capsules of severe combined immunodeficiency (SCID) mice, and the results were expressed as percentages of the numbers in comparable pieces of ungrafted tissue. While only 10% of the total number of follicles was found in the glycerol group compared to controls, significantly higher percentages (44-84%) survived cryopreservation in the other media. The tissues were sterile when frozen and thawed without a cryoprotectant. These results suggest that if comparable results could be achieved by autografting, a patients fertility should be safeguarded from cytotoxic treatment.

Continuous loss of oocytes throughout meiotic prophase in the normal mouse ovary.

The number of germ cells reaches the maximum just prior to entry into meiosis, yet decreases dramatically by a few days after birth in the female mouse, rat, and human. Previous studies have reported a major loss at the pachytene stage of meiotic prophase during fetal development, leading to the hypothesis that chromosomal pairing abnormalities may be a signal for oocyte death. However, the identification as well as the quantification of germ cells in these studies have been questioned. A recent study using Mouse Vasa Homologue (MVH) as a germ cell marker reached a contradictory conclusion claiming that oocyte loss occurs in the mouse only after birth. In the present study, we established a new method to quantify murine germ cells by using Germ Cell Nuclear Antigen-1 (GCNA-1) as a germ cell marker. Comparison of GCNA-1 and MVH immunolabeling revealed that the two markers identify the same population of germ cells. However, nuclear labeling of GCNA-1 was better suited for counting germ cells in histological sections as well as for double labeling with the antibody against synaptonemal complex (SC) proteins in chromosome spreading preparations. The latter experiment demonstrated that the majority of GCNA-1-labeled cells entered and progressed through meiotic prophase during fetal development. The number of GCNA-1-positive cells in the ovary was estimated by counting the labeled cells retained in chromosome spreading preparations and also in histological sections by using the ratio estimation method. Both methods demonstrated a continuous decline in the number of GCNA-1-labeled cells during fetal development when the oocytes progress through meiotic prophase. These observations suggest that multiple causes are responsible for oocyte elimination.

A laparoscopic technique for obtaining ovarian cortical biopsy specimens for fertility conservation in patients with cancer

OBJECTIVE To evaluate the efficacy of a newly designed round biopter as a practical and safe method for collecting ovarian tissue for cryopreservation in young women with cancer before chemotherapy. DESIGN Prospective study of young women volunteering for research (Leeds, United Kingdom) and patients with cancer (Jerusalem, Israel and Leeds, United Kingdom) undergoing laparoscopic ovarian cortical tissue biopsy and cryopreservation before administration of high-dose radiochemotherapy. SETTING Two university-based tertiary referral centers of oncology and gynecology (Hadassah Medical Center, Israel; Leeds General Infirmary, United Kingdom). PATIENT(S) Twenty female volunteers undergoing routine laparoscopic gynecologic procedures (age, 25-34 years) and 20 young women (age, 11-30 years) with advanced cancer requiring potentially sterilizing radiochemotherapy. INTERVENTION(S) Cortical ovarian tissue biopsies performed under laparoscopy with use of the round biopter. RESULT(S) The laparoscopic sampling procedure was uncomplicated in all cases. In treated patients, five to six samples were obtained (5 mm in diameter; 2-3 mm in depth) using the round biopter, and radiochemotherapy was administered without delay. In volunteers, no adhesions were noted at repeat laparoscopy (9 patients). All biopsy specimens were cryopreserved, and histologic examination confirmed the presence of many primordial follicles. CONCLUSION(S) Laparoscopic ovarian biopsy performed with the round biopter is a safe and efficient method for collecting ovarian tissue for cryopreservation in patients with cancer.

In vitro growth of human primordial follicles from frozen-banked ovarian tissue

The rarity of human oocytes frequently limits the success of assisted reproductive technology and delays research progress. Development of technologies to grow mature oocytes from the more abundant small follicles, perhaps after long-term storage at low temperatures, is a theoretically attractive solution to both problems. The length of the follicular growth span from the primordial to Graafian stage and changes in the trophic requirements of the cells, cellular interactions, morphogenesis and the sheer increase in bulk as the antrum forms are major challenges for cell culture technology. Even so, much progress has been made with animal follicles, and has begun with human tissue. A multi-step procedure, which reflects these changes, is perhaps the most likely to succeed. At present, the best strategy appears to be to initiate follicle growth in situ and isolate the follicles or granulosa-oocyte complexes once they have progressed to preantral stages. The final step is to mature the oocytes within their cumulus cells. The prospects of succeeding at each stage, and producing a fertile gamete at the end, are likely to be greater by preserving cellular interactions and the phenotype of follicle cells as these provide the physiological environment in which oocytes develop.

Ovarian aging, follicular depletion, and steroidogenesis

The follicular population. Menopause occurs as a consequence of the continuous utilization of a fixed store of primordial follicles leading to almost total depletion at mid-life or sometimes earlier. The great majority of follicles that disappear are lost by atresia rather than by ovulation, and the rate of loss accelerates in the last decade of menstrual life. The numbers of growing follicles at a given age are correlated with those of the primordial stages, but there are always more being recruited than required for a single ovulation each month. The extent to which a dwindling number is responsible for the character of cycles of the menopausal transition remains unclear. Ovarian secretion. While menstrual cycles remain regular, circulating concentrations of estradiol and progesterone are relatively independent of age. On the other hand, serum levels of inhibin are substantially lower in women approaching menopausal age, probably reflecting smaller numbers of growing follicles at the beginning of the cycle. Alleviation of negative feedback on the pituitary gland results in a greater output of follicle-stimulating hormone (FSH), but the effects of chronic superstimulation on the aging ovary are not known. Follicular aging. Aging of long-lived oocytes could affect the developmental potential of the follicle unit as well as compromising the chances of late pregnancy. Another important field of investigation is therefore to determine the balance of responsibility between cumulated damage to molecules by toxins, on the one hand, and the effects of physiological aging and such epiphenomena as the changing hormonal or paracrine environments, on the other.