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 patient's fertility should be safeguarded from cytotoxic treatment.
In contrast to the many detailed studies of Graafian follicles, the biology of small follicles in the human ovary is poorly understood and the trigger for follicular growth initiation remains unknown. No practical model exists to study preantral follicle growth in the human because of their slow growth rate and lack of an effective culture system. We therefore tested ovarian xenografts as a new strategy to study the early stages of ovarian follicular growth in vivo. Mice homozygous for severe combined immunodeficiency (SCID) and hypogonadism (hpg) received human ovarian xenografts under their kidney capsules. Follicle growth was assessed by morphology and proliferating cell nuclear antigen (PCNA) immunostaining. The grafts were recovered after 11 (short-term) and 17 weeks (long-term), and serially sectioned. During the last 6 weeks of long-term grafting, mice were randomized to receive either placebo or 1 IU of purified follicle stimulating hormone (FSH) s.c. on alternating days. After 11 weeks of grafting, the most advanced follicles had a maximum of two granulosa cell layers. In the absence of FSH administration, follicles did not progress beyond the two-layer stage even after 17 weeks of grafting, and the oestradiol levels remained undetectable. In the FSH-treated long-term grafts, follicles had grown to antral stages and resulted in oestradiol levels as high as 2070 pmol/l. Growth initiation indices did not differ between control and FSH-treated grafts. This study demonstrates that follicles can survive and grow in human ovarian tissue grafted under the renal capsules of immunodeficient mice for at least 17 weeks, and indicate that xenograft models are potentially useful for studying human follicle development. Using this physiological model, we showed that FSH is required for follicle growth beyond the two-layer stage, although growth initiation is independent of gonadotrophin stimulation.
Ovarian cortical tissue cryopreservation with subsequent autografting is a potential strategy for the preservation of fertility in patients undergoing systemic chemotherapy and pelvic radiotherapy. Non-vascular
The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.
A culture system has been designed in which enzymatically isolated oocyte-granulosa cell complexes from fresh and frozen-thawed ovine ovarian tissue can be grown to antral size in vitro. Oocyte-granulosa complexes ranging from 100 to 240 microns in diameter were dissected from stromal tissue and grown individually in serum-free medium for 30 days. Complexes < 190 microns generally excluded their oocytes or lost three-dimensional structure early in the culture period. In contrast, complexes isolated from fresh or frozen-thawed tissue and measuring 190-240 microns on the day of isolation formed antral cavities in 25 +/- 9% and 18 +/- 6% (mean +/- SEM) of cases, respectively. The effect of gonadotrophin supplementation to the culture medium was tested on frozen-thawed oocyte-granulosa cell complexes only. In cultures supplemented with both FSH and LH or FSH alone, there was no significant difference in the number of oocyte-granulosa cell complexes that formed antral cavities (18 +/- 7%). However, antrum formation was significantly less frequent in cultures lacking gonadotrophin stimulation (7 +/- 4%). All oocyte-granulosa cell complexes maintained a three-dimensional structure throughout culture and developed a functional P450 aromatase enzyme complex, as revealed by the induction of oestradiol production during 8 days of culture after antrum formation in serum-free medium containing testosterone. Oocytes recovered after 30 days of culture were viable and had increased in diameter from 78 +/- 2 microns on the day of isolation, to 131 +/- 3 microns at the end of culture. These results show that oocyte-granulosa cell complexes isolated from cryopreserved ovarian tissue can be grown to antral size in vitro with similar efficiency to those isolated from fresh tissue.
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