Preservation of fertility through cryopreservation and in vitro maturation of human ovarian follicles and oocytes
Sammanfattning: One of the most rapidly expanding fields in assisted reproduction is the preservation of fertility for young women at risk of premature ovarian failure. This may be caused by cytotoxic therapy or other reasons. Cryopreservation of follicles in ovarian tissue has been successful in animal models with live young being born. Furthermore, the survival of human ovarian follicles after cryopreservation and thawing has been shown. In vitro maturation of oocytes (IVM) is important for patients having ovarian tissue cryopreserved and stored for further use. This method has also applications in clinical assisted reproduction in order to avoid ovarian hyperstimulation syndrome, which is a serious side effect of ovarian hyperstimulation during IVF treatment. The aim of this thesis was to develop methods for cryopreservation of follicles in human ovarian tissue, to identify new groups of patients who might benefit from such methods and to develop techniques for in-vitro maturation of ovarian follicles and oocytes. The effect of growth differentiation factor-9 (GDF-9) on early human ovarian follicles was studied in an organ culture system. A significantly higher proportion of cultured primordial follicles showed initiation of growth, reaching the secondary stage of development in the presence of GDF-9 compared to controls. Follicle viability was also improved with GDF-9 resulting in a smaller reduction in follicle numbers due to atresia. GDF-9 thus promoted growth, development, and survival of human ovarian follicles in organ culture. In a study comparing the use of serum and human serum albumin (HSA) for cryoprotectant solutions in cryopreservation of human ovarian cortical tissue, good viability of the follicles after thawing was shown using light microscopy, transmission electron microscopy and live/dead assay for analysis. A cryoprotectant solution containing HSA was equally effective as a cryoprotectant containing serum. Recombinant LH and recombinant hCG were equally efficient in promoting the maturation of oocytes in clinical in-vitro maturation (IVM). An IVM programme was established in the process, giving a powerful method to study the final stages of oocyte maturation in addition to clinical applications. Follicular density was analysed in ovarian cortical tissue from nine out of ten adolescent girls with Turner's syndrome, coming for preservation of ovarian tissue for possible fertility treatment later in life. Follicles were found in the biopsy tissue from eight subjects, the highest numbers being seen in the youngest girls and in those with mosaicism where not all of the cells in the body have the XO karyotype. Follicular density was negatively correlated with serum levels of FSH. The finding that adolescent girls with Turner's syndrome still have follicles in their ovaries runs contrary to previous assumptions that these women in most cases have no hope of having their own genetic offspring and raises the possibility of future fertility through cryopreservation of ovarian tissue. A culture system for human ovarian follicles and oocytes has been established through these projects and the process of cryopreservation of follicles in ovarian tissue has been studied. These methods are of great importance for women at risk for premature ovarian failure and cryopreservation of ovarian tissue is now offered for these women at Huddinge University Hospital. The need to further develop these methods is evident and more studies along these lines are being conducted.
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