Human ovarian follicles and oocytes : Collection, cryopreservation, culture and gene expression

Sammanfattning: Infertility affects approximately 15% of couples globally. The most effective treatment today is in vitro fertilization. However, not all couples can be helped with the current techniques. To achieve better success, techniques of storage and in vitro maturation of ovarian follicles have to be improved. Meanwhile, understanding of the molecular mechanisms of follicle/oocyte maturation and embryo development would doubtlessly facilitate technique development. Nonetheless, little work has been done in these areas due to the limited availability of human research material. Therefore, we performed five studies on the collection, cryopreservation, culture and gene expression of human ovarian follicles/oocytes respectively. In Article I, we examined if follicular aspirates obtained during oocyte retrieval for IVF were a good source of human ovarian follicles. The presence of follicles in the aspirates was examined by mechanical/enzymatic isolation, culturing and histological analysis. We found only 14 follicles in 86 aspirates. The results indicate that follicular aspirates are not a reliable source of human ovarian follicles. In Article II, we evaluated whether serum-free cryoprotectants could be used for cryopreservation of human ovarian cortical tissue. Biopsies of ovarian cortical tissue donated by healthy women were frozen and thawed using two kinds of cryoprotectants containing either human serum or human serum albumin. Light microscopy, transmission electron microscopy and live/dead fluorescence assay were performed to evaluate the structure and viability of the follicles. The results showed that the majority of the follicles retained normal structure and viability after thawing. Cryoprotectants containing human serum albumin were equally effective as those containing human serum. Therefore, serum free cryoprotectants are suitable for the cryopreservation of human ovarian cortical tissue. In Articles III and Article IV, we tested the effect of two secondary messengers, cGMP and cAMP, on human ovarian follicles cultured in ovarian cortical slices. Donated ovarian cortical biopsies from healthy women were cut into slices and cultured in parallel in the presence and absence of 8-br-cGMP or 8-br-cAMP for 1-3 weeks. Oestrdiol production, developmental stage, size and viability of the follicles were recorded. The results showed that both 8-br-cGMP and 8-br-cAMP enhance the survival and development of human early follicles cultured in ovarian cortical tissue. In Article V, to expose the gene expression profile of human germinal vesicle oocytes (hGVO) and reveal different gene expression patterns between hGVO, embryonic stem cells and foreskin fibroblasts, we performed microarray (Affymetrix U133 plus 2.0) analysis and RT-PCR. In total, 11,191 unigenes were expressed in normal human GV oocytes. Forty-nine percent of these genes are as yet unclassified by biological function. A few oocyte specific genes that are obligatory for oocyte maturation/early embryo development in animals were found expressed in hGVO for the first time. Furthermore, known components of MOS-MPF, TGF-beta superfamily, and WNT pathway were identified in hGVO. Last, twelve gene expression patterns were found between hGVO, embryonic stem cells and fibroblasts, suggesting potential candidate genes involved in oocyte maturation and embryonic development. Our findings will help improve the technique of cryopreservation and culture of human ovarian follicles. Further, our last study provides a rich source for continued research in elucidating the molecular mechanism of oocyte maturation and embryo development in humans.