In Vitro Growth and Apoptosis Incidence, valuation of in Vitrified Human Ovarian Tissue Following Treatment with Growth Differentiating Factor 9B (GDF- 9B) and Leukemia Inhibitory Factor (LIF)

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Keywords: Vitrification, Human Ovarian Tissue Culture, GDF 9B, LIF, Apoptosis Related Genes

Background: The conventional freezing and vitrification are different cryopreservation protocols for fertility preservation in cancer patients. The high effectiveness of vitrification for human oocytes and embryos is shown, whereas data on human ovarian tissue are limited.

Follicular growth, ultrastructure, and apoptosis incidence in human ovarian tissue following vitrification/warming and after culture in the presence of GDF-9B and or LIF evaluated.

Material and Methods: Ovarian cortex biopsies from normal pregnant women fragmented and then divided to 2 main groups: vitrified and non-vitrified. Ovarian fragments in each main group cultured in 3 groups: in presence GDF-9B or LIF or absence any factor. Then the morphology, ultrastructure and incidence of apoptosis using biochemical analyses and analysis of apoptosis related genes expression in ovarian tissue fragments in all 6 experimental groups were evaluated before and after 2 weeks culture.

Results: Morphology and ultrastructure of vitrified human ovarian tissue were better preserved in vitrified group in presence of LIF compared to GDF-treated and non-treated groups. Apoptosis biochemical evaluations in all non-vitrified and vitrified treated groups were same. Apoptotic genes expression in both non-vitrified and vitrified groups at the end of culture period showed significant differences compared to non-treated groups.

Morphological studies of ovarian tissue in all groups showed better conservation of ovarian follicles in presence of GDF9B and LIF treated groups (P<0.05). But there were no significant differences between non-vitrified and vitrified ovarian tissue in all treated groups.

The levels of hormones at the end of culture period in treated groups were higher compared to non-treated groups.

Apoptosis evaluation techniques showed that apoptosis incidence in treated groups were lower than non-treated cultured groups and non-cultured ovarian tissue (P<0.05).

Conclusion: This study showed that culture of vitrified human ovarian tissue has not increased the incidence of apoptosis and GDF-9B and LIF could improve survival and development of cultured vitrified ovarian fragments and reduce incidence of apoptosis in human ovarian tissue.