Vitrification and in vitro culture had no adverse effect on the follicular development and gene expression of stimulated human ovarian tissue.

Abstract

The study assesses the effect of the vitrification procedure on the integrity, morphology, follicular development and gene expression of stimulated human ovarian tissue after warming and two weeks of in vitro culture. Ovarian specimens were divided into non-vitrified and vitrified groups and were cultured for two weeks. Morphological analysis and immunohistochemistry were performed. The 17-β estradiol and anti-Müllerian hormone levels in collected media were assessed. Gene expression was analyzed using real-time reverse transcription polymerase chain reaction. The morphology and immunohistochemistry of bcl-2-like protein 4 and B-cell lymphoma 2 of human stimulated ovarian tissue were similar in both groups. There was no significant difference in the percentage of normal follicles between the groups before and after in vitro culture. In spite of an increase in the percentage of growing follicles in cultured tissues compared to the non-cultured groups, the rate of normal follicles was significantly decreased in both cultured groups (P < 0.05). Gene expression was no different in vitrified tissues compared to the control; however, the expression of growth differentiation factor 9 and follicle stimulating hormone receptor genes were increased and factor in germ line alpha and kit ligand genes were decreased during in vitro culture (P < 0.05). In the two cultured groups, the level of 17-β estradiol was increased (P < 0.05), but the anti-Müllerian hormone concentration was not statistically altered. These results showed that the integrity of stimulated human ovarian tissue after vitrification/warming was well preserved; however, the in vitro culture condition needs improvement.