The effect of agar substrate on growth and development of cryopreserved-thawed human ovarian cortical follicles in organ culture.

OBJECTIVE: To preserve human ovarian tissue structure and improve follicular growth and survival during in-situ culture, various biomaterials are used. In this study we aimed to compare agar as a cultivation substrate with matrigel-coated insert in order to achieve an optimum system for in-situ human follicle culture. STUDY DESIGN: Frozen-thawed human ovarian cortical tissues were cultured on either matrigel-coated inserts or agar-soaked substrates. The proportion of morphologically viable and degenerated follicles at different developmental stages, secreted hormonal levels, and apoptotic and proliferation gene expressions were compared between the cultured groups after 7-days of culture. RESULTS: The follicular growth was not significantly different between the two cultured groups, although showing higher percentage of growing follicles in agar cultured group. The secreted hormonal levels didn't have any difference between two cultured groups. Although the apoptotic gene expressions didn't show any difference between the cultured groups, the apoptotic index was lower in agar cultured group. In addition, Ki67 gene expression, a proliferative marker, showed a significantly higher expression in agar cultured group. CONCLUSION: Based on the results, agar is as suitable as matrigel-coated inserts for the survival and growth of follicles during culture. Therefore, agar can be an inexpensive alternative substrate for culturing frozen-thawed human ovarian cortical strips.

The combination of basic fibroblast growth factor and kit ligand promotes the proliferation, activity and steroidogenesis of granulosa cells during human ovarian cortical culture.

Different factors, such as basic fibroblast growth factor (bFGF) and kit ligand (KL), are used in ovarian cortical culture to promote activation of primordial follicles. In the present study, the effects of bFGF and KL, alone and in combination, were evaluated on human follicular activation and growth during in-situ cortical culture. Slow frozen-thawed human ovarian cortical tissues (n = 6) were cultured in 4 different groups: 1) control (base medium), 2) KL (base medium; BM + 100 ng/ml KL), 3) bFGF (BM + 100 ng/ml bFGF) and 4) bFGF + KL (BM + 100 ng/ml KL + 100 ng/ml bFGF) for a week. The proportion of morphologically normal and degenerated follicles at different developmental stages, secreted hormonal levels and specific gene expressions were compared. Although the proportion of growing follicles was higher than primordial counterpart in all cultured groups, no significant differences were observed among the cultured groups. In all cultured groups, anti-Müllerian hormone (AMH), progesterone and estradiol hormones levels increased after 7 days of culture; however, this increase was only significant for estradiol in the bFGF + KL group. The expression of Ki67 gene indicated an increase in ovarian cell proliferation in the three experimental groups compared to the control group, however this increment was only significant for the bFGF + KL group. It can be concluded that KL and bFGF factors individually have no beneficial effects on in-situ follicular growth, but their combination positively influences steroidogenesis of granulosa cells without significantly increasing the number of growing follicles.