In vitro development of human primordial follicles to preantral stage after vitrification

Abstract

Purpose: The aim was to culture primordial follicles in vitro to reach preantral stage in vitrified human ovarian tissue.

Methods: Ovarian tissue samples were obtained from six women. Tissue strips were vitrified by infiltration with a cryoprotectant followed by mounting on a stainless steel carrier. After culturing for 7 days the morphology and developmental stages of follicles enclosed in fresh and vitrified groups were analyzed.

Results: High proportion of viable follicles in vitrified ovarian strips was obtained. After culturing for 7 days the percentage of secondary and preantral follicles increased significantly (P < 0.05) whereas primordial and transitory follicles showed a significant decrease (P < 0.05) compared to their respective counterparts at day 0 of culture.

Conclusions: Vitrification of ovarian strips with an improved carrier device and culturing of follicles in ovarian strips after warming yielded developed follicles with high viability and morphological integrity that may be suitable for use in fertility preservation among cancer patients.

Fig. 1

Photographs showing the actual dimensions of a stainless steel mesh for carrying ovarian strips for vitrification (a) and how they appear in a petri-dish (b)

Fig. 2

Light photomicrographs showing hematoxylin and eosin stained human ovarian follicles in fresh ovarian strip and after vitrification/warming. a and b: fresh follicles; c: follicles after IVC; d and e: primordial follicle after vitrification; f: preantral follicle after vitrification and 7 days of IVC. Abnormal follicles are indicated by arrows in c and d. Bars: a = 100 μm, b and f = 20 μm, c-e = 50 μm

Fig. 3

Histogram showing the number of follicles (mean ± SD) in non-vitrified and vitrified ovarian strips. There is no significant difference between the vitrified and fresh (non-vitrified) groups. ^a p = 0.503, ^b p = 0.140, ^c p = 0.496, ^d p = 0.260 and ^e p = 0.11

Fig. 4

Histogram showing the number of developed and undeveloped follicles (mean ± SD) at day 0 and day 7 after IVC. a: fresh (non- vitrified group); b: vitrified group. * indicates significant difference (p < 0.05), **indicates significant difference (p < 0.01) and ***indicates significant difference (p < 0.001) between day 0 and day 7

Fig. 5

Histogram showing the number of isolated follicles (mean ± SD) in non-vitrified and vitrified ovarian strips after 7 days of IVC. There is no difference between the isolated follicles in non-vitrified and vitrified groups except for primary follicles (^c p = 0.011). ^a p = 0.360, ^b p = 0.096, ^d p = 1.000 and ^e p = 0.206

Fig. 6

Light photomicrographs showing ovarian strips (a and b) and isolated follicle (c) stained with Neutral Red after 7 days of IVC, and isolated follicles (d and e) as well as vitrified/warmed ovarian strips (e) stained with Calcein AM and Ethidium homodimer-1 after 7 days of IVC. a: fresh ovarian strips; b: vitrified/warmed ovarian strips; c: isolated follicle; d: viable isolated follicle (green) and dead stromal cells (red); e: an isolated follicle with several dead granulosa cells (red); f: ovarian strip showing numerous live stromal cells and follicles within the strip. Bars = 50 μm