Schematic experimental setup for the procedure of vitrification in the study.

Follicles within the cultured ovarian tissue in the vitrified group (B) showed similar morphological features as in the non-vitrified tissue (A). Few of pyknotic follicles were seen in both groups.

Image of the nuclei (N) of the oocytes at a high magnification. The nuclear membranes containing nuclear pores (arrows) are clearly shown in both non-vitrified (A) and vitrified (B) groups. The Golgi apparatus (G) is well defined (A). Scale bars; 5A and 5B = 1 µm.

Morphology of follicles at different developmental stages within human ovarian tissue in non-vitrified (A) and vitrified (B) group. Well-preserved morphology of pre-antral follicles within vitrified tissue (B) was seen, similar to follicles in non-vitrified tissue which showed normal morphology.

Transmission electron microscopy of follicles within human ovarian tissues in two groups of non-vitrified (A) and vitrified (B) tissues. Overview of non-vitrified tissue showing a transitional primary follicle containing an oocyte (OC) in contact with granulosa cells (GC) surrounded by stromal cells (S) (A1). Higher magnification of the follicle showing the contact between OC and GC and the basement membrane (BM). Gap junctions (arrows) as well as microvilli (MV) are well defined and the contact with BM is uniform. Mitochondria (M) and endoplasmic reticulum (ER) are well defined (A2). An arrow shows the contact between OC and GC (A3). Stroma in non-vitrified tissue contained collagen fibres (CF) and spindle-shaped S (A4). Scale bars; A1 = 10 µm, A2 = 1 µm, A3–A4 = 2 µm. An overview of a primordial follicle within well-preserved ovarian stroma in a vitrified tissue (B1). An irregular pattern seen in the follicles in both vitrified (*) and non-vitrified group (not shown here). Contact between OC and GC and BM in a follicle of vitrified tissue. The homogenous cytoplasm of the oocyte contains uniform M and some ER (B2). Projections of MV are filling the space and connecting the OC and GC (B2), which is seen also in the non-vitrified tissue (A3). Higher magnification of the follicle showing the intact contact between the OC and GC containing gap junctions (arrows). The MV is also clearly seen (B3). Image from the stroma containing abundant CF and spindle-shaped S (B4). Scale bars; B1 = 10 µm, B2 = 2 µm, B3 = 1 µm, B4 = 2 µm.

Ultrastructure of follicles within human ovarian tissue after culture for 24 h in non-vitrified (A) and vitrified (B) groups. The oocyte nuclei (N) surrounded by nuclear membrane in a primordial follicle is shown (A1). Cytoplasm contains well-defined M. The intact contact between GC and OC is seen (arrows). The N and cytoplasmic organelles, M and ER are well defined. The nuclear pores (arrows) are clearly seen (A2). The contact between GC, OC and BM is shown (A3). GC contains hetero- and euchromatin in their nuclear. Well-defined M scattered in the cytoplasm are seen (A3). Image of stromal tissue composing bundles of CF and S (A4). Scale bars; A1 = 2 µm, A2–A3 = 1 µm and A4 = 5 µm. Overview of a primary follicle with OC and GC showing well-defined M and ER and intact contact between GC and the OC (arrows) (B1). Detail of the border between N and cytoplasm in OC showing well-defined nuclear membrane (arrows), ER and M containing cristae (B2). Granulosa cell in contact with OC and BM is shown (B3). Numerous M are scattered in the cytoplasm of the GC (B3). A sample of stroma containing numerous CF and flattened S is shown (B4). Scale bars; B1 = 2 µm, B2–B3 = 1 µm and B4 = 2 µm.