Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue

Abstract

At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5-2.0×1.0-1.2×0.8-1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips.

Figure 1. Cryopreserved ovarian medulla-free and medulla-containing ovarian pieces after thawing and 8 days in vitro culture.

(a, b) medulla-free piece, (a) just after thawing, (b) the same piece after culture, (c, d) medulla-containing piece, (c) just after thawing, (d) the same piece after culture. Bar = 1 mm.

Figure 2. Cryopreserved ovarian medulla-free and medulla-containing pieces before and after 5 days culture with chorioallantoic membrane (CAM) system.

(a, b, c, d, e) medulla-free piece, (a, b) just after thawing and seeding on CAM marked by silicone ring, (c, d, e) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (f, g, h, i, j) medulla-containing piece, (f, g) just after thawing and seeding on CAM marked by silicone ring, (h, i, j) the same piece after culture, (c) piece on CAM, (d, e) piece in Petri dish; (e) outer CAM-layer with medulla-free piece, (j) inner CAM-layer with medulla-containing piece. Different intensiveness of the avian vascularisation in the place of the seeding of pieces was noted: (e) versus (j). Bar = 1 mm.

Figure 3. Quality of follicles (expressed as quantity and percentage of normal follicles) after in vitro and CAM-culture of cryopreserved ovarian pieces.

Respective rates are not statistical different (P>0.1).

Figure 4. Histological micrographs of follicles from cryopreserved ovarian tissue after in vitro- and CAM-culture.

(a, A) in vitro culture, (b, B) CAM culture. Bar = 25 µm.

Figure 5. Vascularisation (von Willebrand factor expression) in cryopreserved medulla-containing ovarian tissue after in vitro and CAM-culture.

Immonostained after in vitro (a, A) and CAM (b, B) culture. Bar = 0.25 mm.

Figure 6. Neo-angiogenesis (desmin expression) in cryopreserved medulla-containing ovarian tissue after CAM-culture.

(rectangles) places where neo-angiogenesis was detected, (head arrow) CAM. Bar = 25 µm.