Attempts to improve human ovarian transplantation outcomes of needle-immersed vitrification and slow-freezing by host and graft treatments

Abstract

Purpose: To investigate if needle-immersed vitrification or slow-freezing yields better implantation results for human ovarian tissue and which method benefits more when combined with the "improvement protocol" of host melatonin treatment and graft incubation with biological glue + vitamin E + vascular endothelial growth factor-A.

Methods: Human ovarian tissue was preserved by needle-immersed vitrification or slow-freezing and transplanted into immunodeficient mice, either untreated (groups A and C, respectively) or treated with the improvement protocol (groups B and D, respectively). Grafted and ungrafted slices were evaluated by follicle counts, apoptosis assay and immunohistochemistry for Ki67 and platelet endothelial cell adhesion molecule (PECAM).

Results: Follicle number in the recovered grafts was limited. The number of atretic follicles was significantly higher after vitrification with/without the improvement protocol and slow-freezing than that after slow-freezing + the improvement protocol. Stroma cell apoptosis was the lowest in the group D. PECAM staining showed a peripheral and diffuse pattern in the group D (mostly normal follicular morphology) and a diffuse pattern in all other groups (few follicles, mostly atretic), with significantly higher diffuse levels in the vitrification groups. Ki67 staining was identified in all normal follicles. Follicles did not survive transplantation in the vitrification groups.

Conclusions: Ovarian sample preparation with slow-freezing + the improvement protocol appears to yield better implantation outcomes than needle-immersed vitrification with/without the improvement protocol. The real quality of frozen tissue can be assessed only after grafting and not after thawing/warming.

Keywords: Human ovarian tissue; Ki67; Needle-immersed vitrification; PECAM; Slow-gradual freezing; TUNEL; “Improvement protocol”.

Fig. 1

Needle-immersed vitrification. a Thinly sliced human ovarian slices placed on an insulin needle connected to a syringe and dipped in a cryotube containing the equilibration solution. b Thinly sliced human ovarian tissue placed on insulin needles attached to a syringe.

Fig. 2

Representative photographs of ungrafted controls and grafted samples. a Section of slow-frozen/thawed ungrafted ovarian sample from a 16-year-old girl (patient 2, as numbered in Tables 1 and 2). Note the primordial-primary follicle (arrow). Hematoxylin and eosin, original magnification ×400. b Section of vitrified/warmed ungrafted ovarian sample from an 18-year-old woman (patient 4, as numbered in Tables 1 and 2). Note the numerous primordial and primary follicles. Hematoxylin and eosin, original magnification ×400. c Section of grafted ovarian sample from a 21-year-old woman after slow-freezing + the improvement protocol (group D) (patient 6, as numbered in Tables 1 and 2). Note the secondary and primary follicles (arrows) and the red-brown Ki67 staining in the granulosa cells and the oocytes. Original magnification ×400. Note the negative control at the upper right hand side: a blue-stained (hematoxylin) secondary follicle without any traces of red-brown color and the positive control at the bottom left hand side with red-brown staining (arrows) between blue (hematoxylin) background staining. d Section of grafted ovarian sample from a 19-year-old woman after vitrification/warming + the improvement protocol (group C) (patient 5, as numbered in Tables 1 and 2). Note the atretic antral follicle. Hematoxylin and eosin, original magnification ×400. e Section of a slow-frozen/thawed ovarian sample from the same patient as in panel a. Note the overall blue staining and lack of brown TUNEL staining, indicating lack of apoptosis. Original magnification ×400. Note the positive control at the bottom left hand side of positively stained (red-brown) epithelium cells, a specimen from a rat mammary gland lobule taken 4 days after weaning. The negative control at the upper left hand side: blue-stained (hematoxylin) primordial follicle without any traces of red-brown. f Section of a grafted ovarian sample from the same patient as in panel d after vitrification/warming + improvement protocol. Note the brown TUNEL staining in the stroma cells and the follicle (arrows), indicating apoptosis. Original magnification ×400. The positive control for TUNEL is shown in panel e. g Section of a grafted ovarian sample from the same patient as in panel c after slow-freezing/thawing + improvement protocol. Note the peripheral red-brown PECAM staining pattern in the graft’s border. Original magnification ×400. The negative control is at the upper right hand side, blue (hematoxylin)-stained stroma cells without any traces of red-brown. The positive control for PECAM is shown in panel h. h Section of grafted ovarian sample from the same patient as in panels c and g after vitrification/warming. Note the scattered red-brown PECAM staining throughout the whole graft. Original magnification ×400. Note the negative control at the upper right hand side, blue (hematoxylin)-stained stroma cells without any traces of red-brown, and the positive control at the lower left hand side with strong red-brown staining within blue background staining

Fig. 3

Percents of atretic follicles in all the study groups The x axis represents the four experimental groups and ungrafted fresh, vitrified/warmed, and slow-frozen/thawed controls. The y axis represents the percent of atretic follicles. Black bar with white dots = ungrafted fresh controls. White bar with black dots = ungrafted vitrified/warmed controls. Black and white perpendicular line bar = ungrafted slow-frozen/thawed controls. Black and white diagonal lines = grafted vitrified/warmed samples (group A). Black and white square bar = grafted vitrified/warmed samples + the improvement protocol (group B). Black and white brick bar = grafted slow-frozen/thawed samples (group C). Black and white parallel line bar = grafted slow-frozen/thawed samples + the improvement protocol (group D). Superscript number one (¹) indicates significantly higher than the fresh, vitrified, and slow-frozen ungrafted samples (Table 1) and grafted samples after slow-freezing + improvement protocol (group D) (p < 0.0001). Superscript number two (²) indicates significantly higher than the fresh (p < 0.0001), slow-frozen (p = 0.03), and vitrified (p < 0.0001) ungrafted controls

Fig. 4

Apoptosis expression in the ungrafted and grafted samples. Results are presented as mean ± SD. The x axis represents the four experimental groups and ungrafted fresh, vitrified/warmed, and slow-frozen/thawed controls. The y axis represents the level of TUNEL staining. Apoptosis (TUNEL) levels were graded according to intensity: 0 = no apoptosis, 1 = low staining intensity, 2 = medium staining intensity, and 3 = high staining intensity. Black bar with white dots = ungrafted fresh controls. White bar with black dots = ungrafted vitrified/warmed controls. Black and white perpendicular line bar = ungrafted slow-frozen/thawed controls. Black and white diagonal lines = grafted vitrified/warmed samples (group A). Black and white square bar = grafted vitrified/warmed samples + the improvement protocol (group B). Black and white brick bar = grafted slow-frozen/thawed samples (group C). Black and white parallel line bar = grafted slow-frozen/thawed samples + the improvement protocol (group D). Superscript number one (¹) indicates significantly higher than the fresh ungrafted (p = 0.01) and slow-frozen grafted (p = 0.01) groups. Superscript number two (²) indicates significantly higher than the fresh (p = 0.0009), slow-frozen (p = 0.0009), and vitrified (p = 0.01) ungrafted samples, the samples grafted after slow-freezing only (p = 0.04), and the samples grafted after slow-freezing + improvement protocol (p = 0.002)

Fig. 5

PECAM expression in the ungrafted and grafted samples. a Diffuse PECAM expression in the ungrafted and grafted samples. Results are presented as mean ± SD. The x axis represents the four experimental groups and ungrafted fresh, vitrified/warmed, and slow-frozen/thawed controls. The y axis represents the level of PECAM staining. PECAM levels were graded according to intensity: 0 = no PECAM staining, 1 = low staining intensity, 2 = medium staining intensity, and 3 = high staining intensity. Black bar with white dots = fresh ungrafted controls with diffuse pattern. White bar with black dots = vitrified/warmed ungrafted controls with diffuse pattern. Black perpendicular line bar = ungrafted slow-frozen controls with diffuse pattern. Black diagonal line bar = grafted vitrified/warmed samples (group A) with diffuse pattern. Black and white square bar = grafted vitrified/warmed samples + improvement protocol (group B) with diffuse pattern. Black and white brick bar = grafted slow-frozen/thawed samples (group C) with diffuse pattern. Black and white dark parallel line bar = grafted slow-frozen/thawed samples + the improvement protocol (group D) with diffuse pattern. Superscript number one (¹) indicates significantly higher than the slow-frozen grafted (p = 0.01). Superscript number two (²) significantly higher than the slow-frozen (p = 0.006), vitrified (p = 0.015), ungrafted, and the samples grafted after slow-freezing (p = 0.0002) ungrafted. Superscript number three (³) indicates significantly higher than the grafted after vitrification + improvement protocol (p = 0.01) b Peripheral PECAM expression in the fresh ungrafted and grafted slow-frozen/thawed samples +the improvement protocol (group D). Black bar with white dots = fresh ungrafted fresh controls. Black and white dark parallel line bar = grafted slow-frozen/thawed samples + the improvement protocol (group D).