Novel extra cellular-like matrices to improve human ovarian grafting

Abstract

Purpose: To investigate if human ovarian grafting with pure virgin human recombinant collagen type-1 from bioengineered plant lines (CollPlant™) or small intestine submucosa (SIS) yields better implantation results for human ovarian tissue and which method benefits more when combined with the host melatonin treatment and graft incubation with biological glue + vitamin E + vascular endothelial growth factor-A.

Methods: Human ovarian tissue wrapped in CollPlant or SIS was transplanted into immunodeficient mice with/without host/graft treatment. The tissue was assessed by follicle counts (including atretic), for apoptosis evaluation by terminal deoxynucleotidyl transferase assay and for immunohistochemical evaluation of neovascularization by platelet endothelial cell adhesion molecule (PECAM) expression, and for identification of proliferating granulosa cells by Ki67 expression.

Results: Human ovarian tissue transplanted with CollPlant or SIS fused with the surrounding tissue and promoted neovascularization. In general, implantation with CollPlant even without additives promoted better results than with SIS: significantly higher number of recovered follicles, significantly fewer atretic follicles, and significantly more granulosa cell proliferation. Moreover, results with CollPlant alone seemed to be at least as good as those after host and graft treatments.

Conclusions: CollPlant is a biomaterial without any potential risks, and grafting ovarian tissue with CollPlant is easy and the procedure may be easily modified, with limited or no foreseeable risks, for auto-transplantation in cancer survivors. Further studies are needed using other novel methods capable of enhancing neovascularization and reducing apoptosis and follicle atresia.

Keywords: Atretic follicles; CollPlant; Immunodeficient mice; Neovascularization; Ovarian transplantation; SIS; Stroma cell apoptosis.

Fig. 1

Representative photographs of matrices. A—CollPlant matrices. B—SIS matrix

Fig. 2

Illustration of the experimental groups. A thawed ungrafted control specimen was fixed immediately for histological evaluation in both SIS and CollPlant groups. The remaining thawed samples were divided for implantation into 12 treatment subgroups, as follows: (a) CollPlant subgroups. A—Engrafted without further treatment, B—engrafted with the improvement protocol only: both host and graft treatment (host treatment with melatonin and graft treatment with HA + VEGF-A + vitamin E), C—engrafted with ovarian tissue encapsulated with CollPlant, D—engrafted after graft incubation with HA + VEGF-A + vitamin E and encapsulated with CollPlant, E—engrafted with host treatment with melatonin and graft encapsulated with CollPlant, F—engrafted with host treatment with melatonin and graft treatment with HA + VEGF-A + vitamin E and encapsulated with CollPlant. (b) SIS subgroups. G—Engrafted without further treatment, H—engrafted and treated as in group B, I—engrafted with ovarian tissue encapsulated with SIS, J—engrafted with graft treatment with HA + VEGF-A + vitamin E and encapsulated with SIS, K—engrafted with host treatment with melatonin and graft encapsulated with SIS, L—engrafted with host treatment with melatonin and graft treatment with HA + VEGF-A+ vitamin E and graft encapsulated with SIS

Fig. 3

Representative photographs of ungrafted controls and grafted samples. A—Thawed ungrafted ovarian section from a 9-year-old girl (patient 5). Note the primordial follicle (arrow). Hematoxylin and Eosin, Original magnification × 400. Note also at the lower left hand side the same section with several primordial follicles but at original magnification of × 200. B—Ovarian section from a 13-year-old girl (patient 1) after grafting with CollPlant alone (group C). Note the two secondary follicles (arrows). Hematoxylin and Eosin, Original magnification × 400. Note also at the bottom the same section with a visible part of the CollPlant matrix (C) and follicles (arrow) at lower magnification, original magnification × 200. C—Ovarian section from the same patient as in panel B, after grafting with CollPlant alone (group C). Note the primordial follicles (arrow head) and the CollPlant matrix that attached the human ovarian tissue to the murine muscle (not shown in this photo). Hematoxylin and Eosoin, original magnification × 100. D—Ovarian section from the same patient as in panel B, after grafting with CollPlant combined with the improvement protocol (group F). Note the atretic follicle (arrow). Hematoxylin and Eosoin, original magnification × 400. Note also at the bottom left hand size the same section at a lower magnification, original magnification × 200. E—Ovarian section from the same patient as in panel B, transplanted without treatment (group A). Note the brown staining indicating apoptosis in the stroma cells. Original magnification × 400. Note also at the upper right hand corner the red-brown staining in the positive control from a rat mammary gland four days after weaning and in the lower right corner the totally blue negative control. F—Thawed ungrafted ovarian section from the same patient as in panel A. Note the light brown staining indicating apoptosis in the stroma cells (arrow head) and lack of apoptosis in a primordial follicle (arrow). Original magnification × 400. Note also at the lower right corner the totally blue negative control. The positive control is the same as in Fig. 3e. G—Ovarian section from the same patient as in panel B from group C. Note the primordial and primary follicles with positively stained red-brown Ki67 staining (arrow), indicating proliferation. Original magnification × 400. Note also at the upper right side the positive control with its blue and red-brown staining and at the lower right side the negative control with a primary follicle and totally blue staining. H—Ovarian section from the same patient as in panel B from group F. Note the red-brown PECAM staining mostly in the borders of the graft but also in its center. The human ovarian tissue is fully fused with the mouse tissue. Original magnification × 400. Note also at the upper right corner the positive control with its blue and red-brown staining and at the lower right corner the totally blue negative control. I—Ovarian section from the same patient as in panel A, after host treatment with melatonin and graft transplantation with CollPlant (group E). Note the PECAM staining mainly in the border of the graft and in the CollPlant matrix but also in the graft’s center (blood vessels). The human ovarian tissue is fully fused with the CollPlant and the mouse tissue. Original magnification × 100. Note also at the lower right corner the totally blue negative. The positive control is shown in Fig. 3h. J—Ungraffted-thawed ovarian section from patient 7. Note the normal primordial follicles. Hematoxylin and Eosin. Original magnification × 400. Note also at the lower right corner the same section at lower magnification, original magnification, × 200. K—Ovarian sample from patient 9 grafted with SIS (S) after improvement protocol (group L). Note that SIS envelops the ovarian slice and the follicles in the graft (arrow) and graft is attached to the mouse tissue. Hematoxylin and eosin. Original magnification × 100. L—Ovarian sample as in K. Note the normal primary follicles with the red-brown. Ki67 staining in the granulosa cells and oocytes (arrows). Original magnification × 400. Note also at the upper right corner the negative control with totally blue follicles. The positive control with Ki67 staining is shown in Fig. 3g. M—Ovarian sample from patient 1 from group L. Note the atretic follicles (arrow). Hematoxylin and eosin. Original magnification × 400. Note also at the lower right corner the same section at a lower magnification, original magnification × 200. N—Ovarian sample from patient 1 grafted with SIS after pre-incubation with HA + VEGF-A + vitamin E (group J). Note the follicle (not midsection, arrow) and the overall blue staining and lack of brown apoptosis staining. Original magnification × 400. Note also at the lower right corner the totally blue negative control. The positive control is shown in Fig. 3e. O—Ovarian sample from patient 6 grafted with SIS (group I). Note the strong brown staining in the stroma cells (arrow), indicating apoptosis. Original magnification × 400. Note also at the lower right corner the totally blue negative control. The positive control is shown in Fig. 3e. P—Ovarian sample as in K. Note the red-brown PECAM staining in the SIS matrix (S) and the ovarian graft (arrow). The SIS matrix is between the graft and the mouse tissue. Original magnification × 100. Note also at the left lower corner the totally blue negative control. The positive control is shown in Fig. 3h. Q—Ovarian sample from patient 8 grafted in group J. Note the red-brown PECAM staining in the ovarian graft and in its blood vessels (arrow). Original magnification × 400. Note also at the right lower corner the totally blue negative control. The positive control is shown in Fig. 3h. R—Partially loose SIS matrix (S) after implantation of ovarian tissue from patient 7 in group I. Note the darker PECAM staining in the SIS matrix (arrow). Original magnification × 400. Note also the totally blue negative control at the right lower corner. The positive control is shown in Fig. 3h

Fig. 4

Follicle numbers. Results are presented as mean ± standard deviation (SD). The x axis represents the six experimental groups and the thawed ungrafted control. The y axis represents the mean follicular numbers. (a) CollPlant groups: the bars represent the different subgroups, as follows: Single asterisk indicates significantly higher than all groups (P > 0.0001 than all groups but group C, P = 0.006). Double asterisks indicate significantly higher than group BC (P = 0.04), group D (P = 0.005), group E (P = 0.04), and group F (P = 0.04). (b) SIS groups: single asterisk indicates significantly higher than in group H (treated host and treated graft without SIS, P = 0.02 and P = 0.0061, respectively), group I (untreated host and graft with SIS, P = 0.01 and P = 0.0033, respectively) and group K (treated host and untreated graft with SIS, P = 0.04 and P = 0.0058, respectively). Double asterisks indicate significantly higher than in groups G (untreated grafts without SIS, P = 0.013), I (untreated host and treated graft with SIS, P = 0.033) and J (untreated hosts and treated grafts with SIS, P = 0.0072)

Fig. 5

Atretic follicle percent. The x axis represents the six experimental groups and the thawed ungrafted control (for both CollPlant and SIS groups).The y axis represents the percent of atretic follicles. The bars represent the different subgroups as in Fig. 4. (a) CollPlant groups: single asterisk indicates significantly higher than group B (P = 0.008, P = 0.03 and P = 0.0018, respectively). Double asterisks indicate significantly higher than group E (P = 0.008, P = 0.03, P = 0.001, respectively). Triple asterisks indicate significantly higher than group C (P = 0.03, P = 0.0002, respectively). (b) SIS groups: single asterisk indicates significantly higher than the thawed ungrafted group, groups K and L (all P < 0.0001) and group IH (P = 0.04). Double asterisks indicate significantly higher than the thawed ungrafted group (P = 0.002) and group IH (P = 0.003). Triple asterisks indicate significantly higher than the thawed ungrafted group and group L (both P < 0.0001), groups IH (P = 0.03) and K (P = 0.049). Quadruple asterisks indicate significantly higher than the thawed ungrafted group (P = 0.002) and group H (P < 0.0001)

Fig. 6

Apoptosis expression in thawed controls and grafts. Results are presented as mean ± standard deviation (SD). The x axis represents the six experimental groups and the thawed ungrafted control. The y axis represents the level of TUNEL staining. The apoptosis level grading scores are described in the “Methods” section. (a) CollPlant groups: single asterisk indicates significantly higher than group B (P = 0.03, P = 0.0015, P < 0.0001, respectively). Double asterisks indicate significantly higher than group C (P = 0.03, P = 0.0015, respectively). Triple asterisks indicate significantly higher than the thawed ungrafted group (P = 0.015, P = 0.001, respectively). Quadruple asterisks indicate significantly higher than group E (P = 0.02). (b) SIS groups: single asterisk indicates significantly higher than group H (P = 0.001 for the ungrafted controls and groups K and L, P = 0.002 for group B and P = 0.02 for group I).

Fig. 7

Percent of Ki67 staining in the CollPlant subgroups. Single asterisk indicates significantly more Ki67 positively stained follicles in ungrafted controls and group C (P < 0.0001 for both), and group D (P = 0.02) for group D than in group A. Double asterisks indicate significantly more Ki67 positively stained follicles (P < 0.0001) than group F