Fibrin-mediated delivery of an ovarian follicle pool in a mouse model of infertility

Abstract

The cryopreservation and autotransplantation of ovarian tissue is emerging as a powerful approach for preserving fertility. However, for cancer patients, it may not be possible to transplant ovarian tissue due to the risk of re-seeding disease. We investigated strategies for transplantation of individually isolated follicles to minimize the risk of re-introducing cancer cells present within the vasculature of ovarian stroma. Procedures for large-scale isolation of early-stage follicles and their encapsulation into fibrin hydrogels were developed. For in vivo validation studies, mice were ovariectomized and transplanted with encapsulated follicles into the ovarian bursa. A substantial increase in the number of secondary follicles was observed in the graft at 9 days after transplantation, and antral follicles by day 21, demonstrating primordial follicle recruitment into the growing pool. Initially, elevated follicle-stimulating hormone levels declined substantially by day 21, indicating feedback from the graft; presence of corpora lutea showed the graft's capability of restoring hormone cyclicity. Taken together, the transplanted follicles were able to engraft, mature, and restore ovarian function in an infertile mouse. This biomaterial may, thus, provide a platform for follicle transplantation with a low risk of cancer contamination and for developing strategies that preserve fertility for women facing a cancer diagnosis.

FIG. 1.

Maintenance of the ovarian orthotopic site with an alginate bead. (a) An alginate bead (Alg) was placed in the ovarian bursa (arrows) so that ovarian tissue could be transplanted at 14 days after ovariectomy. The incision is denoted by the presence of sutures (arrow head). (b) Neither cellular infiltration nor inflammation is observed at the host material interface of the alginate (Alg) and ovarian bursa (arrows). Blood vessels (BV) are observed within the bursa, which may initially support the transplant. (c) The absence of an alginate bead leads to collapse of the bursa, resulting in dense connective tissue. Sutures that identify the bursal tissue are denoted by arrow heads. (d) Magnification of the sutures (S) used to identify connective tissue as the collapsed bursa. Scale bars are 250 μm (a, c) or 25 μm (b, d), F denotes fat tissue, Color images available online at www.liebertpub.com/tea

FIG. 2.

Encapsulation of an ovarian follicle pool in a fibrin hydrogel. (a) Representative image of a cross-section of follicles encapsulated within the fibrin hydrogel. The edge of the fibrin hydrogel is indicated with black arrows. (b) A secondary follicle (open arrow head) and primary follicle (closed arrow head) with a centrally located oocyte (Oo) and surrounding cuboidal granulosa cells (GC). (c) Primordial follicles with an oocyte and squamous granulosa cells. Scale bars are 250 μm (a) or 25 μm (b, c). Color images available online at www.liebertpub.com/tea

FIG. 3.

DiI staining confirms origin of grafted tissue. (A) CM-DiI stained follicles in retrieved tissue at day 3 (A), day 9 (B), and day 21 (C). The corresponding hematoxylin counterstaining is shown in (D–F). DAPI and CM-DiI are blue and red, respectively. Graft is denoted by γ. (G) Hematoxylin and eosin staining of histological sections identified primordial follicles (denoted by an *) within the graft at day 3 h. At day 9, primary follicles (**) are observed within the graft. (F) After 21 days, some antral follicles are present (***). Scale bars are 100, 25, and 50 μm in (A–F, G), and (H–I), respectively. Color images available online at www.liebertpub.com/tea

FIG. 4.

Follicle development and attrition. (A) Number of follicles as a function of time in the graft. Different letters above bars indicate significant difference (p<0.05). (B) Percentage of follicles in each developmental stage within the graft. The n was 6, 6, 10, and 12 for day 0, 3, 9, and 21, respectively.

FIG. 5.

Graft survival. Graft survival was determined by the presence of transplanted follicles within the harvested bursa. Note that samples were harvested at each time point for analysis by histology, and the n for each time point was 6, 6, 10, and 12 for day 0, 3, 9, and 21, respectively.

FIG. 6.

Graft function. (A) Follicle-stimulating hormone (FSH) levels within the serum were measured pre-ovariectomy, post-ovariectomy but pre-transplantation, and post-transplantation. Statistical analysis was performed using a one-way ANOVA. (B) Large antral follicles and (C) corpora lutea were observed at 21 days post-transplantation in four of five mice, in agreement with the FSH levels. The corpus (dashed outline) luteum supports the occurrence of ovulation. Scale bars=100 μm. Color images available online at www.liebertpub.com/tea