Primordial Follicle Transplantation within Designer Biomaterial Grafts Produce Live Births in a Mouse Infertility Model

Abstract

The gonadotoxic effects of chemotherapy and radiation may result in premature ovarian failure in premenopausal oncology patients. Although autotransplantation of ovarian tissue has led to successful live births, reintroduction of latent malignant cells inducing relapse is a significant concern. In this report, we investigated the design of biomaterial grafts for transplantation of isolated ovarian follicles as a means to preserve fertility. Primordial and primary ovarian follicles from young female mice were extracted and encapsulated into biomaterials for subsequent transplantation into adult mice. Among the formulations tested, aggregated follicles encapsulated within fibrin had enhanced survival and integration with the host tissue following transplantation relative to the fibrin-alginate and fibrin-collagen composites. All mice transplanted with fibrin-encapsulated follicles resumed cycling, and live births were achieved only for follicles transplanted within VEGF-loaded fibrin beads. The extent to which these procedures reduce the presence of metastatic breast cancer cells among the isolated follicles was evaluated, with significantly reduced numbers of cancer cells present relative to intact ovaries. This ability to obtain live births by transplanting isolated primordial and primary follicles, while also reducing the risk of re-seeding disease relative to ovarian tissue transplantation, may ultimately provide a means to preserve fertility in premenopausal oncology patients.

Figure 1. In Vitro Follicle Culture in Fibrin and Alginate.

(a) Average absolute total number of follicles present in the bead are quantified for post-encapsulation and post-culture in 40 mg/ml and 20 mg/ml fibrin and 2% and 1% alginate (d), where error bars represent propagated error, with p-value determined by two-tailed Student’s t-test and n is as indicated in (b,c,e,f); follicle quantification in terms of their maturation stage (primordial, primary or secondary) vs. average normalized follicle density with (b) 40 mg/ml fibrin (n = 4); (c) 20 mg/ml fibrin (n = 4); (e) 2% alginate (n = 8); (f) 1% alginate (n = 4) over days 0, 1, 2, where error bars represent s.e.m. with p-value determined by two-tailed Student’s t-test; * indicate p ≤ 0.05, for (b,c) and (e) comparison is between day 0 and day 2.

Figure 2. In Vitro Follicle Culture in Combination Biomaterials.

(a) Average absolute number of follicles present in the bead are quantified for post-encapsulation and post-culture in fibrin-alginate (20 mg/ml–0.05%), fibrin-alginate (20 mg/ml–0.05%), and fibrin-collagen (15 mg/ml–0.44 mg/ml), where error bars represent propagated error, with p-value determined by two-tailed Student’s t-test and n is as indicated in (b,c) and (d); follicle quantification in terms of their maturation stage (primordial, primary or secondary) vs. average normalized follicle density with (b) fibrin-alginate (20 mg/ml–0.05%) (n = 4); (c) fibrin-alginate (10 mg/ml–0.05%) (n = 4); (d) fibrin-collagen (15 mg/ml–0.44 mg/ml) (n = 4) over days 0, 1, 2, where error bars represent s.e.m. with p-value determined by two-tailed Student’s t-test; * indicate p ≤ 0.05, for (d) comparison is between day 0 and day 2.

Figure 3. Follicles Survive Transplantation: follicles are present in beads that were prepared by using extra sedimentation procedure to aid in follicle aggregation.

The contrast is seen with much sparser follicle population present where follicle-to-follicle contact is minimal in pre-(a) and post-(c) transplantation (Day 3) beads in fibrin-alginate, as opposed to sedimented tissue pre-(b) and post-(d) transplantation beads; an example of modified procedure for transplant fabrication where follicles are readily visible within fibrin-alginate transplant surrounded by fatty tissue (e,f), within fibrin (g) and fibrin-collagen (h).

Figure 4. Follicle Population Data for In Vivo Studies.

(a) Average number of follicles (normalized by number of ovaries used per transplant) 9 days post-transplantation represented by secondary, primary and primordial stages for fibrin-alginate (10 mg/ml–0.05%) (FA) (n = 4), fibrin-collagen (15 mg/ml–0.44 mg/ml) (FC) (n = 6), and fibrin (20 mg/ml) (F) (n = 7) conditions; error bars indicate s.e.m. with p-value determined by two-tailed Student’s t-test as indicated; (b) % primordial follicles in FA, FC and F conditions 9 days post-transplantation, error bars indicate propagated error; * indicates p ≤ 0.05 for comparison of primary follicles, and ** for secondary follicles.

Figure 5. Follicle Transplant Results in a Live Birth.

(a) Vaginal Cytology Demonstrates Cyclicity in Transplantation Mice: % cycling mice is shown as a function of time over the course of 2 weeks for both fibrin and fibrin-VEGF conditions, where n = 12. 0 time point indicates the onset of cytology measurements, which occurred 4 days post-transplant surgery, when the mice were fully recovered; (b) photograph of the mixed litter with both white and grey pups present, arrows indicate white pups, * indicates grey pup.

Figure 6. Tumor Burden Evaluation within Unprocessed Ovary and Prepared Transplantation Bead.

Cancerous ovarian tissue extracted from a mouse 28 days post-inoculation (phase contrast in (a)) is showing a strong red fluorescent signal (b); (c) flow cytometry results for TdTomato + MD-MBA-231BR cells within unprocessed ovary and transplant bead made from processed ovary, where tissue was extracted 5 days post-inoculation; data shown represent average cell number ± s.e.m., n = 5 and p = 0.03 by two-tailed Student’s t-test, * indicates statistical significance, where p ≤ 0.05.