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Objectives Ovary tissue engineering can be an approach to preserve the fertility of prepubertal girls and women who cannot utilize the available strategies due to the presence of malignant cells in their cryopreserved ovarian fragments. The main challenge of preparing an engineered ovary is providing an appropriate substrate to maintain the 3D structure of follicles and allowed them a radial growth [1-3]. Fibrin is a potential candidate for ovarian tissue engineering, but its main drawback is fast degradation, which results in implant volume loss within days and subsequently loss of physical support of follicles [4-6]. To overcome this obstacle, physical or chemical modifications such as PEGylation have been developed [7-10]. Methods PEGylation process was employed to improve fibrin stability. Moreover, a sequence of designed experiments was programmed by Response Surface Methodology to achieve a hydrogel formulation with similar mechanical property to the reproductive-age ovarian cortex. Afterward, isolated human preantral follicles were encapsulated in the optimized formulation of PEGylated fibrin and cultured for 4 or 7 days to assess cell-cell communication and follicle growth, respectively. Results The biomechanically optimized PEGylated fibrin formulation was developed using mathematical modeling with specific targeting of Young’s modulus of the human ovarian cortex at reproductive age (3178 ± 245 Pa [11]). The PEGylated fibrin hydrogel containing 39.06 mg/ml of PEGylated fibrinogen and 50.36 IU/ml of thrombin with Young's modulus of 3512.31±656.97 pa was found to be the most desirable condition (desirability of 97.5%). The viability of follicles was analyzed morphologically, which was 87% and 83% on days 1 and 7, respectively. The diameter of isolated follicles cultured in our hydrogel significantly increased from 48.5±10.8µm (day 1) to 222.5±84.5µm (day 7). The follicles’ growth was confirmed by the presence of Ki67-positive granulosa cells in follicles analyzed by confocal microscopy on day 7. Moreover, our results from cell-cell communication analyses on day 4 showed that 100% of these follicles were stained positive for connexin 43. On the other hand, 41.67% of follicles had complete transzonal projections (TZPs), while TZPs were partially and completely absent in 45.83% and 12.5% of the follicles, respectively. Conclusion Our finding suggested a PEGylated fibrin formulation for encapsulation of human preantral follicles that could support the survival and growth of follicles by resembling the mechanical property of the natural human ovary at the reproductive-age stage.
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Dadashzadeh, A., Moghassemi, S., Peaucelle, A., Dolmans, M.-M., & Andrade Amorim, C. (2022). Encapsulation of human preantral follicles in PEGylated fibrin hydrogel with similar mechanical strength of reproductive-age ovarian tissue. Bioregate Forum, Belgium.