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Study question: Could a hydrogel with similar mechanical properties of reproductive age ovarian cortex support the growth of human preantral follicles? Summary answer: Yes, our optimized PEGylated fibrin hydrogel with tailored mechanical strength provides a suitable substrate for follicle survival and growth. What is known already: The most challenging part of assembling an engineered ovary is developing an appropriate scaffold that can maintain follicle spatial architecture and allow them to have radial growth and development1-3. Fibrin is a promising biomaterial due to its biocompatibility and bioactivity. Although fibrin has been used as a scaffold in fabricating different tissues such as the ovary, it has a fast degradation rate resulting in loss of physical support for the isolated follicles4-6. Therefore, various approaches such as physical or chemical modifications have been investigated to overcome the fibrin limitation7-10. Study design, size, and duration: PEGylation as a chemical reaction was used to control fibrin degradation. Moreover, to create an appropriate scaffold for encapsulating human preantral follicles, the Response Surface Methodology was used to find a hydrogel formulation with similar mechanical property to the reproductive-age ovarian cortex. The chosen hydrogel was used to in vitro culture isolated human preantral follicles for 7 days. Participants/materials, setting, and methods: Design-Expert® software was employed to optimize PEGylated fibrin formulation to match mechanical properties of human ovarian tissues. Preantral follicles isolated from reproductive age patients were embedded in the hydrogel and in vitro cultured for 7 days. Follicle viability and diameter were evaluated on days 1 and 7. Moreover, confocal microscopy analysis was performed on whole follicles to confirm their growth by Ki67 staining of granulosa cells. Main results and the role of chance: In this study, the biomechanically optimized PEGylated fibrin formulation was obtained by mathematical modeling. The hydrogel formulation was established by setting the specific goal of targeting 3178 ± 245 Pa, the Young’s modulus of ovarian tissue fragments in reproductive-age women11, in order to obtain the optimized concentration of PEGylated fibrinogen and thrombin. Our results showed that the optimum condition with the desirability of 97.5% occurred by the PEGylated fibrin hydrogel containing 39.06 mg/ml of PEGylated fibrinogen and 50.36 IU/ml of thrombin with the Young’s modulus of 3512.31±656.97 pa. Isolated follicles cultured in our hydrogel significantly increased in their diameter from 48.5±10.8µm (day 1) to 222.5±84.5µm (day 7), indicating the suitability of the optimized PEGylated fibrin formulation to support radial follicle growth. Moreover, follicles survival on days 1 and 7 did not significantly differ (86.9±8.9% and 83.4±6.4%, respectively). Follicle growth was confirmed by the presence of Ki67-positive granulosa cells from follicles at day 7. Limitations, reasons for caution: In this study, the optimized hydrogel was first evaluated only in vitro, which is different from the actual physiological condition. Therefore, it is essential to perform a study analyzing the follicles after their encapsulation in the optimized hydrogel and transplantation, which will be the next step of our study. Wider implications of the findings: Our results introduced an appropriate biomaterial, which resembles Young’s modulus of ovarian tissues in reproductive age, for encapsulating human preantral follicles. The optimal concentration of PEGylated fibrin allowed follicles to maintain their viability and have radial growth. Moreover, PEGylation enhanced the fibrin stability and physical support of follicles. Study funding/competing interest(s) This study was supported by grants from the Fondation Louvain (PhD scholarship awarded to A.D.) and Fonds National de la Recherche Scientifique de Belgique (the Excellence of Science (FNRS-EOS), number 30443682 (PhD scholarship awarded to S.M.), FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., and grant 5/4/150/5 awarded to M.M.D). The authors declare no competing interests.
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Dadashzadeh, A., Moghassemi, S., Peaucelle, A., Dolmans, M.-M., & Andrade Amorim, C. (2022). Encapsulation of human preantral follicles in an optimized hydrogel with similar mechanical strength of reproductive-age ovarian tissue. The 38th Annual Meeting of the European Society of Human Reproduction and Embryology (ESHRE), Italy (Online).