Mind the mechanical strength: tailoring a 3D matrix to encapsulate isolated human preantral follicles

Abstract

STUDY QUESTION Would a hydrogel with similar mechanical properties to the human ovarian cortex support preantral follicle development? SUMMARY ANSWER Yes, our tailored PEGylated fibrin hydrogel was shown to significantly improve follicle growth in vitro. WHAT IS KNOWN ALREADY One of the main challenges in developing an engineered ovary is to provide a 3D matrix that supports the follicle architecture and the interaction between granulosa cells and the oocyte as they are essential for folliculogenesis. Thanks to its biocompatibility and bioactivity, fibrin has been employed to fabricate a 3D matrix to encapsulate ovarian follicles. However, follicles lose their physical support within a few days owing to rapid fibrin degradation. Therefore, different strategies, including physical and chemical modifications, have been developed to enhance the stability of fibrin. STUDY DESIGN, SIZE, DURATION By developing a matrix made of a synthetic (polyethylene glycol: PEG) and natural polymer (fibrin), we aimed to overcome fibrin degradation by the chemical reaction of PEGylation and tailor a PEGylated fibrin hydrogel formulation with mechanical strength similar to the ovarian cortex in women of reproductive age. To this end, response surface methodology was employed to obtain a tailored formulation of PEGylated fibrin. This hydrogel was then tested to encapsulate and support isolated human preantral follicles in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS A PEGylated fibrin formulation was tailored using mathematical modeling software to mimic the mechanical properties of human ovarian tissue at reproductive age. Human preantral follicles were isolated from 11 patients of reproductive age and encapsulated in the tailored hydrogels, which were cultured in vitro for 4 or 7 days. Follicle survival and diameter were assessed on days 1 and 7. Furthermore, the follicles were subjected to confocal microscopy to evaluate their growth (Ki67 staining) on day 7 and analyze cell-cell communication (connexin 43 and transzonal projection staining) on day 4. MAIN RESULTS AND THE ROLE OF CHANCE In this study, mathematical modeling was applied to achieve the biomechanically tailored PEGylated fibrin formulation by targeting the specific goal of 3178 ± 245 Pascal, Young’s modulus of ovarian cortical tissue in reproductive-age women. Our results demonstrated that the PEGylated fibrin hydrogel consisting of 39.06 mg/ml of PEGylated fibrinogen and 50.36 IU/ml of thrombin was the optimum condition with the desirability of 97.5%. This tailored hydrogel yielded a high follicle survival rate (83%) after 7 days of in vitro culture and supported its development up to the secondary stage. Follicle growth was confirmed by the presence of Ki67-positive granulosa cells on day 7. Additionally, connexin 43 and Phalloidin staining indicated the retention of connections between granulosa cells and the oocyte. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION In this study, our tailored hydrogel was only tested in vitro, which is not the same as the physiological environment. It is crucial to conduct a study assessing the follicles following their encapsulation in the tailored hydrogel and transplantation, which will be the next step of our investigation. WIDER IMPLICATIONS OF THE FINDINGS The findings from this study introduced a suitable biomaterial similar to the ovarian cortex in reproductive-age women in terms of biomechanical properties for encapsulating human preantral follicles. This biomaterial allowed the radial growth of follicles and preserved their viability. Furthermore, PEGylation improved the stability of fibrin and the physical support of follicles. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by a grant from the Fondation Louvain (awarded to C.A.A.). The authors declare no competing interests. WHAT DOES THIS MEAN FOR PATIENTS? Survival rates of leukemia patients have been steadily increasing but, owing to cancer treatment, some survivors will face infertility at a very young age. One of the strategies under development to restore fertility in these patients is the transplantable ‘engineered ovary’, which should mimic the normal ovary as closely as possible. To assemble the engineered ovary, it is necessary to encase isolated preantral follicles (follicles at an early stage of growth that contain one immature egg) and ovarian cells in a 3D matrix. Developing this matrix is one of the main challenges in creating an engineered ovary, as it should support the follicle architecture as well as the interaction between granulosa cells (which produce estrogen, progesterone) and the oocyte (egg), as they are essential for follicle development. This study demonstrates that a matrix made of a synthetic (polyethylene glycol) and natural polymer (fibrin), with mechanical strength similar to that of the outer layer of the ovary in women of reproductive age, can successfully be used to encapsulate human preantral follicles. Indeed, we have shown that our 3D matrix yielded a high follicle survival rate and supported follicle growth to the secondary stage.

Comprehensive Review of In Vitro Human Follicle Development for Fertility Restoration: Recent Achievements, Current Challenges, and Future Optimization Strategies

“…This is not surprising as, during intraovarian development, follicles migrate from the rigid cortex towards the less dense medulla. Secondary follicle isolation can be performed either enzymatically [ 56 , 58 , 104 , 111 ], mechanically by microdissection [ 10 , 21 , 23 , 34 , 52 , 53 , 107 , 109 , 110 ], or a combination of both [ 39 , 105 , 106 ] ( Table 2 ). The microdissection approach, using fine-gauge needles, has been established as the most appropriate, as it maintains an intact follicle basement membrane, thereby preserving oocyte-GC communications [ 10 , 21 ].…”

“…Among these bio-matrices, natural compounds like alginate and Matrigel (a commercialized solubilized basal membrane matrix) were found to support IVG of isolated human secondary follicles [ 56 , 105 ]. Moreover, synthetic components such as polyethylene glycol (PEG)-ylated fibrin hydrogels were successfully utilized to promote human secondary follicle development in vitro [ 58 ]. Other approaches using decellularized ovarian tissue [ 113 ] and 3D microporous scaffolds [ 114 ] were also shown to support follicle IVG.…”

“…Medium composition is also crucial to IVG. The most commonly used media in human IVG systems are αMEM [ 23 , 56 , 105 , 107 , 108 ], McCoy’s 5a [ 10 , 21 , 34 , 39 ], Dulbecco’s MEM (DMEM) [ 104 ], or mixed media (DMEM+F12) [ 58 ], while McCoys’ 5a [ 109 , 110 ] and TCM-199 [ 53 ] are typically utilized in bovines ( Table 2 ).…”

Making a good egg: human oocyte health, aging, and in vitro development

“…Novel scaffolds to support human follicle growth in vitro have been developed; these include decellularized ovarian tissue ( 566 , 574 , 575 ) and three-dimensional microporous scaffolds ( 576 , 577 ). The use of these scaffolds may enhance in vitro development of follicles but will be important in developing artificial ovaries for transplantation ( 569 , 578 ).…”

Liposomal oxygen-generating hydrogel for enhancing cell survival under hypoxia condition

“…After procurement, ovaries were immediately frozen [30]. For ovarian cell isolation, the ovarian samples were thawed and digested using our routine protocols [31][32][33]. Briefly, the tissue fragments were mechanically minced and then digested in DPBS, 0.28 Wünsch units/mL Liberase DH, and 8…”