# Technologies for the Production of Fertilizable Mammalian Oocytes
## 5 Towards a Biosynthetic Mammalian Ovary
### The New Ovaries  
The possibility to use fibrin gel, formed by high concentration of fibrin and thrombin, as scaffold was tested by [PERSON] and her colleagues in 2016 [PERSON] and her colleagues in 2016 [PERSON] and others (2016). They recovered human primordial and primary follicles that were encapsulated into fibrin clots before being xenotransplanted into nude mice. The grafted follicles were viable and increased in size. In 2017, [PERSON] and colleagues [PERSON] and others (2017) published the possibility of creating a mouse bioprosthetic ovary capable of restoring ovarian functions in sterilized animals. If compared with the encapsulation within hydrogels (Figure 5a), now the strategy is to produce a structure capable of assuring not only follicle growth but also long-term function of the bioconstruct after transplantation. Therefore, the authors focused particular attention on scaffold printing (Figure 5b), because it was necessary to obtain scaffold designs with similar struts (about 250 \(\upmu\)m) and distance between struts (about 350 \(\upmu\)m) to reproduce a defined micropore architecture, that assures follicle development. By using a 3D printable gelatin, they formed a hydrogel scaffolds with optimized pore geometry either to seed follicles or to allow nutrient diffusion. They reported that maintenance of follicle's spherical shape required scaffolds with corners surrounding follicles on multiple sides but with limited follicle-scaffold interactions. Follicles cultured for eight days expressed laminin, thus confirming the formation of a basal lamina, and produced steroids. The enclosed oocytes reached normal final diameters and were able to complete meiotic maturation up to MII stage and to ovulate. These scaffolds can be implanted without undergoing enzymatic degradation, as required when follicles are encapsulated in gels. When implanted into ovarian bursa of ovariectomized mice, scaffolds were vascularized within few days, showing a distribution and density of vessels comparable to that recorded in normal ovaries. It is remarkable that the lack of any mechanical manipulation better preserves oocyte quality. In fact, after mating and without any hormonal supplementation, recipients with bioprosthetic ovarian fragments produced healthy litters that are nursed because of normal prolactin production. This indicates that following xenografts the normal hypothalamic-pituitary-ovarian axis is well re-established. More recently, three new approaches have been reported in the literature. The first is based on the decellularization of human ovary. In fact, by using sodium lauryl ester sulfate (SLES), organ-specific extracellular matrix-based scaffolds have been used as a native niche for the culture of primary ovarian cells (POC) obtained from human ovaries. These cells show a surprising capability to reconstruct primordial or primary follicle-like structures within the scaffolds. This innovative approach has been validated by in vivo studies that confirm biocompatibility, normal steroid production and neovascularization [PERSON] and others (2016). In the second, innovative fibrous scaffolds (average diameter: \(495\pm 133\)\(\upmu\)m) have been produced by electrospinning of poly (\(\varepsilon\)-caprolactone) (PCL) and PCL/gelatin, which reproduce a structure similar to the ovarian healthy tissue. PCL is a biodegradable polyester that, when blended with gelatin, improves strongly scaffold biocompatibility while reducing biodegradability. Figure 5: Comparison of the experimental design used to in vitro culture of secondary follicles. (**a**) Isolated secondary follicles are transferred in drops of hydrogels (alginate—Matrigel®) on a polypropylene mesh (100 \(\upmu\)m). Then, the mesh is immerged into encapsulation solution. (**b**) Bioprinted scaffold (gelatin) has struts of \(\sim\)350 \(\upmu\)m and pore size of \(\sim\)350 \(\upmu\)m; angles are 30\({}^{\circ}\) or 60\({}^{\circ}\). Secondary follicles are seeded onto scaffold and cultured. Contacts with scaffold and follicles are limited.