Ovarian extracellular matrix‐based hydrogel for human ovarian follicle survival in vivo: A pilot work

Abstract

To successfully assemble a bio-engineered ovary, we need to create a three-dimensional matrix able to accommodate isolated follicles and cells. The goal of this study was to develop an extracellular matrix hydrogel (oECM) derived from decellularized bovine ovaries able to support, in combination with alginate, human ovarian follicle survival and growth in vitro. Two different hydrogels (oECM1, oECM2) were produced and compared in terms of decellularization efficiency (dsDNA), ECM preservation (collagen and glycosaminoglycan levels), ultrastructure, rigidity, and cytotoxicity. oECM2 showed significantly less dsDNA, greater retention of glycosaminoglycans and better rigidity than oECM1.Isolated human ovarian follicles were then encapsulated in four selected hydrogel combinations: (1) 100% oECM2, (2) 90% oECM2 + 10% alginate, (3) 75% oECM2 + 25% alginate, and (4) 100% alginate. After 1 week of in vitro culture, follicle recovery rate, viability, and growth were analyzed. On day 7 of in vitro culture, follicle recovery rates were 0%, 23%, 65%, 82% in groups 1-4, respectively, rising proportionally with increased alginate content. However, there was no difference in follicle viability or growth between groups 2 and 3 and controls (group 4). In conclusion, since pure alginate cannot be used to graft preantral follicles due to its poor revascularization and degradation after grafting, oECM2 hydrogel combined with alginate may provide a new and promising alternative to graft isolated human follicles in a bio-engineered ovary.

Future Challenges and Opportunities of Extracellular Matrix Hydrogels in Female Reproductive Medicine

“…Recent work converted decellularized scaffolds from reproductive organs into ECM hydrogels ( Table 1 ), which, as discussed in Section 2.1 , have the main advantage of being able to conform to the shape of any defect site, since they are first injected as a viscous fluid (pre-gel) and finally polymerize into a 3D structure (hydrogel) at physiological temperature [ 55 ]. The development of ECM hydrogels from the female reproductive tract is still nascent, but promising results include the formation of hydrogels from decellularized uterus [ 40 , 52 , 106 , 107 ], ovary [ 108 , 109 ], oviduct [ 110 ], vagina [ 111 ], and placenta [ 112 , 113 , 114 ] ( Table 1 ).…”

“…Only two studies have developed ovarian ECM (OvaECM) hydrogels. Buckenmeyer et al used them as a follicle carrier in a murine model of premature ovarian failure (POF), and Chiti et al created a support that was based on an OvaECM hydrogel for human and murine follicle growth in vitro [ 108 , 109 ].…”

“…Given its phylogenetic proximity, fragments or whole pig and cow ovaries have been the most used for decellularization techniques. The ovarian ECM of these species presents high protein conservation with respect to humans [ 108 , 115 , 116 ]. In addition, the ovaries have human-like size and anatomy and, in the case of cow, an analogous mono-ovulatory dynamic.…”

“…Immersion and agitation either of the whole organ or of small tissue fragments is the most appropriate protocol for ovarian decellularization to generate OvaECM hydrogels. Rather than using enzymatic agents alone, it is best to use a combination of these agents and detergents, as detergents efficiently disrupt cell-nuclei and membranes and consequently promote better decellularization [ 108 ]. Perfusion of the whole ovary can be a very slow process and requires large volumes of detergents because of the complex anatomy of the blood vessels being cannulized.…”

“…Recently, Chiti et al designed decellularized OvaECM hydrogels that are able to support human and murine ovarian follicle survival and growth in vitro [ 108 ], which is also described in ovarian ECM scaffolds [ 82 ]. Even if OvaECM hydrogels provide more suitable microenvironment than native scaffolds, based on rheological characterization, the hydrogel exhibited lower rigidity than the native ovary.…”