Peri-Ovarian Mitochondrial Administration Improves Surrogate Markers of Ovarian Function in Aged Rats: Free Versus Alginate-Encapsulated Mesenchymal Stromal Cell-Derived Mitochondria

Background: Ovarian aging is driven in part by mitochondrial dysfunction, and transfer of exogenous mitochondria has been proposed as a cell-free alternative to stem cell therapy; whether the delivery format alters efficacy has not been tested directly. Methods: We compared free and alginate-encapsulated mitochondria isolated from rat bone marrow-derived mesenchymal stromal cells (BM-MSCs) in aged rats. Mitochondria retained their membrane potential after encapsulation; approximately 35% of the encapsulated protein was released within 1 h in vitro, and release was complete by 24 h. Thirty-five female Wistar rats were allocated to five groups (n = 7): young control (Y-Ctrl), aged control (O-Ctrl), and three aged subgroups receiving empty capsules (Alg), free mitochondria (MSC-Mito), or encapsulated mitochondria (MSC-Mito@Alg), administered peri-ovarianly and evaluated on day 14. Results: Both treatments were associated with higher primordial and secondary follicle numbers, fewer atretic follicles, higher estradiol, higher Esr1, Fshr, and Sod2 expression, lower Casp3 and Mct4 expression, and lower Caspase-3 and higher Ki-67 immunoreactivity relative to aged controls. Anti-Müllerian hormone, Nrf2, and Tomm20 were higher only with MSC-Mito@Alg; corpus luteum counts were higher only with MSC-Mito. Although empty capsules were not inert, peri-ovarian mitochondrial administration improved follicular, endocrine, and molecular indices of ovarian aging. Conclusions: The encapsulated formulation was associated with additional effects on antioxidant and mitochondrial-mass endpoints but was not uniformly superior to free mitochondria.