Purpose <p>To evaluate the effects of indirect co-culture of fresh or vitrified ovarian fragments (OFs) with Wharton’s jelly–derived mesenchymal stem cells (WJ-MSCs).</p> Methods <p>The ovarian fragments were divided into two groups: fresh and vitrified. Some fresh OFs were immediately fixed (fresh control), while others were cultured in vitro for 14&#xa0;days, either without (monoculture) or with (co-culture) WJ-MSCs. After vitrification and warming, some OFs were immediately fixed (vitrified control), while others were cultured under the same conditions as the fresh OFs. Morphological analysis (classical histology), proliferation (PCNA), senescence (Sudan Black B), expression of genes related to folliculogenesis (FSH-R, LHX8, NANOS3, and FOXL2), apoptosis and anti-apoptosis (BAX and BCL2), and DNA fragmentation (TUNEL) were evaluated in all OFs. Estradiol (E2) levels were measured in the culture medium on days 2, 8, and 14.</p> Results <p>The follicular morphology of fresh or vitrified OFs cultured with WJ-MSCs was similar to that of fresh and vitrified OFs fixed on day 0. Regarding development, a higher proportion of normal primary follicles was observed in fresh and vitrified ovarian fragments co-cultured with WJ-MSCs. The presence of WJ-MSCs increased the proportion of stromal cells and proliferative follicles in fresh OFs, and reduced the number of senescent stromal cells in vitrified OFs. Additionally, in fresh OF, the absence of WJ-MSCs reduced BCL2 expression, while their presence in vitrified OF reduced E₂ secretion only at day 14.</p> Conclusion <p>JW-MSCs provide a favorable microenvironment for the survival and development of pre-antral follicles cultured in vitro.</p>