Do ovarian stromal cells (OSCs) influence the viability and growth of human preantral follicles ? A feeder layer of OSCs promotes the growth and transition of low developmental stage follicles to the primary/secondary stage while maintaining a high proportion of viable follicles. In the ovary, follicles rely on the support of ovarian cells, which secrete essential factors for their survival and development. This phenomenon has also been demonstrated through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of OSCs. This co-culture notably enhances follicle survival and growth. Pre-antral follicles were isolated from human frozen-thawed ovarian tissue biopsies and then encapsulated in 1% alginate scaffolds. These embedded preantral follicles were either placed directly on the OSCs feeder layer or at the bottom of a culture dish for a 7-day culture (control). The study compared follicle viability, growth, and hormone production between the different groups. Primordial/intermediate and primary follicles were isolated from frozen-thawed ovarian tissue of cancer patients (n = 6). OSCs were then isolated from ovarian tissue of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 using an inverted microscope to assess their development based on the increase in diameter. Viability was evaluated by staining a subset of follicles (n = 87) with calcein AM and ethidium homodimer-I, followed by classification into healthy/minimally damaged and damaged/dead follicles using confocal fluorescence microscopy. Additionally, estradiol levels were measured using ELISA. A total of 382 human preantral follicles (370 primordial/intermediate and 12 primary) with a mean diameter of 40.8 ± 9.9 µm (mean ± SD) were isolated, embedded in 1% alginate hydrogel, and placed either on a monolayer of OSCs or directly on the plastic. By Day 7, the preantral follicles showed a significant size increase under both culture conditions ( < 0.0001 for D0 vs D7). The mean diameter of follicles (quiescent and growing) cultured on the feeder layer was 80.6 ± 11.0 μm compared to 67.3 ± 7.2 μm without it ( = 0.07). During the 7-day culture, the viability of the follicles significantly decreased only in the group without an OSCs monolayer compared to the D0 viability ( < 0.05). Additionally, more follicles transitioned to a higher developmental stage in the presence of OSCs (D0 primordial/intermediate: 184, primary: 7 vs D7 primordial/intermediate: 51, primary/secondary: 93) compared to those cultured without OSCs (D0 primordial/intermediate: 186, primary: 5 vs D7 primordial/intermediate: 84, primary/secondary: 65; < 0.001). Specifically, 66 and 44 follicles reached the secondary stage (75< x <200 μm) in the presence and absence of OSCs, respectively. Moreover, the estradiol level was significantly higher ( = 0.006) in the alginate beads containing primordial and growing follicles cultured on the OSCs (54.1 ± 14.2 pg/ml) compared to those cultured without OSCs (29.9 ± 4.0 pg/ml). N/A. This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further studies are required to investigate follicular developmental capacity, physiology, and steroidogenesis in alginate scaffolds with human OSCs. Activating and growing human primordial/intermediate follicles to a secondary stage in short-term culture has posed a longstanding challenge. However, co-culturing with human OSCs has shown the potential to overcome this limitation. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., PhD scholarship awarded to H.V.), Fondation Louvain (awarded to C.A.A.; PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer), Foundation Against Cancer (grant 2018-042 awarded to A.C.), and the European Community Structural Funds and Lithuanian Research Council (Agreement registration No. D-19-0874). The authors have no conflicts of interest to declare.
Vlieghe, Hanne
