Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment.

study questionDo ovarian stromal cells (OSCs) influence the viability and growth of human preantral follicles SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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 STUDY DESIGN SIZE DURATION: 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 PARTICIPANTS/MATERIALS SETTING

methodsPrimordial/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. MAIN RESULTS AND THE ROLE OF CHANCE: 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 ( LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further WIDER IMPLICATIONS OF THE

findingsActivating and growing human primordial/intermediate follicles to a secondary stage in STUDY FUNDING/COMPETING INTERESTS: 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.