In-depth analysis of transcriptomes in ovarian cortical follicles from children and adults reveals interfollicular heterogeneity [miRNA-Seq]

The ovarian cortical reserve of follicles is vital for fertility. Some medical treatments are toxic to follicles, leading to premature ovarian insufficiency. Ovarian tissue cryopreservation is an established method to preserve fertility in adults and even applied in prepuberty despite unproven efficacy. Here, we analyze transcriptomes of 120 cortical follicles from children and adults for detailed comparison. We discover heterogeneity with two main types of follicles in both age groups: one with expected oocyte-granulosa profiles and another with predicted role in signaling. Transcriptional changes during growth to the secondary stage are similar overall in children and adults, but variations related to extracellular matrix, theca cells, and miRNA profiles are found. Notably, cyclophosphamide dose correlates with interferon signaling in child follicles. Additionally, morphology alone is insufficient for follicle categorization suggesting a need for additional markers. Marker genes for early follicle activation are determined. These findings will help refine follicular classification and fertility preservation techniques across critical ages. Overall design: Parallel analysis of the RNA content, including both long RNAs containing a poly-A-tail and small RNAs, was performed on the same individual follicles. To reveal differences between prepubertal and adult follicle gene expression, two groups of patients were included. Ovarian tissue was collected from adult gender reassignment patients (GRP) and from children undergoing fertility preservation. Ovarian cortical samples were collected and cryopreserved from both patient groups. The cryopreserved cortical pieces were then thawed, and enzymatically digested to collect ovarian follicles. The quality of follicles was assessed by neutral red staining, and only those follicles that stained positively were collected. Each follicle was individually photographed, lysed, and frozen at -80°C until RNA library preparation.