P-424 Effects of in vitro activation vs fragmentation on human ovarian tissue in culture

Abstract

Abstract Study question What is the added value of in vitro activation (IVA) protocol compared with fragmentation only in human ovarian tissue culture? Summary answer Although histological assessment shows that the IVA increases follicle survival and growth, IVA and fragmentation stimulate extensive and nearly identical transcriptomic changes in cultured tissues. What is known already Treatments based on activation of the PTEN/PI3K pathway in ovarian tissue in vitro have been administrated to refractory primary ovarian insufficiency (POI) patients and resulted in live births following auto-transplantation of the tissue. However, some studies have shown that mere fragmentation of the tissue produces comparative effects, questioning the added value of chemical stimulation protocol which could potentially activate oncogenic pathways. Study design, size, duration Thirty-three ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section at Karolinska University Hospital Huddinge. The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic) +740Y-P (IVA group) during the first 24 h of culture while half were exposed to vehicle only (fragmentation only group). Subsequently, both groups were cultured for additional 6 days. Media change was performed every other day. Participants/materials, setting, methods Effects on follicles were evaluated by counting and scoring follicles before and after the 7-day culture via HE-stained serial sections. Follicle function was assessed by quantification of steroids by UPLC-MS/MS at the end of the culture. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the 24-h initial culture step. Selected differentially expressed genes (DEGs) were validated by qPCR and immunofluorescence in independent culture experiments. Main results and the role of chance Compared to fragmentation only group, significantly higher follicle survival rate, increased secondary follicle number and sizes were found in IVA group. No significant differences were detected in levels of steroids in culture media on day 7 between the two groups. In our RNA-seq data, when comparing the IVA group to fragmentation only group, only 110 DEGs were found with the relaxed cutoff of FDR<0.1. The signaling pathways affected by IVA involved follicle growth but also inflammation and DNA damage. However, we found that the gene expression was profoundly affected in both IVA and fragmentation only groups compared to the fresh tissue: in total 3,676 and 4,223 DEGs were found (FDR<0.001), respectively. The top enriched gene sets in both groups included several pathways that are known to modulate follicle growth, e.g. PI3K/AKT, MTORC1. We also found that classical steroidogenesis genes (e.g. CYP11A1, CYP17A1) were significantly downregulated in both groups after 24-h culture. Moreover, upregulation of genes related to glycolysis, a process that was shown to promote activation of primordial follicles, and its upstream regulator were shown in the RNA-seq data. This effect was validated by qPCR (ENO1, PKM, LDHA, p < 0.0001). Future validation will be performed using western blot and immunofluorescence. Limitations, reasons for caution The study was performed in an in vitro model where tissue was isolated from the regulation of hypothalamic-pituitary-ovarian (HPO) axis. And thus, further experiments with xeno-transplantation may be needed to explore the effect of IVA in vivo in the future. Wider implications of the findings The impact of 24-h culture on gene expression in ovarian tissue far exceeds the effect of IVA. Yet, follicle growth was stimulated by IVA, which may suggest effects on specific cell populations that are diluted in bulk transcriptomics. Cell type-specific impacts need further studies to conclude about effectiveness of IVA. Trial registration number Not applicable