Effects of chemical

study questionWhat is the effect of the chemical SUMMARY ANSWER: Although histological assessment shows that cIVA significantly increases follicle survival and growth compared to Frag, both protocols stimulate extensive and nearly identical transcriptomic changes in cultured tissue compared to freshly collected ovarian tissue, including marked changes in energy metabolism and inflammatory responses. WHAT IS KNOWN ALREADY: Treatments based on cIVA of the phosphatase and tensin homolog (PTEN)-phosphatidylinositol 3-kinase (PI3K) pathway in ovarian tissue followed by auto-transplantation have been administered to patients with refractory premature ovarian insufficiency (POI) and resulted in live births. However, comparable effects with mere tissue fragmentation have been shown, questioning the added value of chemical stimulation that could potentially activate oncogenic responses. STUDY DESIGN SIZE DURATION: Fifty-nine ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section (C-section). The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic)+740Y-P (Frag+cIVA group) during the first 24 h of culture, while the other half were cultured with medium only (Frag group). Subsequently, both groups were cultured with medium only for an additional 6 days. Tissue and media samples were collected for histological, transcriptomic, steroid hormone, and cytokine/chemokine analyses at various time points. PARTICIPANTS/MATERIALS SETTING

methodsEffects on follicles were evaluated by counting and scoring serial sections stained with hematoxylin and eosin before and after the 7-day culture. Follicle function was assessed by quantification of steroids by ultra-performance liquid chromatography tandem-mass spectrometry at different time points. Cytokines and chemokines were measured by multiplex assay. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the initial 24-h culture. Selected differentially expressed genes (DEGs) were validated by quantitative PCR and immunofluorescence in cultured ovarian tissue as well as in KGN cell (human ovarian granulosa-like tumor cell line) culture experiments. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to the Frag group, the Frag+cIVA group exhibited a significantly higher follicle survival rate, increased numbers of secondary follicles, and larger follicle sizes. Additionally, the tissue in the Frag+cIVA group produced less dehydroepiandrosterone compared to Frag. Cytokine measurement showed a strong inflammatory response at the start of the culture in both groups. The RNA-seq data revealed modest differences between the Frag+cIVA and Frag groups, with only 164 DEGs identified using a relaxed cut-off of false discovery rate (FDR) <0.1. Apart from the expected PI3K-protein kinase B (Akt) pathway, cIVA also regulated pathways related to hypoxia, cytokines, and inflammation. In comparison to freshly collected ovarian tissue, gene expression in general was markedly affected in both the Frag+cIVA and Frag groups, with a total of 3119 and 2900 DEGs identified (FDR < 0.001), respectively. The top enriched gene sets in both groups included several pathways known to modulate follicle growth such as mammalian target of rapamycin (mTOR)C1 signaling. Significant changes compared to fresh tissue were also observed in the expression of genes encoding for steroidogenesis enzymes and classical granulosa cell markers in both groups. Intriguingly, we discovered a profound upregulation of genes related to glycolysis and its upstream regulator in both Frag and Frag+cIVA groups, and these changes were further boosted by the cIVA treatment. Cell culture experiments confirmed glycolysis-related genes as direct targets of the cIVA drugs. In conclusion, cIVA enhances follicle growth, as expected, but the mechanisms may be more complex than PI3K-Akt-mTOR alone, and the impact on function and quality of the follicles after the culture period remains an open question. LARGE SCALE DATA: Data were deposited in the GEO data base, accession number GSE234765. The code for sequencing analysis can be found in https://github.com/tialiv/IVA_project. LIMITATIONS REASONS FOR CAUTION: Similar to the published IVA protocols, the first steps in our study were performed in an WIDER IMPLICATIONS OF THE

findingsThe general impact of fragmentation and short (24 h) STUDY FUNDING/COMPETING INTERESTS: This study was funded by research grants from European Union's Horizon 2020 Research and Innovation Programme (Project ERIN No. 952516, FREIA No. 825100), Swedish Research Council VR (2020-02132), StratRegen funding from Karolinska Institutet, KI-China Scholarship Council (CSC) Programme and the Natural Science Foundation of Hunan (2022JJ40782). International Iberian Nanotechnology Laboratory Research was funded by the European Union's H2020 Project Sinfonia (857253) and SbDToolBox (NORTE-01-0145-FEDER-000047), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. No competing interests are declared.