P-272 Ovarian follicular fluid: liquid biopsy insights via cfChIP-seq in IVF and oocyte preservation

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Abstract Study question How can cfChIP-seq analysis be used to characterize functional gene expression signatures in follicular fluid and be used to assess ovarian function and oocyte competence? Summary answer CfChIP-seq analysis revealed intact modified nucleosomes with distinct active promoters, including hallmarks of granulosa cells and signs of immune modulation in unexplained infertility samples. What is known already While follicular fluid analysis offers potential insights into oocyte quality, traditional approaches have been limited to proteomics, metabolomics, and hormone levels as well as quantitative cell-free DNA measurements. Cell-free chromatin immunoprecipitation followed by sequencing (cfChIP-seq) moves beyond standard DNA detection to reveal functional signatures of gene expression and active biological processes. Study design, size, duration An ongoing prospective clinical study in a single university-affiliated IVF unit. So far, over the past two years, 47 patients have been recruited. Participants/materials, setting, methods Patients included were either diagnosed with infertility or were patients performing oocyte cryopreservation from choice. Patients were approached before oocyte retrieval procedure and were asked to provide a 5-ml serum sample along with a uni-follicular sample of follicular fluid, collected after oocyte separation. The samples underwent cfChIP-seq assay, including immunoprecipitation to enrich nucleosomes with active chromatin marks, followed by DNA sequencing of these nucleosomes. Collected patient data included patient clinical characteristics and treatment details. Main results and the role of chance We analyzed follicular fluid (FF) samples from 47 patients: 20 were presumably fertile (controls), 8 had polycystic ovarian syndrome (PCOS), 9 had unexplained infertility (UE), and 6 had diminished ovarian reserve (DOR). CfChIP-seq analysis revealed intact modified nucleosomes with distinct gene expression signatures. When comparing serum samples to FF, there was a unique signature pattern to FF. These active promoter patterns showed hallmarks of granulosa cells, including FSHR and HSPG2 (Perlecan) and even active promoters not previously reported in ovarian samples, such as GLP1R. Analysis of gene activation showed no remarkable alterations in PCOS and DOR, but UE demonstrated significant changes: SLC17A8 and GAB4 upregulated, ARHGEF35 and LIPF downregulated. Non-negative matrix factorization successfully distinguished between clinical conditions, revealing unique molecular signatures in patients with unexplained infertility. Furthermore, enrichment analysis revealed distinct patterns of immune modulation in UE samples. Limitations, reasons for caution To the best of our knowledge, qualitative analysis of cell-free DNA was not previously done on follicular fluid, and we have yet to determine the clinical implications of our results, thus caution is mandatory. Since the sample size is limited, our findings require further validation. Wider implications of the findings This study introduces a novel qualitative tool for analyzing follicular fluid. Future applications of this methodology could enable more personalized approaches to medical care tailored to specific patient populations. Trial registration number Yes

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