O-159 Impact of open and closed methods of vitrification on oocyte mitochondria function and RNAseq transcriptome profile: a preclinical screening study

Abstract

Abstract Study question Do current methods of open vitrification (OV) and closed vitrification (CV) impact on oocyte mitochondria function and transcriptome? Summary answer Oocyte vitrification confers unique mitochondria distribution patterns and alters the expression of stress response genes and mitochondria function relative to control (fresh) oocytes. What is known already Mitochondria are essential for maintenance of metabolic function and developmental competence at physiological conditions. Conversely, open and closed methods of oocyte vitrification are associated with physical, chemical and non-physiological changes, and exposures to cryoprotecting agents which could be harmful at extremely high and low temperatures. Study design, size, duration Ninety-four unfertilised Metaphase II (MII) oocytes were donated to research between May 2018 and August 2021. Fifty-seven oocytes were randomly allocated to the different study groups. Similarly, thirty-seven oocytes were allocated to the different study control groups. Participants/materials, setting, methods Fourteen women underdoing IVF/ICSI treatment cycles were involved in the study. cDNA was obtained for qPCR and the Smart-Seq2 protocol and single-cell RNA-Seq was performed (illumina NextSeq2000). Mitochondrial staining on live-oocytes was performed using Mito-tracker Deep Red stain, and images evaluated using fluorescence microscopy. Differentially expressed genes were identified using the R package edgeR and functional gene ontology was performed using an over-representation analysis via WebGestalt. Correction for multiple testing was calculated using Benjamini-Hochberg method Main results and the role of chance Mean mitochondrial intensity/density showed similarities between open (OV) and closed vitrification (CV) and control (fresh oocytes FO): 25.8 (12.7-44.9, standard deviation [SD] 10.2) for FO oocytes (n = 15), 30.3 (16.2-44.8, SD 10.1) for OV oocytes (n = 11), 26.6 (13.3-64.6, SD 15.3) for CV oocytes (n = 12) (p = 0.617). Distinct mitochondria distribution patterns (cytoplasmic and peripheral) were associated with OV and CV oocytes. Twice as many peripheral to cytoplasmic patterns were observed in FO and OV oocytes in contrast to CV oocytes (p = 037). Mean expression of the gene SDHB, which is associated with the TCA cycle and the mitochondrial electron transport chain was reduced in vitrified oocytes (n = 13) (0.0879 ± 0.0216 vs 0.0593 ± 0.0479), as was the anti-apoptotic gene BCL2 (p = 0.0061), relative to control oocytes (n = 13), (p = 0.01). Conversely, genes associated with apoptosis and stress response (BAX (p = 0.002) and BCL2L1 (p = 0.016)) were significantly enriched relative to control oocytes. RNAseq transcriptome analysis revealed a total of 71 differentially expressed genes following vitrification relative to control oocytes. Genes associated with critical metabolic processes (death domain binding, BCL-2 homology 3 (BH3) domains, and exopolyphosphatatse activities) were enriched in vitrified oocytes relative to the control group (FDR>0.05). Limitations, reasons for caution The use of unfertilised metaphase II (MII) oocytes on day 1 as a preclinical application screening model for vitrification may not reflect the molecular status and integrity of clinical grade MII oocytes on day 0. Wider implications of the findings This study emphasizes the inherent impact of vitrification on oocyte mitochondria function and transcriptome relative to control oocytes. Importantly, unintended perturbations may be detrimental to oocyte health and developmental competence, suggesting the need for a continuous safety screening and monitoring of ART methods of cryopreservation and offspring long-term health. Trial registration number National Institute for Health Research (NIHR). Grant Reference: ICA-CDRF-2015-01-068