Abstract Study question Can centromeres be labelled in live human oocytes using Cas9 system and is the labelling efficiency affected by advancing female age? Summary answer Centromeres in human oocytes can be labelled using Cas9 system. Signal intensity decreases in oocytes from older patients, suggesting centromeric DNA unravelling as oocytes age. What is known already Aneuploidy in human oocytes is associated with infertility, pregnancy loss or genetic diseases in offspring. It arises due to abnormal chromosome segregation during meiosis, with the risk increasing with advancing female age. This is attributed to age-dependent loss of pericentromere-localised cohesion which holds sister chromatids together. Our work revealed that shugoshin-2 (Sgo2), a protein involved in protection of sister chromatid cohesion is also lost with age. This can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. This hints at more extensive alterations to centromere organisation with advanced maternal age. Study design, size, duration This study uses human oocytes deselected from fertility treatment donated for research purposes with informed consent (HFEA RO155). The oocytes were collected fresh at germinal vesicle (GV), metaphase I (MI) or metaphase II (MII) stage, injected with Cas9 labelling system and live imaged or fixed. Participants/materials, setting, methods Centromeres were labelled in human oocytes using a dead (nuclease-deficient) (d)Cas9-EGFP protein and alpha satellite-specific small guide RNAs (sgRNA). Human oocytes were injected with dCas9-EGFP:sgAlphaSat and live-imaged using dual colour confocal microscopy with SPY555-DNA dye to also visualise chromosomes. High resolution imaging results were analysed with in-house automated kinetochore tracking software. Additional oocytes were fixed and processed for immunofluorescence to determine the high resolution localisation of dCas9-EGFP:sgAlphaSat and Sgo2 molecules. Main results and the role of chance We found that centromeres in human oocytes are efficiently labelled when using Cas9 system. During both meiosis I and II the dCas9-EGFP:sgAlphaSat probe co-localised with pericentromeric bridge and subcentromeric pools of Sgo2. We observed that the dCas9-EGFP:sgAlphaSat signal fades with advancing female age. Quantification reveals that in meiosis II the loss is abrupt at around 35 years of age, resembling Sgo2 behaviour. Initial live imaging indicates that already compromised signal in older oocytes is completely lost about 4hrs after germinal vesicle breakdown, suggesting that advancing age renders centromeres more vulnerable as meiosis I progresses. We have successfully tracked the centromeres in high resolution imaging using the automated software, which allows for detailed investigation of human meiosis chromosome dynamics. This method has been previously validated and successfully used for other cell systems. Limitations, reasons for caution Availability of human oocytes for research purposes is low, limiting our sample size. Moreover, most oocytes used for this study were deselected from treatment of patients diagnosed with infertility, potentially affecting their quality. Wider implications of the findings Our results reveal a large-scale change in centromeric DNA structure in human oocytes with increasing age. Live centromere labelling is a first successful attempt to bivalent tracking in human meiosis with potential to provide insight into age-related loss of fertility in women. Trial registration number n/a