Communication Dans Un Congrès Année : 2025

P-226 OWLO's new three-dimensional label-free imaging technology, applied to cumulus-oocyte complexes, reveals subcellular structures for quality assessment

Résumé

Study question Can three-dimensional (3D) imaging improve oocyte evaluation for assisted reproductive technologies (ART)? Summary answer OWLO's high-resolution, label-free, ultrafast imaging allows the assessment of oocyte maturity through the identification of polar bodies and intracytoplasmic organites such as lipid droplets. What is known already There is a need to develop robust assessment tools, as higher quality oocytes have been shown to have higher blastocyst rates but also a higher chance of producing euploid embryos. Oocyte nuclear maturity cannot currently be assessed in non-ICSI IVF because cumulus cells prevent visualization of the polar body (PB). Morphological abnormalities in oocyte and zona pellucida (ZP) shape, PB and perivitelline space size or ooplasmic granularity, and changes in the lipid profile are known to be detrimental. However, phase-contrast microscopy used in clinical practice does not allow proper visualization of these structures, making it difficult to identify cytoplasmic abnormalities. Study design, size, duration In this proof-of-concept study, bovine oocytes were imaged for the first time using OWLO's 3D imaging prototype. The imaging prototype is a new technology under development that allows non-invasive 3D imaging without moving the sample in depth. It is based on a low energy near-infrared laser source and is label-free. Participants/materials, setting, methods Cumulus-oocyte complexes were collected from bovine ovaries obtained from a slaughterhouse, cultured in supplemented in vitro maturation medium for 22 hours, and fixed in 2% PFA. Some samples were centrifuged (7 min, 13400g) prior to fixation. Images were captured using the OWLO prototype, at a resolution of 0.3 × 0.3 × 1.74 µm, with an acquisition window large enough to capture the entire oocyte. Acquisition takes a few seconds and physics-based algorithms perform 3D reconstruction. Main results and the role of chance OWLO's imaging prototype and physics-based algorithms achieve high-resolution images in depth (500µm). These are the first label-free 3D images of oocytes done in an ultra-fast acquisition. The technology is successfully used to image through complex oocyte cumulus and identify the polar body. This is a straightforward marker of oocyte maturity that is in general not available in clinics. The imaging system's resolution also enables visualization of intracellular structures without requiring fluorescent labelling. In particular, the zona pellucida (ZP), trans-zonal projections (TZP) and lipid droplets were identified as the brightest elements of the image. To validate this, some samples were imaged after centrifugation, which concentrates lipids at the centripetal pole. Imaging both centrifuged and not centrifuged oocytes shows that the lipid profile can be identified on OWLO's images. Moreover the thickness of the ZP can be measured accurately, and TZPs can be identified and quantified. Vacuoles also appear as hypoechoic and can be easily identified. Further work will aim to gather evidence of mitochondrial abnormalities and presence of refractile bodies. Non-invasively visualizing and quantifying these intracellular structures opens new opportunities to assess oocyte quality in IVF treatments. Limitations, reasons for caution This is a preliminary study that provides promising results. Fluorescence imaging will be used to validate the identification of lipids and other subcellular structures. This study used bovine models only and will be extended to human oocytes in the future to validate the potential of this method. Wider implications of the findings OWLO's imaging technology is the first to allow non-invasive 3D imaging of oocytes without decoronization in a few seconds. This technological breakthrough can remove bottlenecks in oocyte quality assessment. It has also been successfully used to image early-stage embryos and blastocysts and could serve to assess their quality before embryo transfer. Trial registration number No

Dates et versions

hal-05297686 , version 1 (04-10-2025)

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Citer

V Barolle, E Giraudat, F Bureau, N Guigui, Vincent Brochard, et al.. P-226 OWLO's new three-dimensional label-free imaging technology, applied to cumulus-oocyte complexes, reveals subcellular structures for quality assessment. European Society of Human Reproduction and Embryology, Jun 2025, Paris, France. ⟨10.1093/humrep/deaf097.534⟩. ⟨hal-05297686⟩
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