Oral Presentation ESA-SRB-NZSE-CaSR 2026 in conjunction with ENSA

Non-invasive light-sheet metabolic imaging detects persistent metabolic alterations in mouse blastocysts following zygotic oxidative stress (143640)

Yaoxi Xiong 1 , Sandra Fok 2 , Ewa M Goldys 3 , Robert B Gilchrist 1 , Fabrizzio Horta 1
  1. Fertility & Research Centre, Discipline of Women’s Health, School of Clinical Medicine and the Royal Hospital for Women, University of New South Wales, Sydney, New South Wales, Australia
  2. Katharina Gaus Light Microscopy Facility, Mark Wainwright Analytical Centre, Division of Research and Enterprise, University of New South Wales, Sydney, New South Wales, Australia
  3. Graduate School of Biomedical Engineering, University of New South Wales, Sydney, New South Wales, Australia

Background

Preimplantation embryo development is highly sensitive to oxidative stress, yet morphology alone may not detect metabolic abnormalities in visually normal embryos. This study used non-invasive light-sheet fluorescence microscopy (LSFM) to determine whether transient zygotic oxidative stress impairs developmental competence and induces persistent metabolic alterations in morphologically normal blastocysts.

Methods

Super-ovulated Swiss mice (n=100) produced 558 presumptive zygotes for embryo culture experiments. Mouse cumulus-oocyte-complexes were fertilized in vitro with F1 spermatozoa. Zygotes were randomly assigned to control or 10, 30, or 50 μM H₂O₂ treatment groups for 30 min at 6 h post-insemination, cultured for 96 h in traditional or time-lapse (Geri®, Genea Biomedx) incubation systems at 37°C, 5% CO₂. Fertilisation rates, blastocyst formation rates, and embryo quality were compared between groups. Embryo quality was assessed by LSFM (InVi SPIM Lattice Pro, Luxendo) imaging of endogenous NAD(P)H and FAD and by immunocytochemistry for ROS (DCFDA), mitochondrial membrane potential (JC-1), lineage allocation (SOX2, GATA3, SOX17), and DNA damage (γH2AX).

Results

Transient H₂O₂ exposure significantly reduced both 2-cell and blastocyst formation rates at 30 μM (p < 0.05) and 50 μM (p < 0.0001), whereas 10 μM had no effect. Among morphologically comparable blastocysts, intracellular ROS increased at 30 μM (p < 0.01) and 50 μM (p < 0.0001), while JC-1 ratios decreased across all treatment groups (p < 0.0001). The inner cell mass (ICM)/trophectoderm (TE) ratio and ICM/total cell index showed decreasing trends, but differences were not statistically significant. NAD(P)H intensity demonstrated a downward trend, while 50 μM H₂O₂ significantly reduced FAD intensity (p < 0.01) and the optical redox ratio (p < 0.05).

Conclusion

Zygotic oxidative stress induces persistent metabolic and mitochondrial alterations despite normal blastocyst morphology. Non-invasive LSFM detects sub-lethal metabolic deviations in morphologically similar blastocysts, providing an objective optical approach to identify embryos with altered metabolic status.