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

Non-invasive metabolic imaging reveals developmental metabolic signatures and aging effects in mouse embryos (144087)

Fabrizzio Horta 1 2 , Ananya Vuyyuru 1 , So Yan Tung 1 , Yaoxi Xiong 2 , Sally Catt 2 , Bettina Mihalas 1 , Sandra Fok 3 , Alex Macmillan 3 , Lindsay Wu 4 , Ewa Goldys 5 , Robert Gilchrist 1
  1. Fertility & Research Centre, UNSW Sydney, Randwick, NSW, Australia
  2. Monash University, Melbourne, VIC, Australia
  3. Katharina Gaus Light Microscopy Facility, Mark Wainwright Analytical Centre, Division of Research and Enterprise, UNSW , Sydney, NSW, Australia
  4. Ageing Research, UNSW, Sydney, NSW, Australia
  5. ARC Centre of Excellence for Nanoscale Biophotonics, UNSW, Sydney, NSW, Australia

Background
Embryo selection remains predominantly morphology based, although embryo metabolism is closely associated with developmental competence and changes with reproductive ageing. Morphologically similar embryos may therefore exhibit distinct metabolic states that are not detectable through conventional assessment. We characterised developmental metabolic signatures, tested whether non-invasive imaging detects age-related differences not evident from blastocyst morphology, and validated responsiveness to NAD(P)H modulation.

Methods
Mouse embryos from Swiss and C57BL/6 females were generated by in vitro fertilization. For ageing analyses, young females aged 5–8 weeks (n=14; 304 oocytes) and older females aged 45–52 weeks (n=35; 351 oocytes) were compared; 121 young and 30 older blastocysts underwent metabolic imaging. For metabolic modulation experiments, zygotes were cultured under four different conditions: (1) control medium (n=74); (2) FK866 (metabolic inhibition; n=72); (3) NMN (nicotinamide mononucleotide; n=74) and (4) NMN + FK866 (n=73) and monitored for 120 hours by time-lapse imaging. NAD(P)H, flavin adenine dinucleotide autofluorescence and optical redox ratio were assessed using confocal, fluorescence lifetime and light sheet microscopy. Development, morphokinetics, morphology and imaging safety were analysed using analysis of variance and time-to-event methods.

Results
Metabolic imaging via light sheet microscopy generated three-dimensional metabolic images from the two-cell stage to blastocyst using less than 50 J cm−2, without detectable effects on development (p>0.05). Older females had reduced blastocyst formation (p<0.05), despite comparable blastocyst morphology (p>0.05). Their blastocysts showed lower inner cell mass NAD(P)H autofluorescence and higher optical redox ratios than young blastocysts (937.0±7.3 versus 901.5±6.2 arbitrary units; p<0.05), with metabolic heterogeneity among cells. FK866 reduced blastocyst formation to 68.5% versus 85.3% in controls and increased arrest (p<0.005). NMN maintained blastocyst formation, advanced early development by approximately six hours (p<0.05) and partially mitigated FK866-associated impairment.

Conclusion
Non-invasive metabolic imaging resolves developmental signatures and age-related alterations not captured by morphology alone, supporting metabolic phenotyping as an adjunct for embryo assessment.