Background: Embryo-secreted EVs have been identified in spent culture media (SCM) and shown to affect endometrial cells. Studies have investigated EV concentration, size and cargo; however, predominantly in bovine models and using embryo morphology rather than clinical outcomes. No study has assessed associations between embryo-secreted EVs and live birth. This study investigated the relationship between EV size, concentration and live birth.

Methods: SCM from ICSI cycles following days 1-5 of embryo culture were collected and stored at -20°C. Samples were processed by differential centrifugation (2000 × g and 20,000 × g). EV concentration and size were measured using nanoparticle tracking analysis for three isolates per sample – 2000 × g pellet, 20,000 × g pellet and 20,000 × g supernatant. SCM samples from live birth (n=21), negative pregnancy (n=22) and media-only control (n=25) groups were analysed.

Results: EV concentration was not significantly associated with live birth - ranging from 2.0 × 10⁹ to 3.7 × 10⁹ particles/mL across isolates. EV size was also not significantly associated with live birth. However, mean EV size in live birth SCM samples showed a trend toward larger EVs in 20,000 × g supernatant (122.1 vs 121.1 nm, p=0.80), 20,000 × g pellet (148.1 vs 144.6 nm, p=0.59) and 2000 × g pellet (167.9 vs 159.0 nm, p=0.19). EV concentration and size in media-only controls were not significantly different to SCM samples, indicating substantial background particles.

Conclusion: No significant associations were identified between EV concentration or size and live birth. However, a trend toward larger EVs was observed in SCM from embryos resulting in live birth. Given the small sample size, these findings warrant evaluation in a larger cohort. Similarity between SCM and media-only controls highlights the challenge of distinguishing embryo-secreted EVs from background particles, potentially limiting EV-based biomarker detection in SCM using this methodology.