Procedure-specific environmental microbiological monitoring in IVF laboratories: the SMILE multicenter study

PURPOSE: Environmental microbiological monitoring is mandatory in IVF laboratories; however, procedure-specific risk assessment across the entire medically assisted reproduction workflow remains limited. This multicenter study, named "SMILE" (Study of Environmental Microbiological Monitoring in IVF Laboratories), aimed to assess settle plate positivity as a surrogate marker of environmental microbial contamination risk, in compliance with the guidelines of the European Directorate for the Quality of Medicines & HealthCare (EDQM). Passive air monitoring using settle plates was performed during individual IVF laboratory procedures under different environmental cleanliness conditions. METHODS: Nine IVF laboratories in Northern Italy prospectively collected environmental microbiological monitoring data between 2023 and 2025. Passive air monitoring using settle plates was performed during seven routine IVF procedures. Monitoring was conducted either under GMP Grade A laminar airflow or at the micromanipulation workstation in the validated GMP Grade D background environment (ICSI and embryo biopsy). A total of 2146 sentinel settle plates were exposed and incubated according to EDQM recommendations. Contamination probability was defined as the proportion of plates with ≥ 1 colony-forming unit (CFU), with exact 95% confidence intervals. Step-specific and cumulative contamination risks were estimated assuming independence, and associations with environmental cleanliness class and exposure time were analyzed. RESULTS: Procedures performed in GMP Grade D environments showed a significantly higher percentage of settle-plate positivity than those performed in GMP Grade A (26.0% [95% CI 21.5-31.0%] vs 2.2% [95% CI 1.6-3.0%], p < 0.001). Intracytoplasmic sperm injection (ICSI) and embryo biopsy were associated with the highest contamination rates. The cumulative probability of detecting at least one contaminated settle plate was 11.2% (95% CI 7.8-14.6%) for procedures performed exclusively in GMP Grade A environments, increasing to 35.1% (95% CI 30.0-40.1%) including ICSI, and to 47.4% (95% CI 38.2-56.5%) when all procedures were considered. Exposure time was associated with an increased risk of settle plate positivity in the complete procedural set, which included Grade D procedures. CONCLUSIONS: Environmental microbiological burden in IVF laboratories is procedure-dependent and strongly associated with environmental cleanliness class. These findings support a procedure-specific, risk-based approach to environmental monitoring in MAR laboratories using settle plate-based air monitoring.