Microbial signatures in follicular fluid and their association with fertilization success.

Background: Emerging evidence suggests that the upper female reproductive tract is not sterile and that microbial signals within follicular fluid (FF) may influence oocyte competence. However, previous studies have largely relied on pooled FF samples or dominant follicles, limiting insight into follicle-specific associations with fertilization outcomes. Methods: In this exploratory paired study, follicular fluid samples were collected from 24 women undergoing IVF/ICSI treatment. For each patient, two FF samples were analyzed individually: one associated with a fertilized oocyte, and one associated with an oocyte that failed fertilization. Bacterial DNA and total bacterial load (TBL) were assessed using quantitative real-time PCR targeting predefined microbial taxa. Results: Bacterial DNA above the predefined detection threshold was identified in 39.6% of all FF samples. Notably, within this exploratory cohort, FF samples associated with fertilization failure were more frequently TBL-positive compared with FF samples linked to successful fertilization (70.8% vs. 8.3%). Follicles from the same patient often differed in bacterial DNA presence, indicating substantial intra-individual variability. Several bacterial taxa, including Conclusion: In this paired follicle-level analysis, the absence of detectable bacterial DNA in follicular fluid was associated with fertilization outcome. These findings highlight follicle-level heterogeneity in microbial DNA detection and underscore the importance of follicle-specific analyses in reproductive microbiome research. Larger prospective studies are required to validate these observations and to clarify the biological mechanisms underlying follicular microbial signals.