Abstract Study question Does vaginal microbiome have an impact on vaginal absorption of exogenous progesterone (P intake) in artificial cycles? Summary answer Results suggest that the presence of lactobacillus in the vagina may positively impact the uptake of P. What is known already Low serum P levels on ET day decrease significantly live birth rates. About 20% of patients receiving micronized vaginal P show inadequate levels. It is of interest to find out which intrinsic factors might influence on the capacity of absorption (P intake) of vaginal P, and if they could be treated to prevent this situation. Vaginal microbiome hast been suggested as a possible factor, although this has not been addressed yet. The aim of this study was to explore if there is a correlation between vaginal microbiome and pH, and P intake. Study design, size, duration A prospective single-centre cohort pilot study including 92 ART patients was conducted from February 2022 to January 2023 in IVI-RMA Valencia. Embryo transfer was conducted in the context of a hormonal replacement therapy cycle with use of vaginal P (400mg/12h) for luteal phase support. “Progesterone intake” capacity was evaluated according to the vaginal microbiome status (lactobacillus dominant (LDM, >90%) or non-lactobacillus dominant (NLDM) and pH levels determinations (considered normal when moderately acidic ( < =4.8). Participants/materials, setting, methods Samples were taken twice: A) proliferative phase (day of initiation of exogenous P) and B) mid-luteal phase (day of ET, P + 5). Serum E2 and P levels as well as vaginal microbiome and pH were analysed. A validated qPCR methodology was used to accurately assess the presence and relative abundance of the four major lactobacillus species associated with eubiosis (crispatus, gasseri, iners, and jensenii) and the most common bacterial species implicated in dysbiosis. Main results and the role of chance Distribution of LDM and NLDM profiles were comparable between Samples A and B. No significant difference between any of the potential confounding factors (age, BMI,baseline P) between the lactobacillus CST groups (CST-I,-II,-III,-V & lactobacillus) and the dysbiotic CST group (CST-IV) in Samples A and B were observed. Serum P levels on the ET day were significantly higher in LDM profiles both in Sample A (15.2 vs. 12.9ng/mL in NLDM;p=0.009) and B (15.0 vs. 12.8ng/mL;p=0.014). A positive significant correlation was found between serum P levels and Lactobacillus abundance in Samples A (r = 0.28; p = 0.008) and B (r = 0.30; p = 0.004). The relative abundance of Gardnerella vaginalis in Sample A was negatively related to serum P levels on the ET day (r=-0.24;p=0.02). Vaginal pH was significantly lower in LDM profiles both in Sample A (4.9 vs. 5.6 in NLDM; p = 0.002) and B (4.7 vs. 5.1;p<0.001). pH levels were negatively correlated with Lactobacillus abundance in Samples A (r=-0.34;p=0.0012) and B (r=-0.41;p<0.001). Additionally, serum P levels were negatively correlated with pH levels in Sample A (r=-0.14;p=0.191) and B (r=-0.27; p = 0.010). Indeed, when patients are grouped based on a cut-off of P = 8.8ng/mL, patients with a pH ≤ 4.8 have significantly higher P levels than patients with a pH > 4.8. Limitations, reasons for caution This is a pilot study, thus further trials with larger sample sizes should be performed in order to confirm these results. Additionally, vaginal microbiome alone does not explain the total variability in serum P levels measured in artificial cycles when using MVP. Wider implications of the findings We might have found one of the causes of the high heterogeneity on vaginal absorption of MVP in artificial cycles. This finding may help us with patient management when programming an ET in this type of cycles, improving personalised patient care and progressing on luteal phase support individualisation. Trial registration number 2110-VLC-095-EL