Quantitative determination of vaginal diamines as a biomarker for vaginal dysbiosis and IVF-ET outcome

BACKGROUND: Lactobacillus species generally predominate in a healthy vaginal microbiota, and alterations of this population correlate with increased risk of genital infections and poor reproductive outcomes. Composition and functions of the vaginal microbiota are influenced by a variety of factors, among which hormones represent a major player. As therapy with gonadotropins is a key step in controlled ovarian hyperstimulation (COH) before in vitro fertilization and embryo transfer (IVF-ET), in this work the effect of hormone therapy for COH on the vaginal microbiota and embryo implantation outcome was evaluated in a cohort of women undergoing IVF-ET. METHODS: A cohort of 108 infertile females were prospectively enrolled in the study. Hormone therapy consisted of recombinant follicle stimulating hormone and recombinant luteinizing hormone in a gonadotropin-releasing hormone antagonist regimen. Two vaginal swabs, before and after therapy, were collected from patients. Vaginal swabs were analyzed by microbiological standard culture methods for the presence of genital pathogens and lactobacilli. Changes in the vaginal microbiota were also assessed by using a vaginal diamine assay for the quantification of diamines, which are considered as a biomarker of the catabolic activity of bacteria involved in bacterial vaginosis. RESULTS: COH significantly impacted on both vaginal diamine levels and microbiota composition. Median concentration of diamines in vaginal fluids increased from 0 µM (IQR = 0-8.96) to 21.86 µM (IQR = 5.11-46.74) before (pre-HT) and after (post-HT) hormone therapy, respectively (p < 0.0001). The diamine value of 9.5 µM was found to be the threshold between eubiotic and dysbiotic microbiota. Microbiological analysis of pre-HT and post-HT samples showed a post-HT enrichment of the vaginal population in streptococci, enterococci, enterobacteria, staphylococci and fungi, which are prevalent in aerobic vaginitis. The frequency of vaginal samples positive for genital pathogens and with elevated concentrations of diamines (> 9.5 µM) was significantly increased in post-HT compared to pre-HT specimens (p < 0.0001). Levels of diamines were significantly lower in patients with successful IVF-ET compared to those with failed implantation (p = 0.015), and 56.25% of patients who achieved embryo implantation had diamine levels below 9.5 µM. Finally, multivariate analysis of factors associated with implantation outcomes showed that younger maternal age, absence of genital pathogens and low levels of vaginal diamine post-HT were all associated with positive reproductive outcomes. CONCLUSIONS: The data from this study demonstrate that, following COH, an increase in diamine levels and genital pathogens occurred, leading to vaginal dysbiosis. Diamine concentrations higher than 9.5 µM post-HT significantly correlated with embryo implantation failure and may be used as a predictive biomarker of vaginal dysbiosis and IVF-ET outcome.