Abstract:

Pregnancy is a complex biological process marked by significant physiological, immunological and hormonal changes. Recent advances in sequencing technologies have deepened our understanding of the role played by the vaginal microbiota in pregnancy. Studies suggest that imbalances in the vaginal microbiota can increase the risk of complications such as preterm birth. Incorporating these microbial signatures into a molecular diagnostic tool could significantly improve the prediction and the management of these complications. However, challenges remain: (i) identified microbial signatures are controversial and lack robustness; (ii) current molecular diagnostic tools do not provide sufficient levels of multiplexing or quantification to effectively characterize the vaginal microbiota and its imbalances.

The first phase of this work focused on culturomics approaches to robustly identify microbial signatures associated with premature rupture of membranes (PROM), preterm labor or preterm birth. We analyzed an observational cohort of 1,999 patients that included more than 1,000 controls and almost 500 cases of threatened preterm labor (PTL) and preterm PROM. Vaginal samples analysis revealed significant correlations between the presence of specific microbial species and these pregnancy complications at their onset. Multivariate analysis, considering demographic, clinical, and microbiological variables highlighted, regarding microbiological aspects, increased risks i) of PTL in case of lactobacilli depletion and presence of enterobacteria and Gardnerella vaginalis, ii) preterm PROM in the absence of lactobacilli and presence of enterobacteria.

The second part of this work focused on developing innovative detection solutions: high content PCRTM (hcPCR) and Molecular Petri DishTM (MPD). These complementary technologies respectively enhance the number of detectable and quantifiable targets per qPCR reaction, aiming to offer a diagnostic tool able to better characterize the vaginal microbiota and its imbalances. High content PCRTM has demonstrated its capability to enhance per reaction: (i) the level of multiplexing to 2 DNA targets per fluorescence channel; (ii) the overall level of multiplexing by simultaneously identifying 2 DNA targets out of 6 potential targets when 3 fluorescence channels are activated. Moreover, optimization efforts on Molecular Petri DishTM led to the development of a highly sensitive molecular diagnostic tool prototype, capable of simultaneously identifying and quantifying the DNA of Lactobacillus crispatus, E. coli and Streptococcus agalactiae - key players of early-onset neonatal bacterial infections - within 11 minutes.

These preliminary findings and developments pave the way for new non-invasive preventive strategies, including rapid monitoring of vaginal microbiota changes throughout pregnancy and evaluating the effectiveness of probiotic-based preventive approaches in high-risk populations.