MSACL 2023 Abstract

INTRODUCTION
We recently developed Direct Swab analysis by desorption electrospray ionisation – mass spectrometry (DESI-MS). This method permits rapid and direct metabolic profiling of mucosal swab samples whereby spectra can be acquired in less than 2 minutes and without any sample preparation, making it suitable for point-of-care applications. The utility of this method was recently demonstrated in a study of almost 1000 vaginal swabs collected from women during pregnancy which showed that swab profiling by DESI-MS enables rapid and robust prediction of microbiota composition and inflammatory status. This has important clinical implications as the vaginal microbiome and host inflammatory response are modulators of preterm birth risk, which remains the primary cause of death in children under 5 years of age, worldwide. However, the strongest risk factor for preterm birth is a previous preterm delivery. Ultrasound measurement of cervical length in early pregnancy is used clinically as a predictive tool for preterm birth risk. Women with a cervical length ≤ 25mm are considered to be at increased risk of preterm birth and can be treated with a cervical cerclage, which is a procedure whereby a stitch is inserted into the cervix to provide mechanical and biochemical support. This procedure is effective in only subset of women and in some cases, can lead to disturbance of the vaginal microbiome. We hypothesized that metabolic profiling of the cervicovaginal mucosal interface permits assessment of preterm birth risk phenotypes associated with cervical shortening and may facilitate prediction of cervical cerclage effectiveness.

METHODS
Vaginal swabs (BBL CultureSwab MaxV Liquid Amies swabs, Becton, Dickinson and Company, Oxford, UK) were collected longitudinally throughout pregnancy from two study cohorts (VMET, n=160 pregnancies; 437 swabs; VMET II, n=201 pregnancies; 581 swabs). Upon collection, swabs were placed on either Amies transport media or a sterile microcentrifuge tube for transfer and stored at -80 °C until analysis. DESI-MS analysis was performed on an LTQ-Orbitrap Discovery mass spectrometer (Thermo Scientific, Bremen, Germany) with a custom rotating swab holder and source interface designed for direct swab analysis. The DESI-MS sprayer solvent used was a methanol/water (95:5, v/v, HPLC grade Sigma-Aldrich, St. Louis, MO) mixture, with a flow rate of 10 μL/min, a nebulising gas pressure of 7 bar, and a sprayer voltage of 4.3 kV. For each swab, approximately 30 spectra were acquired in positive and negative ionisation model with a m/z window from 50–1000. MS spectra were pre-processed (peak picking and grouping) in R with the maldiquant package. Multivariate partial least squares-discriminant analysis and random forest models with K-fold cross-validation were used to assess the ability of DESI-MS metabolic profiles to predict clinical outcomes.

RESULTS
Multivariate analysis performed on both VMET and VMET II samples indicated that DESI-MS cervicovaginal metabolic profiles are weakly predictive of previous pregnancy history (previous PTB n=204, vs previous cervical treatment n=109; AUC=0.65 negative mode, AUC=0.64 positive mode). Similarly, DESI-MS profiles provided a weak prediction of subsequent treatment for cervical shortening (n=85) compared to controls matched for gestational age and microbial composition (n=85) (positive ion mode AUC=0.65, negative ion mode AUC=0.67). DESI-MS profiling of samples collected following intervention indicated capacity to distinguish women who went on to subsequently shorten their cervix despite cerclage (n=14) from those who maintained cervical length (n=37) (AUC =0.74 negative mode, AUC=0.62 positive mode). Finally, the capacity of DESI-MS to predict vaginal microbial composition in women with a cervical cerclage in situ was found to remain strong at genera level (AUC=0.92 negative mode, AUC=0.93 positive mode) and for major species-level community state types (Lactobacillus crispatus dominated v Lactobacillus iners dominated, AUC= 0.81 negative mode, AUC=0.84 positive mode; Lactobacillus crispatus dominated v dysbiotic, AUC=0.98 negative mode, AUC=0.98 positive mode and Lactobacillus iners dominated v dybiotic, AUC=0.93 negative mode, AUC=0.94 positive mode).

DISCUSSION
Our data indicate that early pregnancy cervicovaginal fluid metabolic profiles obtained using Direct Swab Analysis by DESI-MS are not strongly reflective of background preterm birth history risk phenotypes. However, they offer potential for stratifying women who will subsequently experience cervical shortening and require clinical intervention. This indicates that early biochemical changes associated with pathophysiological shortening of the cervix are detectable in the cervicovaginal metabolome prior to clinically overt shortening detectable by ultrasound. Early detection of women at risk of cervical shortening may improve efficacy of current strategies for its treatment and prevention. Moreover, our results suggest that swab profiling by DESI-MS may also permit monitoring of patient response and prognosis following cervical cerclage. Insertion of cervical cerclage does not hamper the ability of the method to robustly predict vaginal microbiome composition, which is an important mediator of maternal and neonatal health outcomes during pregnancy. Collectively, these findings highlight Direct Swab analysis by DESI-MS as an innovative approach for the stratification of preterm birth risk phenotypes associated with cervical shortening and as a potential tool for the monitoring and prediction of response to commonly used preventative interventions.