Methods
GDM
GDM, DIABETES AND PREGNANCY, COMPLICATED PREGNANCIES, GESTATIONAL DIABETES
Food frequency questionnaires were completed in a subset of 108 women with uncomplicated pregnancies enrolled in the Study of Probiotics IN Gestational Diabetes (SPRING), an RCT of probiotics to prevent GDM in pregnant women with overweight and obesity (ACTRN12611001208998) [Demographic data were collected for each woman including ethnicity, maternal age at delivery, maternal BMI at the 28 week-gestation antenatal visit, and intervention group in the study (probiotic or placebo). The study population by design has a higher BMI than the general obstetric population in Australia; it has a higher proportion of women with white ethnicity and who have completed tertiary education (as a proxy for socioeconomic status) [Mass spectrometry was used to analyse serum B-OHB levels. Analytical reagents DL-β-hydroxybutyric acid sodium salt (β-HBA, catalogue no. H6501), 3-nitrophenyl hydrazine (3NPH, catalogue no. N21804), Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis was performed using a Shimadzu Nexera ultraperformance LC system (Kyoto, Japan) attached to a SCIEX 5500 QTRAP mass spectrometer (Framingham, MA, USA). Chromatographic separations were carried out using a Kinetex 2.6 μm 150×2.1 mm C18 column (Phenomenex, Lane Cove, NSW, Australia), with a total flow of 0.3500 ml/min at 40°C. The pressure range was 0–12,000 psi for Pump A/B and 0–900 psi for Pump C. Buffers for mobile phase for gradient elution consisted of Buffer A, made up of (vol./vol.) 1% ACN, 0.1% formic acid and Buffer B composed of 90% ACN, 0.1% formic acid. The mass spectrometer, coupled with an electron spray ionisation source, was operated in the negative multiple reaction monitoring (MRM) mode and included source parameters of ion spray voltage (ISV) at −4200 V; temperature 450°C; GS1 (nebuliser gas) 35; curtain gas 20. Each standard and sample was injected at a volume of 10 μl and subsequently 50 μl. Optimised parameters through manual infusion for maximum sensitivity for each compound transition included the collision energy, declustering potential and collision cell exit potential. Individual fragment ions were detected using the Q3 mass analyser to identify the Q1/Q3 pairs for each analyte. The two most sensitive pairs of Q1/Q3 per analyte were used for the subsequent analyses. The retention time for propionic acid was ~5.94, butyric acid was ~7.71, acetic acid was ∼4.17, β-HBA was ~4.10 and 2EBA was ∼10.14 min. UPLC/MRM-MS data was processed using Multiquant 3.0 (SCIEX) software to optimise Gaussian smoothing to peaks and retrieve area measurements.Obstetric outcomes were obtained for each woman including GDM status defined according to the International Association of the Diabetes and Pregnancy Study Groups (IADPSG) guidelines [