Nmr suite software

NMR was conducted as previously described^{64 (link)}. Briefly, cells were treated with 200 µl of 6% HClO₄ and ground for 30 s with a hand homogenizer. The mixture was vortexed and frozen in liquid nitrogen. The samples were thawed and spun down at 10,000 × g for 15 min. The supernatant was collected, and the pellet was re-extracted with 100 µl of 6% HClO₄. Combined extracts were neutralized with 105 µl of 2 M KHCO₃. The mixture was spun down at 10,000 × g for 15 min and supernatant (300 µl) was collected. Then 200 µL of phosphate buffer (pH 7.4) and 50 µl of TSP-d₄ solution in D₂O (1 mM) were added, and the sample was transferred to a 5 mm NMR tube.
NMR spectra were acquired on a Bruker 600 MHz Avance III HD spectrometer (Bruker, Billerica, MA), using 1D NOESY pulse sequence with presaturation (noesygppr1d). The spectra were analyzed using Chenomx NMR suite software (Chenomx, Edmonton, Canada). Metabolite concentrations were exported as μM in the NMR sample and recalculated as nmoles in the cell extract.

CVF samples (described above) were immersed in liquid nitrogen, lyophilized at −58°C overnight, and resuspended in 550 μL D₂O. CVF metabolite profiles were generated using ¹H-NMR spectra acquisition, processing, and OPLS-DA, done as reported previously (54 (link)). For this study, spectral regions above 8.5 ppm and below 0.5 ppm were excluded for noise content. The water peak and trimethylsilylpropanoic acid reference signals were also excluded. A total of 29 metabolites were identified using the Chenomx NMR suite software (Chenomx Inc.). The signal from propylene glycol was removed from analyses, as it is a known contaminant from the gel used in transvaginal scanning.

All NMR experiments were performed at 25°C on a Bruker Avance III 850 MHz spectrometer (Bruker BioSpin, Germany) equipped with a TCI cryoprobe. One-dimensional (1D) ¹H spectra were recorded on aqueous cell extracts using the pulse sequence NOESYGPPR1D [RD-G1-90°-t1-90°-τ_m-G2-90°-ACQ] with water suppression during the relaxation delay and mixing time. t1 was a short delay (10 μs), and τ_m was the mixing time (10 m). Pulsed gradients G1 and G2 were used to improve water suppression quality. A total of 32 transients were collected into 64 K data points using a spectral width of 20 ppm with an acquisition time (ACQ) of 2.66 s, using an additional relaxation delay (4s). Based on the 1D ¹H spectra, metabolites were identified using a combination of Chenomx NMR Suite software (version 8.3, Chenomx Inc, Canada), the Human Metabolome Data Base (HMDB, http://www.hmdb.ca/), and relevant published references (Xu et al., 2017 (link); Liu et al., 2018 (link)). To confirm the resonance assignments of the metabolites, two-dimensional (2D) ¹H-¹³C heteronuclear single quantum coherence (HSQC) spectra and ¹H–¹H total correlation spectroscopy (TOCSY) spectra were recorded on selected NMR samples.

The final dataset was imported into the SIMCA-P (version 14.0, Umetrics, Sweden) program and was Pareto scaled prior to analysis. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), unsupervised and supervised methods, respectively, were applied to the NMR dataset. PCA is a projection method used to obtain a general overview on the state of samples, highlighting possible clusters, trends, or outliers.
OPLS-DA is a classification method that maximizes the covariance between the measured data of the X variable (peak intensities in NMR spectra) and the response of the Y variable (class assignment) within the groups. The generated R²Y and Q² values described the predictive ability and the fitting reliability, respectively. The model was validated by a permutations test (n = 200). The permutation test was used to check the validity and the degree of over-fit for the model. The importance of the discriminating variables has been indicated as VIPs (variables of importance on the projection). Using the VIP list, the most important variables were translated and identified by means of the Chenomx NMR Suite software (version 7, Chenomx Inc., Edmonton, Canada) and the HMDB database [17 (link)].

Metabolite identification was performed according to Chenomx NMR Suite 8.4 profiler (Chenomx Edmonton, Canada), the available databases such as the Human Metabolome Database (http://www.hmdb.ca), Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu), J-res 2D experiments, and the existing NMR-based metabolomics literature. Τhe 700 MHz library of the Chenomx NMR Suite software was used for the metabolites quantification, urinary creatinine was used as an internal reference, and values were expressed as µmoles of metabolite per mmol (μΜ/mM) of creatinine. For serum samples, an internal standard of known concentration (TSP) was used for the obtained quantitative values of metabolites concentration, which were presented in micromoles per liter (μM).