Study Design and Subject Recruitment

This cohort study was conducted between 2019 and 2021 at the Affiliated Wuxi Maternity and Child Health Care Hospital of Nanjing Medical University. Participants included pregnant women with a single fetus and head presentation who received routine prenatal care and labored vaginally. Preeclampsia, gestational diabetes, malnutrition, multiple gestation, breech presentation, preterm labor experience, short cervical length, preterm labor history, oral or vaginal medication administration within 48 h prior to admission, sexual activity within 48 h prior to admission, and vaginal douching within 48 h prior to obtaining a swab were exclusion criteria for this study.

Ethical Approval

This study was approved by the Ethics Committee of The Affiliated Wuxi Maternity and Child Health Care Hospital of Nanjing Medical University (2019-02-0402-01) and adhered to the Declaration of China and applicable local regulatory requirements, and the Declaration of Helsinki Principles. It was registered in the Chinese Clinical Trial Registry system (ChiCTR2000034721, registration date: 16 July 2020, https://www.chictr.org.cn/showproj.aspx?proj=56635, accessed on 18 July 2020) and all participants gave written informed consent with their privacy being respected and protected.

Sample Collection and Processing

All subjects received routine examinations at the Affiliated Wuxi Maternity and Child Health Care Hospital of Nanjing Medical University from the first to the fortieth week of pregnancy. Between weeks 31 and 36, samples were taken from the posterior fornixes, immediately frozen in liquid nitrogen, and stored at -80 °C within five minutes. The calculated prevalence of PROM was 19.05%. A two-sided binomial test was conducted using PASS 11.0 software with a total sample size of 220 (including 42 PROM cases) to determine the minimum sample size required to reach a statistically significant conclusion (α = 0.05, 1-β = 0.8, sensitivity = 72%, specificity = 75%). According to the sensitivity and specificity test results, the actual significance levels achieved were 0.0436 and 0.0427, respectively (the target significance level was 0.05). Then, targeted metabolomics analysis was performed on 50 PROM cases and 50 matched healthy control (HC) cases.

Targeted Metabolomics

Twenty-three amino acids were chosen for targeted metabolomics. These included Glycine (Gly), L-Alanine (Ala), L-Valine (Val), L-Leucine (Leu), L-Isoleucine (Ile), L-Phenylalanine (Phe), L-Tryptophan (Trp), L-Tyrosine (Try), L-Aspartic(Asp), L-Histidine(His), L-Glutamic (Glu), L-Lysine (Lys), L-Methionine (Met), L-Arginine (Arg), L-Serine (Ser), L-Threonine (Thr), Creatine, L-Proline (Pro), L-Asparagine, (Asn), L-Glutamine, (Gln), 4-Aminobutyric acid (GABA), L-Ornithine hydrochloride (Orn), and Taurine. In brief, the metabolites were extracted from each vaginal cotton swab (adjusted to contain exact 50 mg vaginal secretion proteins), precipitated and mixed with 5 mg/ml amino acid standard for LC-MS analysis. The final results of quantitative analysis for each component were expressed in “μg/mg secretion proteins” (or “μg/mg”, in short).

Cell Culture

Human amniotic epithelial cells (WISH) and mouse monocyte macrophage (RAW 264.7) were purchased at the National Collection of Authenticated Cell Cultures, China. The high-glucose DMEM culture medium was prepared as following: 88% high-glucose DMEM (Gibco, 10313021), 10% FBS (Gibco, 10099141 C), 1% GlutaMax (Thermo, 350050061), and 1% penicillin-streptomycin (Sigma, 15140-122). Cell culture was kept in incubator with 5% CO2 at 37 °C. The RAW 264.7 cell morphology was monitored under a microscope. When the growth state was optimal, the cells were predominantly round and devoid of pseudopods. If more than 10% of the cells showed pseudopods, the M0 phase cells were activated and were, therefore, unsuitable for this portion of the experiment and discarded.

Transwell Cell Co-Culture and Cytokine Panel Assay

Transwell cell co-culture system (24-well inserts, Corning, 3379) was used to understand the cross-talk between amniotic epithelial cells (WISH) and monocytes/macrophages (freshly isolated from human peripheral blood). About 1 × 105/ml monocytes were seeded in the top insert, while 2 × 105 /ml WISH cells were seeded in the bottom chamber. WISH cell culture medium was set in 4 treatment conditions: no treatment, 10 mM Glutamine treated, 1.5 μg/mL LPS treated, and 1.5 μg/mL LPS plus 10 mM Glutamine treated. The co-culture system was then kept in incubator with 5% CO2 at 37 °C for 12 h. After that, the top insert was carefully washed and the membrane was stained with crystal violet to visualize cell migration. The culture medium in the bottom chamber was collected for cytokine panel assay following the manufactory’s instructions (Bio-Rad, 500KCAF0Y). In brief, WISH cell culture supernatant samples were centrifuged at 10,000 rpm for 10 min, and 50 μL of the supernatants were taken for analysis. Incubation of the sample was followed by antibody incubation and detection, color development, and reading on a calibrated Bio-Plex instrument. The detection chip used was 27-plex Bio-Plex Pro Human Cytokine Grp I Panel. The fluorescence values obtained from the standards were used to generate the standard curve and equation under a multi-parameter model. The sample concentration was then calculated using the standard curve and equation.

Western Blot

1 × 106/mL RAW 264.7 cells were seeded in six-well plates. After growing into monolayers overnight, cells were treated with 2.5 mL of fresh serum-free DMEM medium containing various concentrations of LPS and Glutamine, and whole-cell lysates were collected at various time points. The protein concentration was determined using the BCA kit (Solarbio, China), and the lysates with equal amount of total proteins (30 μg) were loaded for western blotting. The following antibodies were used: iNOS antibody (Abcam, ab283668), COX-2 antibody (Abcam, ab179800), β-actin antibody (Abcam, ab8227), IκBα antibody (CST, 4814 S), Phospho-IκBα antibody (CST, 9246 S), p65 antibody (Abcam, ab32536), PCNA antibody (Abcam, ab92552).

ROS Fluorescent Probe

RAW 264.7 cells were seeded in 12-well plates at a density of 1 × 105 /mL for ROS fluorescent probe detection. The cells were cultured overnight to form monolayer before 6-hour treatment as described above. After that, the cells were washed twice with PBS before staining. The ROS fluorescent probe (DCFA-CH, Sigma, USA) was diluted to 20 μmol/L, and 400 μL probe was added dropwise to each well with 30 min incubation at 37 ℃ protected from light. The cells were then washed thrice with PBS to remove the fluorescent probe that did not enter the cells. ROS fluorescence intensity of each well was measured using an inverted fluorescence microscope.

Total RNA Reverse Transcription and Quantitative PCR

RAW 264.7 cells were seeded as described above and treated with LPS and various concentrations of Glutamine for 6 h. Total RNA preparation and reverse-transcription were performed following the manufactory’s standard protocol (Vazyme, China). Quantitative PCR was performed on ABI 7500 (Applied Biosystems) using the following primer sets:

TNF-α: 5′-TTCTCATTCCTGCTTGTGG-3′(F), 5′-ACTTGGTGGTTTGCTACG-3′(R);

IL-6: 5′-GAGGATACCACTCCCAACAGACC-3′(F), 5′-AAGTGCATCATCGTTGTTCATACA-3′(R);

IL-1β: 5′-AGAGCATCCAGCTTCAAAT-3′(F), 5′-CATCTCGGAGCCTGTAGTG-3′(R);

GAPDH: 5′-CCTTCCGTGTTCCTACC-3′(F), 5 ′CAACCTGGTCCTCAGTGTA-3′(R).

Immunofluorescence (IF)

RAW 264.7 cells were seeded in 24-well plate and treated with LPS and various concentrations of Glutamine for 1 h. The culture medium was aspirated and cells washed with PBS once. To fix the cells, 1 ml of 4% paraformaldehyde was added to each well and incubated for 15 min on ice. After fixation, the cells were washed with washing solution thrice for 3–5 min each time. 1 ml of immunostaining blocking solution and was added and incubated for 1 h at room temperature. NF-κB p65 mouse monoclonal antibody was then added and incubated at 4 °C overnight. The cells were washed thrice for 10 min each time. The anti-mouse Cy3 was added and incubated for 1 h at room temperature, followed by thorough washes, 5-minute cell nuclear staining (DAPI) and further washes. Finally, the slides were sealed and observed under a fluorescence microscope. NF-κB staining was red fluorescence, and the DAPI staining of cell nuclei was blue fluorescence.

Quantification of Cellular α-KG Level and NADPH/ NADP + Ratio

Cells were seeded into 12-well plates at a density of 106 cells/mL in complete medium overnight and then changed to fresh Glutamine-free medium for 24-hour starving period. After that, cells were treated under various treatment conditions (control, 1.5 μg/mL LPS, or LPS plus 2 mM Glutamine) for 6 hours and then harvested in lysis buffer (∼ 5 × 106 cells in 1 mL lysis buffer) provided by α-KG assay kit (Aidisheng, China, ADS-W-S012) or NADPH/NADP + assay kit (Aidisheng, China, ADS-W-FM015-48). α-KG concentrations (μg/104cell) and NADPH/NADP + ratios were measured following manufactory’s standard protocol.

Cell Counting Kit-8 (CCK-8) Assay

100μL cells were seeded into 96-well plates at a density of 3 × 104 cells/mL. The medium was changed after 16 h, and LPS and Glutamine of different concentrations were added for stimulation. At 24-hour timepoint, 10μL CCK-8 reagent (Beyotime, China, C0038) was added to each well, and incubated at 37 °C for 30 min. Absorbance at 450 nm was determined using an ELISA microplate reader. The cell proliferation rate was calculated according to the following formula: Cell Proliferation Rate = [(As-Ab)/(Ac-Ab)] × 100%. (As: absorbance of experimental wells, Ac: absorbance of control wells, Ab: absorbance of blank wells).

Mito-Tracker Green Staining

Mito-Tracker Green (Beyotime, China) was dissolved in anhydrous dimethylsulfoxide (DMSO) to a final concentration of 1mM. Cells were incubated with 200nM Mito-Tracker Green working solution at 37 °C for 15–45 min and then washed with culture medium. Fluorescence microscopy was used to collect images.

Scanning Electron Microscopy

WISH cells were cultured overnight at 1 × 106/mL during the logarithmic division phase and then changed to Glutamine-free medium for 24 h. They were then treated under 4 conditions for another 24 h: (1) control, (2) 1.5 μg/mL LPS, (3) 1.5 μg/mL LPS + 2mM Glutamine, or (4) 1.5 μg/mL LPS + 10mM Glutamine. The cell pellets were fixed, dehydrated, embedded and sectioned (60–80 nm) for electron microscopy using standard protocol. The sections were double stained with uranium-lead (2% uranyl acetate saturated aqueous solution and lead citrate) for 15 min each and then dried at room temperature overnight. They were examined using a scanning electron microscope, and images were collected for analysis.

Statistical Analysis

For targeted metabolomics, the results of the quantitative analysis were compared to the median of all amino acid quantification for normalization (the range of normalized data was defined as 0–10), The Wilcoxon test was used to statistically compare the PROM group to the HC group for various between-group differences in the standardized values of the amino acid groups, with P < 0.05 indicating a statistically significant difference. ROC (Receiver Operating Characteristic) curve was made in a plot of detection specificity (X-axis) against detection sensitivity (Y-axis), and was used for MS quantitative software optimization. The area under the curve (AUC) for each ROC curve serves as a quantitative metric. The closer of AUC to 100% represented more accurate detection of that amino acid. All experiments were repeated independently three times.

Western blot grayscale values and fluorescence intensity values were quantified using NIH Image J software. qPCR results were exported to Microsoft Excel and the relative mRNA expressions of TNF-α, IL-6, and IL-1β in cells were calculated by 2-ΔΔCT method. One-way analysis of variance (ANOVA) method (GraphPad Prism 9.0) was applied for all statistical analysis of Cytokine panel, α-KG, NADPH/NADP+, CCK-8 assay and Mito-Tracker Green staining results. P < 0.05 indicates a statistically significant difference.