Study subjects Pregnant women who received regular perinatal health care in the outpatient department of the Third Affiliated Hospital of Zhengzhou University and gave birth in the hospital between July 2019 and January 2020 were randomly selected. They included 30 pregnant women with SCH (SCH group) in late pregnancy and 30 healthy pregnant women (control group) in late pregnancy who met the inclusion criteria.
Inclusion criteria (1) The thyroid function levels of the SCH group met the diagnostic criteria in the 2017 guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and the postpartum[34] and the reference-range criteria developed by the Department of Clinical Laboratory of the Third Affiliated Hospital of Zhengzhou University (11.5<FT4<22.7 pmol/L, TSH>4.0 mIU/L, and thyroid peroxidase antibody (TPOAb) <35 U/mL). The control group included pregnant women who had normal thyroid function and did not have other obstetric complications. (2) All pregnant women were in the late pregnancy period.
Exclusion criteria The exclusion criteria were as follows: (1) Patients who had incomplete data. (2) Patients who had central hypothyroidism. (3) Patients who had other obstetric complications. (4) Patients who were taking antithyroid drugs or thyroid hormone replacement. (5) Patients who had severe systemic diseases or were recovering from thyroiditis. (6) Patients who took drugs that affected thyroid functions in the last 3 months, including metoclopramide, iodine supplements, domperidone, amiodarone, and lithium carbonate tablets. (7) Patients who had a medical history of liver diseases, malignant tumors, diabetes mellitus, and hereditary hyperlipidemia. (8) Patients who took lipid-lowering drugs in the last 3 months, such as fibrates, statins, or Xuezhikang capsules. (9) Patients who had had intestinal surgery. (10) Patients who were aged <18 years.
Note: Central hypothyroidism refers to hypothyroidism caused by reduced production and secretion of thyrotropin-releasing hormone or TSH resulting from a disorder of the hypothalamus or pituitary gland.
Statement: (i) All enrolled subjects volunteered and signed informed consent forms. This study was approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University. (ii) All methods were carried out in accordance with relevant guidelines and regulations.
Specimen collection All pregnant women fasted for 8-12 h before blood collection. A total of 5 mL blood from the median cubital vein was collected in ethylenediaminetetraacetic acid–coated tubes. After collection, the collection tube was gently inverted four times, wrapped in aluminum foil, and temporarily stored in a 4°C refrigerator. Blood samples were centrifuged within 2 h of sample collection in a low-temperature centrifuge at 4°C and 1600×g for 10 min. The supernatant (plasma) after centrifugation was aliquoted into several centrifuge tubes and stored in a -80°C freezer. Later, samples were sent to the BGI Group for lipidomic examination.
Data collection The basic data of the two groups of pregnant women, including body mass index (BMI) before pregnancy, BMI at enrollment,, gravidity, parity, and delivery mode, neonatal sex, birth weight (BW), Apgar scores at 1 and 5 minutes, were collected. In addition, clinical data were collected during sample collection, including serum TSH, fasting blood glucose (FBG), CRP, IL-6, TNF-α.
Major instruments and reagents Instruments used in this study included an ultrahigh-performance liquid chromatograph (UPLC) (Waters 2D UPLC, Waters, USA), a high-resolution mass spectrometer (Q Exactive, Thermo Fisher Scientific, USA), chromatographic column: ACQUITY UPLC CSH C18 (1.7 μm,2.1*100 mm,Waters, USA), a low-temperature ultracentrifuge (Centrifuge 5430,Eppendorf), a vortex (QL-901, Qilin Beier instrument, China), a water purifier (Milli-Q Integral,Millipore Corporation,USA), and a refrigerated vacuum concentrator (Maxi Vacbeta,GENE COMPANY). Reagents used in this study included LC-MS-grade (Thermo Fisher Scientific,USA) methanol (A454-4), acetonitrile (A996-4), isopropanol (A461-4), ammonium formate (17843-250G,Honeywell Fluka,USA), and formic acid (50144- 50ml,DIMKA,USA)and water purified by a water purifier.
Extraction of lipid molecules After samples were slowly thawed at 4°C, 100 μL of each sample was pipetted into a 96-well plate. Next, 300 μl isopropanol (precooled to -20°C) and 10 μl SPLASH Lipidomix internal standard were added and vortexed to homogeneity for 1 min. Samples were kept still at -20 °C overnight and then centrifuged at 4°C and 4000 rpm for 20 min, The supernatant was collected and placed in a sample tube. The supernatant of each sample was collected at 10 μL and mixed together to form the quality control (QC) sample.
UPLC-MS analysis UPLC materials and methods: The CSH C18 column (1.7 μm 2.1*100 mm, Waters, USA) was used for chromatography. The mobile phases of the positive ion mode were a water solution (solution A) containing 10 mM ammonium formate, 0.1% formic acid, and 60% acetonitrile and a solution (solution B) containing 10 mM ammonium formate, 0.1% formic acid, 90% isopropanol, and 10% acetonitrile. The mobile phases of the negative ion mode were a water solution (solution A) containing 10 mM ammonium formate and 60% acetonitrile and a solution (solution B) containing 10 mM ammonium formate, 90% isopropanol, and 10% acetonitrile. The following gradient was used for elution: 0-2 min, 40-43% solution B; 2~2.1 min,43%~50% solution B;2.1~7min,50%~54% solution B;7~7.1 min,54%~70% solution B;7.1~13 min, 70% ~99% solution B;13~13.1 min,99%~40% solution B,13.1~ 15min,40% solution B。 The flow rate was 0.35 mL/min, the column temperature was 55 °C, and the sample load was 5 μL.
MS materials and methods: The Q Exactive mass spectrometer (Thermo Fisher Scientific, USA) was used for primary and secondary MS data acquisition. The range of the mass-to-charge ratio in MS scanning was 200-2000, the primary resolution was 70,000, the automatic gain control was 3e6, and the maximum injection time was 100 ms. According to the precursor ion intensity, Top3 was selected for fragmentation. The secondary MS data were collected. The secondary resolution was 17,500, the automatic gain control was 1e5, the maximum injection time was 50 ms, and the stepped normalized collisional energy was set as 15, 30, and 45 eV. The settings of electrospray ionization were a sheath gas flow rate of 40, an aux gas flow rate of 10, a spray voltage (|kV|) of 3.80 in the positive ion mode and 3.20 in the negative ion mode, a capillary temperature of 320°C, and an aux gas heater temperature of 350°C.
To provide more reliable experimental results during instrument detection, samples were randomly sorted to minimize systemic errors. One QC sample was inserted for every 10 samples.
Statistical methods Raw data generated by the LC-MS/MS detection were input into LipidSearch v.4.1 (Thermo Fisher Scientific, USA) for MS data analysis. The data matrix contained the lipid molecule identification results and quantitative results. Identification and peak extraction of individual samples were first performed using LipidSearch v.4.1, and then peak alignment was performed on all samples. The raw data obtained from LipidSearch were input into metaX for data pretreatment and subsequent analyses. SPSS 25.0 software was used to process basic clinical data of pregnant women in two groups. Normally distributed quantitative data are described as mean ± standard deviation (χ±s), and differences between groups were compared using the independent-samples t-test. Nonnormally distributed quantitative data are expressed as median (M) and quartile (Q), and differences between groups were compared using the Mann-Whitney U-test. Categorical variables are described as frequency, and the differences between groups were compared by the chi-squared test. Correlations were calculated by Spearman analysis.