2.1 Study subjects
In this study, 125 women (aged 22 ± 3 years) with a body mass index (20.83 ± 2.6 kg/m2) working in the same occupation who had lived in the local area for more than half a year were recruited from two regions of North and South China. The recruited women had no smoking or drinking habits and all had the same daily working hours, exercise intensity, and diet structure. The northern region of China is located at 43.88︒N latitude, which is in the northern temperate zone, and the southern region is located at 23.05︒N latitude, which belongs to the tropical zone. Women from the northern region (average annual temperature 0-10°C, summer temperature 15-25°C) were included in the C group (n = 80), and those from the southern region (average annual temperature 20-28°C, summer temperature 28-38°C) were included in the H group (n = 45). The inclusion criteria for subjects were ability to complete the survey correctly; patients with history of pregnancy, hysterectomy, and ovariectomy and without menarche were excluded.
This study was conducted according to the principle of the Declaration of Helsinki. All operational procedures were performed according to ethical principles of the Institute of Environmental and Operational Medicine and approved by the ethics review committee. The subjects were informed about the study’s objective and voluntary participation, and all signed the informed consent form.
2.2 Research questionnaire
A total of three questionnaires, including the Menstrual Status Questionnaire, the Influence Factors of Menstrual Questionnaire, and the Symptom Checklist-90 (SCL-90), were administered . The Menstrual Status Questionnaire inquires about menstrual characteristics and premenstrual symptoms (the coefficient of internal consistency Cronbach’s α = 0.879). The Influence Factors of Menstrual Questionnaire inquires about emotional and psychological status, family history of the disease, medication history, diet, sleep, and environmental conditions (the coefficient of internal consistency Cronbach’s α = 0.703). The SCL-90 consists of nine subscale dimensions, namely, Somatization (SOM), Obsessive Compulsive (OC), Interpersonal-Sensitivity (INT), Depression (DEP), Anxiety (ANX), Hostility (HOS), Phobic-Anxiety (PHOB), Paranoid Ideation (PAR), and Psychoticism (PSY). This study was conducted during summer. All necessary information was collected from the medical records of participants through direct interviews and questionnaires from the last 6 months.
2.3 Sample collection and preparation
On the day before saliva collection, all subjects were allowed to only drink water after 21:00 (UTC+8). Subjects' saliva was collected from 09:00 AM to 11:00 AM according to the saliva collection method specified by Asai  et al. Subjects were not allowed to drink water, smoke, brush their teeth, perform energetic exercise, or apply lipstick before saliva collection. Participants mouth were rinsed gently with clean water during saliva collection, and straws were used to assist in collecting unstimulated saliva. The collected saliva was stored at -80°C until further use. Furthermore, 5 mL of venous blood was collected on an empty stomach, and serum was collected and stored at -80°C.
2.4 Non-targeted saliva metabolomics
2.4.1 Sample pretreatment and analysis
The saliva samples were thawed at 4°C and centrifuged (7000 g, 5 min) to obtain the supernatant. Then, 100 µL of the saliva samples were mixed with 400 µL of precooled methanol acetonitrile solution (1:1, v/v) to remove protein, and then vortex-mixed for 20 min (4°C, 14000 g). The supernatant was freeze-dried and stored at -80°C. For mass spectrometry, 100 μL of acetonitrile aqueous solution (acetonitrile: water = 1:1, v/v) was added to re-dissolution, vortexed, and centrifuged (4°C, 14000 g, 15 min), and the supernatant was collected for analysis.
The procedure was performed in the Agilent 1290 Infinity LC Ultra High-Performance Liquid Chromatography System (UHPLC) Hydrophilic Interaction Liquid Chromatography (HILIC) column. The column temperature was 25°C, flow rate was 0.3 mL/min, and injection volume was 2 μL. The mobile phase comprised A (water + 25 mM ammonium acetate + 25 mM ammonia) and B (acetonitrile). The gradient elution procedure was as follows: 0-1 min, 95% B; 1–14 min, 95%-65% B; 14–16 min, 65%-40% B; 16–18 min, 40% B; 18–18.1 min, 40%-95% B; and 18.1–23 min, 95% B. The samples were placed in an autosampler at 4°C during the entire analysis.
Electrospray ionization (ESI) experiments were executed on the Triple TOF 5600 mass spectrometer (MS) (AB SCIEX) in positive and negative ion modes. The ESI source conditions after HILIC chromatographic separation were set as follows: ion source Gas 1, 60 psi; ion source Gas 2, 60 psi; curtain gas, 30 psi; source temperature, 600°C; ion spray voltage floating ±5500 V; TOF MS scan m/z range, 60–1000 Da; production scan m/z range, 25–1000 Da; TOF MS scan accumulation time, 0.20 s/spectra; and production scan accumulation time, 0.05 s/spectra. The secondary mass spectrum was acquired using information dependent acquisition (IDA) and adopting the high sensitivity mode. Declustering potential: ± 60 V; collision energy, 35±15 eV; IDA was set as Exclude isotopes within 4 Da, candidate ions to monitor per cycle: 6.
2.4.2 Data analysis
The raw data were converted into mzXML format using Proteo Wizard. The XCMS program (http://xcmsonline.scripps.edu) was adopted for peak alignment, retention time correction, and peak area extraction. The software SIMCA-P 14.1 (Umetrics, Umea, Sweden) was used for pattern recognition, and data were reprocessed by Pareto-scaling; then, multi-dimensional statistical analysis was performed. One-dimensional statistical analysis included Student’s t-test and fold change analysis. Data were visualized using R software. Metabolites with significant differences between the groups (variable importance for the projection (VIP), VIP >1; Wilcoxon rank-sum test P < 0.05) were screened to perform qualitative analysis.
2.5 Targeted metabolomics of serum neurotransmitters
2.5.1 Sample pretreatment and analysis
The saliva samples (100 µL) were mixed with 400 µL of precooled pure acetonitrile containing 1% FA, vortex-mixed, and ultrasonicated in ice bath for 20 min. The protein was precipitated ultrasonically at -20°C for 1 h in an ice bath, centrifuged (4°C, 14000 g, 20 min), and taken dry with the supernatant vacuum. For mass spectrometry, 100 μL of ACN/water (1:1, v/v) with 1% FA was added to re-dissolution and centrifuged (4°C, 14000 g, 20 min) , and the supernatant was collected for analysis.
Sample separation was performed using the Agilent 1290 Infinity LC UHPLC system. The mobile phase comprised liquid A (0.1% FA 25 mM ammonium formate aqueous solution) and B (0.1% FA acetonitrile). The sample was placed in an automatic sampler at 4°C, column temperature was 45°C, flow rate was 300μl/min, and injection volume was 2 μL. The relevant liquid phase gradients were as follows: 0-18 min, 90%-40% B; 18-18.1 min, 40%-90% B; and 18.1–23 min, 90% B. The 5500 QTRAP mass spectrometer (AB SCIEX) was used for mass spectrometry in the negative ion mode. The 5500 QTRAP ESI source conditions were set as follows: source temperature, 550°C; ion source Gas 1, 60 psi; ion source Gas 2, 60 psi; curtain gas, 35 psi; and ion spray voltage floating, 5000 V. The multiple reaction monitoring (MRM) mode was applied to detect the ion pair.
2.5.2 Data analysis
The chromatographic peak area and retention time were measured using the Multiquant software. The neurotransmitter standard were used to correct the retention time and identify the metabolites.
2.6 Statistical analysis
SPSS24.0 software was used for statistical analysis. The quality parameters are presented as percentages, and the quality parameters between the two groups were compared using chi-square test. The influencing factors were analyzed using logistic regression analysis. Wilcoxon rank-sum test was used for comparisons between the two groups. Values with P < 0.05 were considered statistically significant.