2.1 Patients and sample collection
Our study recruited 75 females undergoing IVF-embryo transfer treatment between January 2022 and June 2022 in Lanzhou University First Affiliated Hospital. There were 25 females in the normal ovarian reserve (NOR) group, 25 in the PCOS group, and 25 in the DOR group based on the inclusion and exclusion criteria. The study was approved by the Clinical Research and Ethics Committee of the Lanzhou University Affiliated First Hospital, and informed written consent was obtained from all patients (Ethics Number: LDYYSZLL2022-02). The diagnosis of DOR was made according to the Bologna criteria. The diagnosis of PCOS was based on the Rotterdam ESHRE/ASRM-sponsored PCOS consensus. Exclusion criteria for both groups included the following: History of laparoscopic surgery; patients with ages > 40 years; autosomal gene defects; antibiotic treatment within 3 months. The progestin-primed ovarian stimulation technique was used to stimulate the ovaries of all recruited females. The data that were accessible included age, body mass index (BMI), infertility type, menarche age, gravidity, parity, basal blood hormone, number of retrieved oocytes, number of transferred embryos, and embryo quality. Follicular fluid was during oocyte retrieval, and contaminated follicular fluid was not included in this study. Follicular fluid collected in the test tube was centrifuged at 3000 rpm for 10 min, and the supernatant was collected freezing preservation in a refrigerator at -80 ℃. High-quality embryo rate = (1-grade embryo + 2-grade embryo)/Total number of retrieved embryos ×100%.
2.2 Sample preparation
The fluid was thawed at room temperature, and 0.5 mL fluid was collected into a 15-mL centrifuge tube. Two milliliters of Na2CO3 buffer solution (0.25 mol/L), 5 mL tert-butyl methyl ether (MTBE), and 1 mL tetrabutylammonium hydrogen sulfate (0.25 mol/L) were successively added into the centrifuge tube. After capping, it was placed on the thirsty rotor for 2 − 3 min. The tube was placed in the ultrasonic extraction instrument for 5 min and then centrifuged at 2500 rpm for 10 min. The upper MTBE was transferred to a new 15-mL tube once the liquid had stratified. These steps were repeated thrice before extracting the liquid. The solution was heated to 45 ℃, dried with high-quality hydrogen, and dissolved in 1 mL of methanol. The solution was filtered by 0.22-mm nylon and then placed in a sample vial for chromatography.
2.3 Ultra-high performance liquid chromatography-tandem mass spectrometry analysis (UHPLC-MS/MS)
Target PFAS were analyzed using an Agilent Technologies UHPLC-MS/MS, comprised of a 1290 Infinity II high-speed pump (model G7120A) connected to a triple quadrupole (model G6470C) mass spectrometer and Jet Stream ESI source (Agilent Technologies Inc., Santa Clara, CA, USA). The UHPLC pump has a switching valve that minimizes impurities from repeated sample analysis, increases column life, and minimizes ion source contamination. Chromatographic separation was done using a Rapid Resolution High Definition (RRHD) Eclipse Plus C18 column (2.1×100 mm, 1.8 µm; Agilent Technologies, USA) connected to an RRHD Eclipse Plus C18 pre-column (2.1×50 mm, 1.8 µm; Agilent Technologies, USA). A gradient elution procedure was used for the liquid phase separation of the target. Mobile phase A was 5 mM ammonium acetate; mobile phase B was chromatographic grade methanol. The program was as follows: 0 − 1 min, 10 − 40% B; 1 − 4 min, 40 − 95% B; 4 − 4.1 min, 95 − 10% B; 4.1 − 5 min, 10% B. The flow rate was 0.3 mL/min; the column temperature was 30°C; the injection volume was 5 µL.
The qualification and quantitative determination of PFASs were performed under negative heated ESI (HESI) and operated in parallel reaction monitoring mode. The conditions of the mass spectrometer were as follows: The HESI spray voltage was ± 3.5 kV; the S-lens RF level was 50. The capillary temperature and aux gas heater temperature were 350°C and 450°C, respectively. The sheath gas flow rate, aux gas flow rate, and sweep gas flow rate were 25, 5, and 0 arb units, respectively. The MS resolution and targeted MS2 resolution were 70,000 and 35,000. The automatic gain control target value was 5×104, and the maximum injection time adopted an automatic value. Data were processed using the Xcalibur software.
2.4 Metabolomics of follicular fluid
The DOR group was classified into high- and low-concentration groups based on the concentration of PFOA exposure and subjected to metabolomic sequencing. The follicular fluid was collected in a test tube and centrifuged at 3000 rpm for 10 min, and the supernatant was collected for frozen preservation at -80 ℃. The follicular fluid samples were thawed before 200 µL was applied to the extraction procedure; the samples were mixed with 800 µL methanol: acetonitrile (4:1, v/v) solution and an internal standard of 0.02 mg/mL L-2 chlorophenylalanine. The mixture was allowed to settle at -10 ℃ and treated with a high-throughput tissue crusher Wonbio-96c (Shanghai Wanbo Biotechnology Co., Ltd.) at 50 Hz for 6 min, followed by ultrasound examination at 40 kHz for 30 min at 5 ℃. The samples were placed at -20 ℃ for 30 min to precipitate the proteins. Following centrifugation at 13000× g at 4 ℃ for 15 min; the supernatant was carefully transferred to sample vials for LC-MS/MS analyses. A polled quality control sample (QC) was established by combining similar quantities of all samples as a part of the system conditioning and QC procedure. Procedures similar to those applied for sample analysis were used to examine and dispose of the QC samples. It was used to represent the whole sample set, which was injected at regular intervals to monitor the stability of the analysis.
The instrument platform used for LC-MS analysis was the UHPLC-Q Exactive HF-X system (Thermo Fisher Scientific) under the following chromatographic conditions: 2 µL of the sample was separated on an HSS T3 column (100×2.1 mm i.e., 1.8 µm) and further detected using mass spectrometry. The mobile phases comprised 0.1% formic acid in water:acetonitrile (95%:5%, v/v) (solvent A) and 0.1% formic acid in acetonitrile:isopropanol:water (47.5%:47.5%:5%, v/v) (solvent B). The following rates caused a change in the solvent gradient and were used for equilibrating the systems: 0–3.5 min, 0–24.5% B (0.4 mL/min); 3.5–5 min, 24.5–65% B (0.4 mL/min); 5–5.5 min, 65–100% B (0.4 mL/min); 5.5–7.4 min, 100% B (0.4–0.6 mL/min); 7.4–7.6 min, 100–51.5% B (0.6 mL/min); 7.6–7.8 min, 51.5–0% B (0.6–0.5 mL/min); 7.8–9 min, 0% B (0.5–0.4 mL/min); 9–10 min, 0% B (0.4 mL/min). The sample injection volume was 2 µL, and the flow rate was set to 0.4 mL/min. The column temperature was maintained at 40 ℃. During the analysis, all these samples were stored at 4 ℃ under MS conditions. The mass spectrometric data were collected using the Thermo UHPLC-Q Exactive HF-X Mass Spectrometer equipped with an ESI source operating in either positive or negative ion mode. The optimal conditions were set as follows: Heater temperature, 425 ℃; Capillary temperature, 325 ℃; sheath gas flow rate, 50 arb; aux gas flow rate, 13 arb; ion-spray voltage floating, -3500 V in the negative mode and 3500 V in the positive mode, respectively; normalized collision energy, 20 − 40 − 60 V rolling for MS/MS. Full MS resolution was 60000, and MS/MS resolution was 7500. Data acquisition was performed in the Data Dependent Acquisition mode. The detection was conducted over a mass range of 70–1050 m/z.
Following data acquisition, the peaks were first filtered for a. very low signal undetectable, b. detection errors, such as ion suppression or instrument performance instability, and c. algorithmic limitations of peak extraction. Further, the peaks were identified, and the peak areas were calculated. Subsequently, the missing data were filled using the minimum value. Finally, normalization was performed using the median. All these steps were performed using Major BIOS software.
2.5 Differential metabolites analysis
Partial least squares discriminant analysis (PLS-DA) and orthogonal PLS-DA (OPLS-DA) models of high-and low-concentration groups were constructed using R package ropls, while the determination of variable importance in projection (VIP) values of metabolites in each model were calculated. Subsequently, the metabolites with significant differences in high-and low-concentration groups were identified using P < 0.05, difference multiples > 1, and VIP value > 1 as the screening criteria, and the screening process was demonstrated by volcano plots. Further, the top 12 metabolites with the most significant differences between the groups were screened. All these steps were performed using Major BIOS software.
2.6 Statistical analysis
The statistical analyses were performed using IBM SPSS 22.0 and empower stats based on the R language. Continuous variables were presented as means ± standard deviation, and a t-test was used for comparisons. If normality was not satisfied, the Mann − Whitney U test was used for comparisons. Categorical variables were presented as a percentage. P < 0.05 was considered statistically significant. In this study, PFOA exposure concentrations in the DOR group were grouped by quartile method, Q1 (4.45 − 13.96 ng/mL), Q2 (14.29 − 38.67 ng/mL), Q3 (62.86 − 155.82 ng/mL), and Q4 (182.23 − 485.43 ng/mL). Single-factor logistic regression analysis based on a generalized estimating equation was used to study the effect of PFOA in follicular fluid on the rate of high-quality embryos. A curve-fitting analysis based on generalized additive mixed modeling (GAMM) was used to investigate the relationship between PFOA in follicular fluid and the rate of the high-quality embryo.