Experimental chemicals
The internal standards, including isotope-labeled Arachidonate (AA), isotope-labeled 16:1(d7) lysophosphatidylcholine(LPC),isotope-labeled 20:4(d7) LPC,isotope-labeled 20:3 (d7) LPC, isotope-labeled 18:3 (d7) LPC and Isotope-labeled 18:1 (d7) LPC, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Distilled water was obtained from a Milli-Q system (Millipore, MA, USA). Chromatographic grade formic acid, acetonitrile and methanol were purchased from Fisher (Fairlawn, NJ, USA).
Sample collection and preparation
Source of cases: 230 cases of patients undergoing IVF-ET between August, 2016 and June, 2017, at the center of integrated traditional Chinese and western medicine reproductive and genetic center of affiliated hospital of Shandong university of traditional Chinese medicine. The study was approved by the Health Authorities and Ethics Committees of Shandong University of Traditional Chinese Medicine Affiliated Hospital.Diagnostic criteria：Previous pregnancy history. No contraception use for 12 months after pregnancy was regarded as secondary infertility[11].Inclusion criteria: Patients aged between 21 and 48 years meeting the diagnostic criteria. They also had to have no major gynecological diseases or other major diseases, have a body mass index (BMI) ranging between 18.5 and 24.9kg/m2[12] and sign an informed consent.Exclusion criteria: Patients with infertility due to other factors such ascongenital ovarian dysplasia, serious malformation of reproductive organs, those with a major operation history, those with infertility due to male factors, those that have used hormone drugs within 3 months prior to the study. Grouping: Patients who met the inclusion criteria were first divided into two groups: a younger group (140 cases) of those aged between 28 and 34 years, and and an older group (90 cases) of those between 35 and 48 years. (World health care (WHO) and international association for obstetrics and gynecology scientific regulations). After identifying the major differences between the two groups, the 230 cases were reclassified into four groups: group A (54 cases) of those aged between 21 and 27 years old, group B (85 cases) of those aged between 28 and 34 years old, group C (51 cases) of those aged between 35 and 41 years old, and group D (40 cases) of those aged between 42 and 48 years old. This was done to observe the specific changes in follicular fluids between the four groups.
All subjects underwent controlled ovarian hyperstimulation according to our established protocols.All patients were enrolled in the controlled super-stimulatory antagonist program (GnRH-ant program).In the program, patients were first tested for serum sex hormone levels, ultrasonography was then done on the third day of their menstrual cycle to assess ovarian function. They then received recombinant follicle stimulating hormone (r-FSH, cognac) or/and injection with human gonadotropin (HMG) at astarting dose of 300 IU per day for 4 days. After 4 days of follow-up, an ultrasound examination to monitor follicular development was done and adjustments of the r-FSH, cognac/HMG dosage made. Once the dominant follicle had a diameter of 13 to 14 mm, 0.25 mg of leuprorelin Acetate per day was administered through injection. This was done until the day the injection with human chorionic gonadotropin (HCG) / recombinant-human chorionic gonadotropin (r-HCG) was administered.
Serum hormone measurement and follicle calculation
Circulating levels of hormones, including serum FSH, LH, E2, testosterone (detected at day 2), E2 and P (detected at hCG day), were measured using a radioimmunoassay method. The numbers of antral follicles were counted using ultrasonography on day 2.
Measurement of the reproducibility and accuracy of SWATH mass spectrometry
Before analysis, eleven follicular fluid samples were thawed at room temperature for quality control (QC) (one QC after every four follicular fluid samples). First, samples were prepared by mixing 100 μL of each individual follicular fluid samples. To reduce the effect of the solvent and obtain a good peak shape, a total volume of 150 μL of follicular fluid or QC sample was mixed with 450 μL of methanol (v/v, 1:3) containing isotope-labeled Arachidonate(AA),isotope-labeled 16:1(d7) lysophosphatidylcholine(LPC),isotope-labeled 20:4(d7) LPC,isotope-labeled 20:3 (d7) LPC, isotope-labeled 18:3 (d7) LPC and Isotope-labeled 18:1 (d7) LPC. Next, the mixture was vortexed for 10 min and centrifuged at 13000×g for 20 min at 15 °C.The contents of the supernatant were analyzed using UPLC-Q-TOF.
Method conditions
Aliquots of 5 μL of the supernatant were injected into the UPLC tandem Triple TOF 5600 system (SCIEX, CA, USA) in random order. A reverse-phase 2.1*100 mm ACQUITY 1.7 μm C18 column (Waters, Ireland) was used for separation. A gradient mobile phase composed of 0.05% formic acid solution (A) and acetonitrile (B) was used and kept at 90% A for 0.5 min, increased to 95% B over the next 6.5 min, and then returned to 90% A from 8.5 min to 8.6 min. The total running time was 13 min. The mass parameters were as follows: nebulizing gas, 55 psi; TIS gas, 55 psi; source temperature, 500 °C; and ion spray voltage, 5000 V with 35 psi curtain gas in positive mode and−4000 V with 35 psi curtain gas in negative mode. The declustering potential and collision energy were set at 55 V and 40 ± 20 V, respectively, in positive mode (− 55 V and − 40 ± 20 V, respectively, in negative mode). The SWATH method with 20 variable isolation windows was performed in TOF MS full-scan mode and in TOF MS/MS product ion scan mode at m/ z 50–1200 in Analyst TF 1.7.1 software.
Data collection, processing and statistical analysis
Analysis was done using SPSS 22.0 statistical software. P value of less than 0.05 (P < 0.05) was considered statistically significant. The quantitative data of each group was normally distributed and hence the means and standard deviation was used for statistical description. The data was not statistically described using the median and quartile deviation. The changes in values before and after treatment in the two groups (the younger and the older) were compared with the normal distribution using the independent sample t test, and the rank sum test without the normal distribution was compared. The count data of the two groups were statistically described using their frequency (composition ratio)and the chi-square test.
The multi-dimensional chromatographic data obtained were transformed into a matrix using metabolomic analysis methods: partial least squares-discriminant analysis (PCA and PLS-DA). In the scoring plot, each point represented a corresponding sample while in the loading plot, discrete points represented the variables separated in the score plot. Those with higher dispersion appeared more in the score plot. The P value for each variable was determined using the t test method. A variable with a P value of more than 0.01 and less than 0.05,0.01< P < 0.05 was said to be significant while those with a P value of less than 0.01,P < 0.01was said to be very significant. According to the primary and secondary mass spectrum information of the groups’ metabolic pathways, there were differences in charge ratio, mass number and isotope abundance.