Study patients
A total of 118 patients who underwent IVF–ET between February 2018 and March 2019 in the Center for Reproductive Medicine, Affiliated Hospital of Jining Medical University, were recruited for this study. One hundred and eighteen women participated in the study because 14 patients were lost to follow-up. All of participated women had regular menstrual cycles (28 ± 2 days, blood progesterone concen-trations measured between the 18th and 21st days of the menstrual cycle, >10 ng/ml in two consecutive cycles) without clinical or bio-chemical hyperandrogenism or polycystic ovary and with no history of any drug intake for at least 3 months. All of them had either fallopian tube obstruction, or normal reproductive health but an infertile male partner. Sex hormone concentrations and routine biochemical examinations of subjects were normal, and patients had no history of genitourinary diseases, or severe cardiovascular, liver or kidney disease. Additional exclusion criteria were smoking and alcohol consumption. The data for all subjects were obtained from clinical and pathologic records including age and history of menstruation. All subjects provided written informed consent in accordance with Institutional Review Board guidelines for the protection of human subjects. The study received ethical approval from the Institutional Review Board on 16 March 2018.
Ovarian stimulation and supernatant collection
All patients were stimulated by the long protocol. Ovarian follicular development was stimulated with recombinant human FSH (Merck Serono, Switzerland) at doses of 225–450 IU/day. Ovulation was triggered by human chorionic gonadotrophin (HCG 4000–10000 IU) (Livzon Pharmaceutical, China) when at least two follicles were 18 mm and half of the remainder were > 15 mm. Oocytes were recovered transvaginally under ultrasound guidance approximately 34.5 h later. All monitoring of controlled ovarian hyperstimulation (COH) as well as egg retrievals and embryo transfers were performed by one of five physicians. Specific methods refer to previous study [9]. Fertilized eggs were cultured to blastocysts. All embryos were graded microscopically according to Gardner grading standard [10]. All patients chose to transplant the embryo at the blastocyst stage on the fifth day. Each patient selected the embryo with the highest morphological score for single-embryo transfer. All embryo transfers were performed using a Wallace catheter under direct ultrasound guidance 120 h after egg retrieval.
LIF and β-HCG Assay
LIF levels were assayed using a specific sandwich enzyme ELISA kit (RD) using a specific monoclonal antibody (mAb). When embryos were transferred to 50 μL droplets, they were transferred together with a small amount of medium. To ensure that equal volume of embryo supernatant was tested, 100 μL of dilution buffer were then added to the microplate testing wells. Forty microliters of embryo supernatant from each droplet were loaded manually into the wells. The standard substance in LIF kit was diluted to give a calibration curve within the range value of the embryos. embryo culture supernatant LIF concentrations were measured by enzyme-linked immunosorbent assay (R and D Systems, USA). Both inter-assay and intra-assay coefficients of variation for LIF assays were less than 10%. The lower limit of detection for LIF was 8 pg/ml. The LIF concentration was determined by the absorbance at 450 nm on the EL800 Universal Microplate Reader (Bio-Tek Instruments Inc., Winooski, VT). Venous blood (2 ml) was collected on the third day of the menstrual cycle after fasting. If subjects had oligomenorrhoea or amenorrhoea,
fasting venous blood could be collected at any time. The concentrations of serum oestradiol, FSH, LH, pro-gesterone, testosterone and prolactin (PRL), and follicular fluid oestradiol and progesterone, were measured by chemilumines-cence (Roche, Switzerland). The lower limit of detection for oestradiol, FSH, LH, progesterone, testosterone and PRL were 18.4 pmol/l, 0.1 IU/l, 0.1 IU/l, 0.095 nmol/l, 0.087 nmol/l and 0.996 mIU/mL, respectively.
Luteal support and judgment of pregnancy outcome
After fertilization, progesterone (Abbott, the Netherlands) was ad-ministered by intramuscular injection at a dose of 60 mg per day and continued for 14 days for corpus luteum support. If pregnancy was confirmed, progesterone administration was continued until the 10th week of pregnancy. Biochemical pregnancy was defined as blood HCG >10 IU/Lon the 14th day of transplantation. Clinical pregnancy was confirmed with ultrasound examination of the number of gestational sacs and fetal heartbeat on approximately the 64th day after embryo transfer.
Statistical Analysis
Differences between groups were evaluated for statistical significance by Student’s t test or Mann-Whitney rank sum test, depending on whether the data were normally distributed. Receiver operating characteristic (ROC) curve analysis was used to analyze the cut off value of LIF concentration and their sensitivity/specificity. Significant P values were set at <0.05. he data were expressed as mean ± standard deviation (SD). The data between two samples were compared by Mann–Whitney U-test. The relationship between the parameters was analysed by Pearson cor-relation analysis. All calculated P-values were two-sided, and P < 0.05 was used to determine statistical significance. Statistical analyses were performed using GraphPad Prism software (GraphPad Software, USA)and SPSS 18.0 software (SPSS Inc, Chicago, IL, USA).