Reagents
The following reagents were used in this study: rabbit anti-GnRHR (Abcam, UK), mouse anti-c-kit (CST, USA), rabbit anti-c-kit (CST, USA), rabbit anti-phospho-c-kit (CST, USA), rabbit anti-AKT (CST, USA), rabbit anti-phospho-AKT (CST, USA), rabbit anti-Cyclin D1 (CST, USA), mouse anti-β-actin (Santa Cruz, USA), Donkey anti rabbit Alexa flour 488 (Thermo Fisher Scientific, USA), Donkey anti rabbit Alexa flour 594 (Thermo Fisher Scientific, USA), GnRH-a (Decapeptyl; Ferring), GnRH-ant (Cetrotide; Serono) and Imatinib (Biovision, USA).
Patients
The present study retrospectively analyzed the clinical data of 2815 patients undergoing fresh embryo transfer in Reproductive Medicine Center of Jiangxi Maternal and Child Health Hospital from Jan 2016 to Dec 2020. All patients were in good physical and mental condition. The inclusion criteria for all patients included age ≤ 37 years, body mass index (BMI) of 15–25 kg/m2, 1.1 < anti-Müllerian hormone (AMH) < 5.0, 5 ≤ antral follicle count (AFC) ≤ 20, and less than twice IVF-ET experiences. Women with a history of the following procedures or disorders were excluded: uterine malformation, ovarian surgery, radiotherapy or chemotherapy, premature ovarian failure, ovarian dysfunction, adenomyosis, polycystic ovarian syndrome, thyroid dysfunction, recurrent implantation failure (failed to achieve a pregnancy more than three times), submucosal fibroids, intrauterine adhesion, hydrosalpinx, and patients (women or men) with abnormal chromosomes.
Study design and groups
2815 patients were divided into GnRH-ant group (563) and GnRH-a group (2252) according to the different protocols. In GnRH-ant group, recombinant human FSH (rhFSH, Merck-Serono, German) was used on day 2 or 3 of the menstrual cycle. The initial dosage (112.5–225 IU/day) was determined based on age, BMI, AFC, and AMH. The dose of rhFSH were adjusted according to ovarian response as monitored by ultrasonography and serum estradiol (E2) levels. GnRH antagonist (Cetrorelix, Merck Serono, Switzerland) at a daily dose of 0.25 mg was started when the largest follicle exceeded 12 mm. Both GnRH antagonist and rhFSH were stopped and a single injection of 6000–8000 IU of hCG (Merck-Serono, German) was administered when the dominant follicle was ≥ 19 mm in diameter or at least 2 follicles were ≥ 18 mm in diameter. Oocyte retrieval was performed 36–40 hours later under transvaginal ultrasound guidance. In GnRH-a group, a standard full dose of gonadotropin-releasing hormone agonist (3.75 mg, GnRH agonist, Ipsen, France) was used in the second day of menstrual cycle for down regulation. Pituitary down regulation (Endometrial thickness ≤ 5 mm, serum FSH < 5 mIU/mL, LH < 5 mIU/mL, E2 < 50 pg/mL) was confirmed with transvaginal ultrasound and endocrine examination after 28–30 days. The initial dosage (112.5–225 IU/day) was determined based on age, BMI, AFC, and AMH. The dose of rhFSH were adjusted according to ovarian response as monitored by ultrasonography and serum estradiol levels. The HCG trigger process was the same as described above.
Embryo assessment
The embryo assessment criteria were executed as the previously described [18]. A good quality embryo should consist of 7–9 blastomeres with a uniform size, and the fragment proportion should be less than 20% at day 3 for human 3PN embryos after fertilization. The good quality embryo rate refers to the number of good quality embryos divided by the total number of all embryos. Blastocyst formation was determined and graded by using the system of Gardner and Schoolcraft [19]. The blastulation rate refers to the number of blastocysts divided by the total number of all embryos. The good quality blastulation rate refers to the number of good quality blastocyst divided by the total number of all blastocysts. The assessment was made in a blinded manner by two embryologists.
Primary human ESCs isolation, culture and treatment
Primary human decidual ESCs were isolated from the decidual tissue of healthy multipara women (aged 25–32 years) undergoing elective surgical termination of a normal pregnancy at 8 to 10 weeks of gestation. The informed consents from all patients were obtained before the initiation of this study. According to the standard protocol [20, 21], the human decidual tissue was minced and treated with type IV collagenase and DNase type I in a shaking water bath at 37℃ for 90 minutes. The cell digest was then passed through a 70 µm filter, both decidual stromal and epithelial cells were collected in tube. Then, decidual stromal cells were separated from epithelial cells with a 45 µm filter. The stromal cells were subsequently pelleted by centrifugation at 1000 rpm for 5 minutes. The cell pellets were washed once, resuspended, and cultured in Dulbecco modified Eagle medium containing 25 mM glucose, Lglutamine, antibiotics and supplemented with 10% fetal bovine serum at 37℃. ESCs was confirmed by detecting the expression of vimentin protein via immunohistochemical analysis (Supplementary Fig. 1). The cells were used after reaching 70–80% confluence. ESCs were treated for 120 hours with different concentrations of GnRH-a, GnRH-ant (10− 8, 10− 5, and 2x10− 5 mol/L) or imatinib (0 µM, 4 µM, 8 µM, 16 µM, 24 µM, 32 µM). Cells were subsequently collected for cell proliferation assay, flow cytometry and western blot.
Cell proliferation assay
5000 cells/well were planted into 96-well plates in quintuplicate wells. Normally, cells were placed in the culture medium with different concentrations of GnRH analogs for 24, 48, 72, 96 and 120 hours, a total of 10 µL CCK8 reagent (APExBio, USA) was added to each well. After incubating for 2 hours at 37°C, the absorbance at 450 nm per well was measured using microplate reader. Each group of experiment was repeated for three times.
Flow cytometry
The suspended cells were collected by centrifugation. Centrifuge 1000g, centrifugation time 5 minutes at 2–8℃. Cultured cells needed to be digested with EDTA-free trypsin, then terminated with serum-containing medium, centrifuged at 1000g for 5 minutes, supernatant removed, and washed with PBS resuspension. After centrifugal precipitation, the PBS was resuspended, transferred into the flow tube, washed once with PBS, centrifuged at 1000g for 5 minutes, and the supernatant was discarded. Cells were suspended with 400 UL × Annexin binding solution at a concentration of approximately 1×106 cells/ml. 5 UL Annexin V-FITC staining solution (BestBio, China) was added to the cell suspension, gently mixed and incubated for 15 minutes at 2–8℃ in the dark. After adding 10 UL PI staining solution, gently mixed and incubated for 15 minutes at 2–8℃ in the dark. Immediately detected by flow cytometry. Each group of experiment was repeated for three times
Immunofluorescence staining
The methods were described previously [22]. Cells were fixed with 4% paraformaldehyde at 4℃ for 10 minutes and washed with PBS three times. The cells were sealed with 2% triton-100 solution at room temperature for 30 minutes. The primary antibody diluted by blocking solution was added and incubated overnight at 4°C. The next day, cells were washed with PBS three times, 5 minutes each time. The fluorescent secondary antibodies were added and incubated for 1 hour at room temperature in the dark, then the cells were washed with PBS for 3 times, 5 minutes each time. Fluoroshield mounting medium (containing DAPI) with 1:10 dilution was added and stored at 4°C in the dark for fluorescence microscopy observation.
Western blot
Total proteins were extracted from cells using the RIPA lysis buffer containing protease inhibitors (Applygen, China) and phosphatase inhibitors (Sigma, USA). The protein concentrations were determined by NanoDrop 2000c spectrophotometer using BCA protein assay kit (Applygen, China). After loading equal amount of protein samples, SDS-PAGE (12% sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed. The proteins were then transferred to a PVDF membrane (Merck-Millipore, USA). After blocking with Tris buffered saline containing 0.05% Tween-20 (TBST) and 5% non-fat dry milk or 5% BSA for 1 hour, the membrane was incubated with corresponding antibodies at 4℃ overnight, washed in TBST, followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies for 1 hour. Visualization of the proteins was detected with ECL chemiluminescence. Beta-actin was used as a loading control. The intensity values were assessed and analyzed with Image J software. (n = 4 for per lane)
Statistical analysis
Statistical analyses were conducted by using SPSS 24.0 software (SASInstitute Inc.), and all data were expressed as means ± standard errors of the means (s.e.m.s) or percentage (%). Results among experimental groups were analyzed by student’s t-test or one-way ANOVA. For all tests, P-value < 0.05 was considered statistically significant.