Forty women with primary infertility due to male factors were prepared for ART and enrolled during January 2018 to October 2018 at The Reproductive Medicine Center of First Hospital Affiliated to Suzhou University. Inclusion criteria included: i) a normal natural menstrual cycle with normal ovulation, ii) normal baseline endocrine levels, iii) no uterine fibroids or polyps, no endometrial tuberculosis, endometritis, endometriosis, hydrosalpinx and other gynecological diseases, iv) no polycystic ovarian syndrome and other endocrine diseases, v) no history of estrogen or progesterone mediation or uterine surgery in the past 3 months. The women were 22 to 35 years old with an average age of 26.7 years. The average duration of infertility was 3.26 years. All patients provided signed informed consent, and the study was approved by the Medical Ethics Committee of the Institute of Reproductive Medicine Center. The female subjects were randomly divided into the experimental and control groups. There were no significant differences for the baseline parameters (age,duration of infertility, BMI, estradiol and progesterone levels) between the two groups (P > 0.05).
Embryo implantation window model
The selected women were prepared for ART treatment. During the ninth day of the menstrual cycle, follicle development was detected using a diagnostic ultrasound instrument (ALOCK-6). If the average follicle diameter had reached 14 mm, patients were then monitored daily. The ovulation day was defined when the follicle diameter reached 18 mm (a mature follicle) and then suddenly disappeared or was reduced more than 5 mm, and dark liquid areas appeared in the uterine-rectum nest. The implantation window was regarded as 7–10 days after ovulation.
Experimental and control groups
Subjects were successively divided into experimental and control groups according to their order of enrolment. On the seventh day after ovulation, the experimental group received a subcutaneous injection of 0.1 mg of GnRH-a (Diphereline, Ipsen Pty. Ltd.), while the control group received a subcutaneous placebo injection (2 ml of 0.9% saline).
Collection of endometrial specimens
Before injection of GnRH-a (day 0) and after 3 days (day 3), endometrial tissue was extracted from the uterine cavity by a single-use suction device (Ningbo TianyiMedical Instrument Co., Ltd.). Extracted endometrial tissues were divided into two parts, washed with 0.9% physiological saline and dried with absorbent paper. One part was quickly placed into 2.5% glutaraldehyde solution and stored at 4°C for subsequent electron microscope scanning. The second part was stored at −80°C prior to western blot analysis.
Collection of serum samples
Fasting peripheral venous blood (3 ml) was collected on the same day endometrial tissue was extracted, and then centrifuged at 3500 rpm for 15 min. Serum samples were collected and estradiol and progesterone levels were measured by chemiluminescence immunoassays(Beckman Coulter Inc., USA).
Assessment of scanning electron microscopy results
The endometrium sample was dried by gradient ethanol dehydration and plated with metal film by a vacuum coating apparatus, then observed by scanning electron microscopy. According to previously described criteria (Aghajanova et al., 2003), during pinopode formation, the cell surface was smooth, microvilli were reduced and the thin endometrial surface was protruded and folded to a large degree, shaped like a mushroom. During the degeneration of pinopodes, protrusions were reduced and microvilli reappeared, projecting from the endometrial surface, and cell volume was markedly increased. Every specimen was taken from near the opening of each gland, five fields were counted, and the average count selected. According to the percentage of pinopodes estimated in the total uterine endometrium, the expression levels were divided into rich, moderate and micro (>50%, 20–50% and <20%, respectively).
Western blot analysis
Total protein was extracted from endometrial tissue samples in liquid nitrogen.During SDS-PAGE, 30 μg denatured protein was added per lane, and after electrophoresis the proteins were transferred to a PVDF membrane (at a constant flow of 300 mA) at room temperature. The membrane was blocked in 5% skim milk powder (Shanghai Biological Engineering) dissolved in Tris-buffered saline with Tween 20 (TBST) at room temperature for 1 h. The primary antibodies (1: 20) were incubated overnight at 4°C, then rinsed three times with TBST for 10 min. The HRP-labeled secondary antibody was then incubated at room temperature for 1 h, then rinsed 3 times with TBST for 10 min. An ECL chemiluminescence imaging system was used to analyze results (Kit purchased from Santa Cruz Biotechnology).