Participants were enrolled for 18 months from 2019 to 2021 at the Reproductive Medical Center of Ruijin Hospital. We recruited 24 RIF patients aged between 25 and 35 years who had experienced three or more previous failed cycles wherein at least four good quality embryos were transferred. The comparison group included 18 women with infertility due to tubal obstruction, who had achieved a successful clinical pregnancy after the first embryo transfer in IVF. The exclusion criteria were as described previously14. Briefly, individuals with uterus pathology, hydrosalpinx, adenomyosis, polycystic ovary syndrome, autoimmune disease, endometriosis and chromosome abnormalities were excluded. Endometrial samples were obtained through pipe suction curettage (LILYCLEANER; Shanghai Jiabao Medical Healthcare Science and Technology Ltd., China).
Furthermore, 12 women in the early proliferative (days 4–5 of the cycle) matching the same criteria as the control group were enrolled.
Immortalized human endometrial stromal cells (T-HESCs) and Ishikawa cells, common surrogates for human endometrial cells, were acquired from the European Collection of Authenticated Cell Cultures (ECACC; Salisbury, UK). Primary human endometrial epithelial cells (HEECs) and endometrial stromal cells (HESCs) were isolated as previously described. Briefly, endometrial samples were minced and digested for 30 min using 1 mg/mL collagenase type I (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C. The mixture was then passed through 100 and 40 µm sieves successively (MilliporeSigma, Burlington, MA, USA), and the flushing and reverse flushing filtrates from the 40 µm sieve were centrifuged 5 min at 100 × g to isolate HESCs and HEECs,. T-HESCs, Ishikawa cells, primary HEECs, and HESCs were cultured in DMEM/F12 medium (Thermo Fisher Scientific), supplemented with 10% (v/v) fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific), at 37 °C according to standard procedures and harvested using 0.25% (w/v) trypsin-EDTA (Thermo Fisher Scientific).
RNA isolation and RT-PCR analysis
Total RNA was extracted from the samples according to the manufacturer’s instructions [Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China] and reverse transcribed using PrimeScript™ RT Master Mix [Takara Biomedical Technology (Beijing) Co., Ltd.]. Reverse transcription-quantitative PCR was performed using SYBR Green Master Mix [Takara Biomedical Technology (Beijing) Co., Ltd.] and Applied Biosystems 7500 Real time PCR System (Applied Biosystems, Waltham, MA, USA). The gene-specific primer sequences are listed in Supplemental table 1. Relative quantification of mRNA levels was performed using the comparative cycle threshold (Ct) method, with GAPDH as the reference gene. All tests were repeated at least thrice.
Protein isolation and western blotting
Proteins were extracted by lysing endometrial samples and cells with RIPA lysis buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The lysates were then centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was collected. Samples with 30 μg proteins were separated via 10% SDS-PAGE, and the resolved proteins were transferred onto PVDF membranes (MilliporeSigma) that were blocked with 5% non-fat milk in TBST for an hour. The membranes were then incubated overnight at 4 °C with protein-specific primary antibodies . Following washing and incubation with an corresponding HRP-conjugated antibody at room temperature for an hour, the bands were visualized via enhanced chemiluminescence (Millipore Sigma). Primary antibodies against CD44v3 (1 μg/mL; R&D Systems, Minneapolis, MN, USA) and GAPDH (1:1,000; Cell Signaling Technology, Danvers, MA, USA) were used in this study. Quantification was performed using the ImageJ software and normalized to GAPDH levels.
Tissue specimens were fixed with 4% formalin. Paraffin sections (5 μm) were prepared and fixed. Antigen retrieval was performed by incubating the cells in buffered citrate for 15 min at 105 °C. The sections were blocked with 5% w/v bovine serum albumin for 30 min and then incubated with primary antibodies against CD44s (1:100; Abcam, Cambridge, UK), CD44v3 (10 μg/mL; R&D Systems), and CD44v6 (1:100; Abcam) overnight at 4 °C. The slides were then stained with horseradish peroxidase-conjugated secondary antibodies, followed by counterstaining with diaminobenzidine (Agilent Technologies, Santa Clara, CA, USA) and hematoxylin. Images were visualized using a microscope (Olympus Corporation, Tokyo, Japan).
SiRNA knockdown and plasmid over expression studies
Control siRNA (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense: 5′- ACGUGACACGUUCGGAGAATT-3′), CD44v3 siRNA1 (sense: 5′- AGGCAUUGAUGAUGAUGAAUU-3′, anti-sense: 5′-UUCAUCAUCAUCAAUGCCUUU-3′), and CD44v3 siRNA2 siRNA (sense: 5′-UGAAGAUGAAAGAGACAGAUU-3′, anti-sense: 5′-UCUGUCUCUUUCAUCUUCAUU-3′), CD44v3 siRNA3 siRNA (sense:5′-GGCUUUCAAUAGCACCUUGUU-3′, anti-sense:5′-CAAGGUGCUAUUGAAAGCCUU-3′) were purchased from GenePharma (Shanghai, China). Mock and CD44v3 overexpression plasmids were purchased from FulenGen (Guangzhou, China). Cell transfection was conducted using the X-tremeGENE 9 DNA transfection reagent (Roche). Cells were transfected with shRNAs or plasmids using standard procedures.
Cell proliferation was examined using the Cell Counting Kit-8 (CCK-8; Dojindo China Co., Ltd., Shanghai, China). Briefly, cells were seeded in 96-well plates at a concentration of 1,000 cells per well. A total of 10 μL CCK-8 solution was added to each well, and the optical density was measured at a wavelength of 450 nm through a microplate reader. The experiment was repeated thrice.
In vitro decidualization activity assay
HESCs separated from the late proliferative phase of the endometrim of the control group were transfected with CD44v3 siRNA or CD44v3 overexpression plasmids. After 48h transfection, the cells were cultured in a serum-free DMEM-F12 medium with 10 nM β estradiol (Sigma Aldrich), 1 μM progesterone (Sigma Aldrich), and 1 mM 8-Br-cAMP (Abcam) for 72 h. Total RNA and protein were extracted, the decidual markers prolactin (PRL) andinsulin like growth factor binding protein-1 (IGFBP1) were evaluated via RT-qPCR and western blotting.
Ishikawa cells (1 × 106 cells) with or without treatment were seeded in 6-well plates to reach sub confluence overnight. The monolayer cells were then scratched using a pipette tip to create a cell free wound. Cells were washed twice with PBS and then cultured in fresh serum-free medium. Wound healing ability was quantified by measuring the percentage of closure at 0, 24, and 48 h. Three independent experiments were conducted.
Embryo outgrowth analysis
Trophoblast outgrowth analysis was constructed as described. In Brief, HESCs were isolated from the late proliferative phase endometrium of the control group and then cultured in in a 24-well plate. After 48 h transfected with CD44v3 siRNA or CD44v3 overexpression plasmids, cells were decidualized as described above. Hatched mice blastocysts with normal morphology were then co-cultured with confluent monolayers of decidualized HESCs in DMEM/F12 complete medium. The trophoblast outgrowth areas were outlined and calculated using Image J 1.46r.
Data are presented as mean ± SEM and were analyzed using the SPSS software (version 22.0; SPSS Inc., Chicago, IL, USA). Statistical analysis between two groups was performed using the two tailed Student’s t-test when data met the normal distribution criterion. For more than two groups, statistical analysis was performed using a one way analysis of variance (ANOVA) with the Bonferroni test for mean separation or a nonparametric test for non-normal data. Statistical significance was set at p < 0.05.