Participants and tissue collection
The use of human tissues was approved by the Ethical Committee of Changzhou Maternal and Child Health Care Hospital Affiliated to Nanjing Medical University. All participants signed a written consent form before participating in the study.
The participants who underwent in vitro fertilization (IVF) involved in this study were recruited from August 2018 to January 2020. A total of 12 patients without achieving clinical pregnancy after at least four good-quality embryo transfers in a minimum of three fresh or frozen cycles under the age of 40 years were enrolled as the RIF group. The control group consisted of 12 women who achieved live birth within the first three transfer cycles. Women with autoimmune disturbances, endocrine dysfunctions, diminished ovarian reserve, polycystic ovarian syndrome (PCOS), endometrial hyperplasia, endometrial polyps, hydrosalpinx, endometriosis, adenomyosis or intrauterine adhesions were excluded. The details of these participants are summarized in Table 1. Endometrial samples were derived from the endometrial biopsy between days 19 and 23 of menstruation from all women.
Table 1
Demographic details of the participants in this study
Fertile
|
FER (n=12)
|
RIF (n=12)
|
P
|
Age (years)
|
29.9 ± 3.2
|
30.5 ± 3.2
|
ns
|
Body mass index (Kg/m2)
|
20.7 ± 1.8
|
21.1 ± 1.5
|
ns
|
Menstrual cycle (days)
|
29.9 ± 2.5
|
29.5 ± 3.9
|
ns
|
Endometrial thickness (mm)
|
10.2 ± 1.0
|
9.7 ± 1.1
|
ns
|
No. of transferred embryos
|
2 ± 0
|
8.6 ± 4.0
|
s
|
The data are presented as the mean ± SD unless otherwise indicated |
Isolation and culture of primary HESCs
Primary HESCs were isolated from endometrial tissues according to a previously described method[19]. After purification, HESCs were digested and grown into new plates with DMEM/F12 (HyClone, Thermo Scientific, South Logan, UT, USA), 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 1% penicillin and 1% streptomycin. The cells were used to induce decidualization as described previously.
Construction of adenoviruses
Adenoviruses harboring a DNA fragment encompassing the miR-133b gene or full-length KLF12 were generated using the AdMax (Microbix Biosystems, Inc., Toronto, Canada) system. The viruses were packaged, amplified and purified according to a previously protocol.
Western blotting
The proteins extracted from endometrial tissues and cultured cells as previously described[21]. After using the BCA kit (Beyotime, Shanghai, China) to measure the protein concentration, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate the proteins, which were then blotted onto polyvinylidene difluoride (PVDF, Millipore, Billerica, MA, USA) membranes and incubated with the primary antibodies including anti-KLF12 (Santa Cruz Biotechnology, 1:1000), anti- LIF (Abcam, 1:500), anti-STAT3 (Cell Signaling Technology, 1:1000), anti-p(Y705)-STAT3 (Santa Cruz Biotechnology, 1:1000) or anti-β-actin (Abcam, 1:5000), followed by incubations with a horseradish peroxidase (HRP)-conjugated secondary antibody or Flag-HRP. The bands were visualized with an enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA) in ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).
Quantitative Real-time PCR
Total RNA was extracted from endometrial tissues or HESCs using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. cDNA was synthesized by using a PrimeScript RT reagent kit (Takara). Realtime-PCR were employed with SYBR Green kit (Qiagen, Hilden, Germany) on a MyiQ single color Realtime PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The level of RNA was calculated using the 2-△△Ct method. 18S rRNA and U6 served as endogenous controls for mRNA and microRNA, respectively. The specific primers used for quantitative PCR analysis were: miR-133b, F: 5’-ACACTCCAGCTGGGTTTGGTCCCCTTCAAC-3’ and R: 5’-GGTGTCGTGGAGTCGGCAATTCAGTTGAG-3’; U6, F: 5’-CTCGCTTCGGCAGCACA-3’ and R: 5’-AACGCTTCACGAATTTGCGT-3’; KLF12, F: 5’-CCTTTCCATAGCCAGAGCAG-3’ and R: 5’-TTGCATCCCTCAAAATCACA-3’; LIF, F: 5’-AGTCGTGACCTTGGCACCTC-3’ and R: 5’-GTTGACAGCCCAGCTTCTTC-3’; PRL, F: 5’-CACTACATCCATAACCTCTC-3’ and R: 5’-ATGCTGACTATCAAGCTCAG-3’; IGFBP-1, F: 5’-TATGATGGCTCGAAGGCTCTC-3’ and R: 5’-GTAGACGCACCAGCAGAGTC-3’; 18S rRNA, F: 5’-CGGCTACCACATCCAAGGAA-3’ and R: 5’-CTGGAATTACCGCGGCT-3’.
Prolactin measurement in cultured supernatants
Prolactin levels in cell culture media were measured using the Mini-Vidas V.B02.96 System with a Vidas prolactin Kit (bioMerieux, France). Each experiment was performed in triplicates and repeated in three primary HESCs derived from different women.
Dual-luciferase reporter assay
The wild-type KLF12 3’-UTR (KLF12 3’-UTR WT) and mutant KLF12 3’-UTR (KLF12 3’-UTR MUT) were synthesized and inserted into the pGL3 luciferase reporter vector (Promega, Madison, WI, USA). For reporter assays, the preconfluent (70%) HEK293T cells in 24-well plates were transfected with the indicated plasmids using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA). The dual-luciferase reporter assay was performed to analyze the relative luciferase activity after transfection for 48 h.
Statistical analysis
All statistical analysis were performed using Prism version 8 software (GraphPad, LaJolla, CA). The data were presented as the mean ± SEM of at least three independent experiments. Student’s t-test was used for comparisons between two groups. One-way ANOVA was performed for comparisons among more than two groups. Pearson correlation analysis was used to assess the relationship between miR-133b and KLF12. Differences at p < 0.05 were considered significant.