Ten eutopic endometrial tissues were acquired from infertile patients with adenomyosis attending the First Affiliated Hospital of Chongqing Medical University during the mid-secretion phase to form the AG group. Patient age ranged from 27 to 45 years with a mean age of 36.80±8.21 years.
The normal eutopic endometrial group (the CG group) featured 11 healthy women from the First Affiliated Hospital of Chongqing Medical University. Age ranged from 26−45 years with a mean age of 35.91±9.61 years. Endometrial samples were taken during the normal secretion phase.
There was no significant difference in age between the two groups (P > 0.05). Each patient underwent curettage or surgery to obtain endometrial samples, and no endometrial lesions were confirmed by pathology. All specimens were selected from January 2014 to March 2015 in the First Affiliated Hospital of Chongqing Medical University and confirmed by surgery and pathology. The study was approved by the Ethics Committee of Chongqing Medical University and all patients provided informed and signed consent.
The inclusion criteria for the AG were as follows：(1) women of reproductive age, 25−45-years-old, with a normal sexual life, no contraception and no pregnancy for 1 year, or having a history of pregnancy and not becoming pregnant again for 1 year; (2) normal menstrual cycle of 21−35 days with a bipolar basic temperature; (3) no abnormalities with regards to basic endocrinology; (4) no hormone therapy over the previous three months; (5) no organic lesions detected in the ovary by B ultrasound; (6) bilateral oviduct patency was confirmed by salpingography, and (7) there was no abnormality with regards to semen analysis in the spouse. The inclusion criteria for the CG group were as follows: (1) women of reproductive age, 25−45-years-old, with a normal sexual life, a history of pregnancy, and no history of infertility; (2) normal menstrual cycle of 21−35 days with a bipolar basic temperature; (3) no abnormalities with regards to basic endocrinology; (4) no hormonal therapy over the previous three months; and (5) no organic lesions in the uterus and ovary.
Patients were excluded if they (1) had infertility caused by abnormal follicles or other reasons; (2) had serious primary diseases of the cardiovascular system, liver, and kidneys, combined with medical and surgical diseases; (3) were unable to cooperate, such as those with neurological or mental disorders, or were unwilling to cooperate; or (4) had an allergic constitution.
2.2 Specimen collection
Twenty-one samples of eutopic endometrium were obtained by surgery and curettage. All cases were confirmed by laparoscopy or laparotomy and verified by pathological sections. All pathological specimens were collected and divided into three parts: one part was immediately placed into D-Hanks solution pre-packed in an icebox and sent to the laboratory within 1−2 h to prepare for subsequent cell culture tests. One part was quickly placed in liquid nitrogen to prepare for western blotting. The last part was placed in 10% formalin, fixed for 24 h, and then embedded in paraffin. This part was used for HE staining, endometrial staging, and immunohistochemistry.
2.3 Experimental groups
In vitro experiments are divided into two groups: (1) CG: eutopic endometrium from healthy women during the mid-secretion phase (n=11) and (2) AG: eutopic endometrium from infertile women with adenomyosis during the mid-secretion phase (n=10).
In vivo, the endometrial cells were divided into two groups: (1) CG: eutopic endometrial cells in mid-secretion of healthy women. (2) AG: eutopic endometrial cells in mid-secretion of infertility in adenomyosis.
In vivo, AG groups cells treated with LIPUS were divided into two groups: (1) Control: No LIPUS treated AG groups cells. (2) LIPUS: 30 mW/cm2 LIPUS treated AG groups cells for 4 days (20 min/day)
2.4 LIPUS applied to cells
Apply special ultrasonic adhesive to make the bottom of the 6-well plate close to the ultrasonic probe on the support table. The low intensity pulsed ultrasonic instrument is provided by the key laboratory of biomedical engineering college, Chongqing medical university. The ultrasonic frequency is 1.5MHz, the ultrasonic gap time is 800μs, the pulse frequency is 1kHz, and the action time is 200μs. After 24 hours of cell culture, LIPUS was applied to the cultured EEEC from infertile patients with adenomyosis in vitro with ultrasonic intensities of 15, 30, and 60mW/cm2, respectively. Ultrasound stimulation of different intensity (20min/ day) was started for 1, 2, 3, 4, 5, 6, 7 days. Continue to incubate after sonicating the cells every day.
IHC staining methods were carried out following the manufacturer’s instructions (Biotin Labeling Immunohistochemical Kit SP-9000 Sheep Resistance Rabbit /Mouse/Guinea pig/Rat IgG; Beijing Zhongshan Cor, China). Sections (4 μm thick) were incubated with H2O2 to eliminate endogenous peroxidase activity for 10 min and then blocked with 10% goat serum at 37ºC for 30 min. The slices were incubated with rabbit monoclonal HOXA10 antibody (1/1,000, GeneTex, USA), rabbit monoclonal LIF antibody (1/800, Abcam, USA), rabbit monoclonal CK19 antibody (1/800, Abcam, UK) overnight at 4ºC. The next morning, appropriate conjugated Sheep Resistance Rabbit IgG antibodies (Beijing Zhongshan Cor, China) were incubated with the slices at 37ºC for 40 min. The sections were then washed and developed with DAB as a chromogen. Sections were then coverslipped and sealed with neutral resin. Representative images were then acquired by microscopy. The histological integral H-score method proposed by Budwig-Novotny was used for semi-quantitative analysis; the formula was h-score =ΣPi(i+l)(10). Pi represents the percentage of the number of positive cells with the same staining intensity in the immunohistochemical staining range of each tissue relative to the total number of cells to be tested; that is, Pi = the number of positive cells/total number of cells to be tested. Staining intensity was graded as 1 (+), 2 (++), and 3 (+++). The maximum H-score was 4, and 1 is the correction factor.
2.5 Western blotting
The tissues were mixed with RIPA protein lysis buffer (Bi Yuntian Biotechnology Institute, China) and fully homogenized, lysed, and centrifuged. The supernatants were then collected. Protein concentration was determined by the BCA method (BCA Protein Concentration Quantitative Kit; Biyuntian Biotechnology Institute China). Adjust the protein concentration to 10ug/ul, and the protein loading volume is 10ul/well. Protein samples were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (upper glue: 90v, 20min, lower glue: 120v, 90min), and then transferred to polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). After being blocked with 5% non-fat milk in Tris-buffered saline with 0.05% Tween-20 for 1 h. The membranes were incubated with primary antibodies at 4℃ overnight as follows: rabbit monoclonal HOXA10 antibody (1/1,000; GeneTex, USA); Rabbit monoclonal LIF antibody (1/800, Abcam, UK). The next morning, membranes were washed and incubated with secondary antibody (sheep anti-rabbit IgG, Abcam, UK; 1:2,000). Positive binding was detected with an ECL Kit (Biyuntian Biotechnology Institute, China) and a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA). The optical density of each band was analyzed by ImageJ.
2.6 Cell adhesion experiment
Matrigel (BD Bioscience, USA) was used to cover 96-well plates and sealed with 2% BSA. For experimentation, we selected EEECs from the CG and AG that had entered the logarithmic growth phase. Samples from the cell cultures were digested and centrifuged. This allowed us to count the number of live cells, adjust the cell density to 5 × 104 cells/mL; we then added 200 µL of the cell suspension to each well for culture. After incubation for 4 h, 200 µL of serum-free DMEM-F12 culture medium and 20 µL of MTT (Sigma-Aldrich, USA) solution were added for 4 h. The culture medium was then drained from each well and 200 µL of DMSO solution was added. Then, the 96-well plates were placed on an oscillator for 10 min until fully dissolved. The absorbance (A570) value of each well at 570 nm wavelength was then measured by an automatic enzyme reader and the adhesion rate was calculated. The experiment was repeated three times. Cell adhesion rate = experimental well A570/ control well A570 × 100%.
The concentration of ICAM-1 in the supernatant of EEEC in vitro cultured of infertile patients with adenomyosis was determined by ELISA. The specific steps were carried out step by step according to the instructions [human intercellular adhesion molecule-1, ICAM-1 ELISA Kit; Abcam, UK]. Final determination (within 15 min after adding stop solution): set the blank well to zero, and then measure the absorbance (OD) of each well in sequence at 450 nm using an ELX800 microplate reader (Bio-Tech, USA). The actual concentration of each target factor was calculated according to the standard curve. The experiment was repeated three times.
2.8 RT-PCR and agarose gel electrophoresis
Cells were lysed by Plus (Invitrogen, USA). RNA was then isolated, precipitated, washed, and dissolved. The ratio of RNA A260/A280 was then determined. Samples with a ratio of 1.8-2.0 were used for RT-PCR experiments. A GeneQuant was used to determine the concentration of each sample. RNA concentration (μg/μL) = concentration × dilution ratio. Reverse transcription was performed according to the instructions of the reverse transcription PCR kit (TAKARA, Shiga Japan). The primers were designed and synthesized by TAKARA. Next, 5 μL of the reaction products were mixed with sample loading buffer for electrophoresis. All RT-PCR results were captured by a gel imager and analyzed by QuantityOne software. Results were expressed as the ratio of the optical density value of a given band for the target mRNA to GAPDH.
The mRNA expression levels of HOXA10 and LIF in EEECs cultured in vitro from infertile patients with adenomyosis during the mid-secretion phase after LIPUS treatment was detected by RT-PCR. The sequences of the forward (F) and reverse (R) primers were as follows: HOXA10, F-5′-ACTGCTCGAAATGGCCCCTGTC-3′, R-5′-CGGCTCCTTTGCACCA TTGACC-3′; LIF, F-5′-GCCCACCCTGTCCTTGTTTCTG-3′, R-5′-CGGGCCACAGGAGGAGCTT-3′, GAPDH, F-5′-TGGGGTGATGCTGGTGCTGAGT-3′, R-5′-AGGTTTCTCCAGGCGGCA TGTC-3′. The product length was as follows: HOXA10 (132 bp), LIF (117 bp), GAPDH (500 bp).
2.9 Statistical analysis
SPSS version 22.0 statistical software (IBM Cor, USA) was used for analysis, and values are expressed as mean ± standard deviation. The Mann-Whitney test was used to compare age between the CG and AG groups. The chi-square test, rank-sum test, analysis of variance, and t-test were used to compare data between groups. P<0.05 was considered statistically significant.