B cell activation factor (BAFF) induces inflammation in the human fallopian tube leading to tubal pregnancy

Seventy patients undergoing salpingectomy for salpingitis (n = 35) and tubal pregnancy (n = 35) were recruited into salpingitis group and tubal pregnancy group, respectively. Twenty patients with benign uterine diseases undergoing complete hysterectomy and salpingectomy were recruited into control group. The information about the clinical characteristics of the enrolled patients was reported in Table 1. There was no significant difference between the three groups. The diagnosis and inclusion criteria in the three groups were showed separately in Table 2. The exclusion criteria for all patients were: ① using exogenous hormone or pharmacologic treatment within the last 3 months before surgery; ② having comorbidities, such as hypertension, diabetes, tuberculosis, tumors or disease of immune system; or ③ combined gynecological disorders, such as endometriosis, polycystic ovarian syndrome (PCOS) or gynecological tumors. This study has been performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Zhuhai Municipal Maternal and Children’s Health Hospital. Written informed consents from all patients were obtained prior to the study.

Seventy-two patients were treated with laparoscopic surgery under general anesthesia. Panoramic laparoscopic pelvic view showed the uterus, fallopian tubes, ovaries, and uterovesical. Eighteen patients underwent abdominal hysterectomy and salpingectomy under continuous epidural anesthesia. The patients in salpingitis group and tubal pregnancy group were suffered from mono/bilateral salpingectomy, and the patients in control group underwent hysterectomy and bilateral salpingectomy excluding mono/bilateral oophorectomy. The tissue samples of fallopian tube were collected during the surgical operation, immediately frozen in liquid nitrogen and subsequently stored at − 70 °C until further process for quantitative real-time polymerase chain reaction (qPCR) and Western blotting. Peripheral blood samples from all patients were collected in the morning (between 8 am and 10 am) in fasting status. All samples were allowed to clot for 2 h at room temperature before centrifugation for 20 min at approximately 1000×g. Aliquots of serum from each sample were collected and stored at − 70 °C until analyzed for ELISA.

Total RNA was isolated from tissue samples using the TRIzol reagent (Roche, Basel, Switzerland) according to the manufacturer’s instructions, and complementary DNA was synthesized using superscript II (Invitrogen, Carlsbad, CA, USA). The forward and reverse primers of BAFF gene were 5′-CTGATAAGACCTACGCCAT-3′ and 5′-GCTACAGACATGGTGTAAGT-3′. A 40 μl reaction mixture containing 2 × SYBR Green PCR Master Mix (Toyobo, Osaka, Japan), and forward and reverse primers is performed. This was followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s and extension at 72 °C for 30 s after conducted at 50 °C for 2 min and a 10-min incubation at 95 °C. The data were collected and analyzed using the ABI PRISM 7300 sequence detection system (ABI, Foster City, CA, USA). The relative quantitation method was used to estimate the levels of messages encoded by this gene in each sample, with 18S ribosome mRNA serving as the endogenous normalization control. The data from the qPCR was converted to 2^-Ct, where Ct represents the threshold cycle. The mean Ct value of the triplicate PCRs was determined, and the mean 2^-∆∆Ct was calculated from the triplicate cDNAs [13].

Cell lysis buffer and protease inhibitor cocktail were used to lyse the tissue samples on ice, and then the supernatants were collected as the total cell lysates. Protein concentrations were measured using the bicinchoninic acid protein assay kit (Pierce, Rockford, USA). Total proteins from each sample were denatured in Laemmli buffer, fractionated by 10% SDS-PAGE, and transferred to nitrocellulose membrane. The membrane was blocked in 5% non-fat milk in TBST (Tris buffered saline plus 0.1% Tween20) for 1 h, and then incubated with antibodies against human BAFF (1:1000, Abcam, England) overnight at 4 °C. Endogenous β-actin expression served as an internal control. The immunoreactive protein complexes were detected using an enhanced chemiluminescence detection system (ECL, Beyotime, Shanghai, China) according to the manufacturer’s protocol. Band intensities were quantified by scanning densitometry by means of the Bio-Rad Quantity One software (Bio-Rad Laboratories, CA, USA).

Serum levels of BAFF, TNF-α and IL-6 were measured by ELISA using kits purchased from Biomatik (Canada). Briefly, 96-well microtiter plates pre-coated with BAFF, TNF-α and IL-6 antibody were incubated with suitable dilutions of the serum. Bound cytokines were detected using enzyme-linked detection antibodies as per the manufacturer’s protocol. The optical density (OD) in the wells was measured at 450 nm, and background values were subtracted. The concentration of BAFF, TNF-α and IL-6 in the samples was then determined by comparing the OD of the samples to the standard curve. The average of duplicate measurements was taken.

Statistical Package of Social Science Software program (SPSS, Chicago, IL, USA), Windows version 20.0 was used to analyze data. Data were presented as the mean ± SD or percentage. One-way ANOVA and Chi-square tests were used. The number of subjects in this study was calculated assuming α = 0.05. P values less than 0.05 were considered significant.