[AQP5 gene silencing inhibits proliferation and migration of ectopic endometrial glandular epithelial cells in endometriosis].

OBJECTIVE: To investigate the effect of aquaporin 5(AQP5) on proliferation and migration of ectopic endometrial epithelial cells. METHODS: AQP5 shRNA interference fragments were designed and transfected into ectopic endometrial epithelial cells stably by lentivirus technology. Fluorescence quantitative RT-PCR and Western blotting were used to detect the AQP5 mRNA and protein expression, respectively. The cell proliferation and migration were determined by using MTT method and Transwell system, respectively. Levels of phosphorylated AKT(p-AKT) and total AKT were examined by Western blotting. The nude mice model of endometriosis was constructed and the endometrial cell nodule formation was observed. RESULTS: AQP5 shRNA transfection inhibited cell proliferation and migration compared with control group (both P<0.05). The activation of AKT in AQP5 shRNA transfected cells was lower than that in control cells (P<0.01). Compared to control group, the endometrial cells nodule formation was suppressed in mice inoculated with AQP5 shRNA-silencing ectopic endometrial epithelial cells. CONCLUSION: Down-regulation of AQP5 expression can suppress the proliferation and migration of ectopic endometrial epithelial cells and endometrial cell nodule formation in nude mice, in which AKT pathway may be involved.

Efficacy and Safety of Sanjie Analgesic Capsule in Patients with Endometriosis-Associated Pain: A Multicenter, 3:1 Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: To assess the efficacy and safety of Sanjie Analgesic Capsule (SAC) in Chinese patients with endometriosis-associated pain. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled trial conducted at 15 centers between November 2013 and July 2017 in China. Eligible 323 patients with endometriosis were randomized at a 3:1 ratio to the SAC group (241 cases) and placebo group (82 cases) by stratified block randomization. Patients in the SAC or placebo groups were given SAC or placebo 1.6 g 3 times per day, orally, respectively since the first day of menstruation for 3 consecutive menstrual cycles. The primary endpoint was clinical response to dysmenorrhea evaluated using a 10-point Visual Analogue Scale at 3 and 6 months. The secondary endpoint was the pain score evaluated by VAS (chronic pelvic pain, defecation pain, and dyspareunia) at 3 and 6 months, and the pain recurrence rate at 6 months. Adverse events (AEs) were recorded during the study. RESULTS: A total of 241 women were included in the SAC group, and 82 were in the placebo group. Among these women, 217 (90.0%) and 71 (86.6%) completed the intervention, respectively. At 3 months, overall response rate (ORR) was significantly higher in women administered SAC (80.1%) compared with those who received a placebo (30.5%, P<0.01). Six months after treatment, the ORR for dysmenorrhea was 62.7% in the SAC group and 31.7% in the placebo group (P<0.01). Chronic pelvic pain and defecation pain were significantly improved by SAC compared with placebo (both P<0.05). The incidence rates of total AEs events in the SAC and placebo groups were 6.6% and 9.8%, respectively, and no significant difference was shown between the two groups (P=0.339). CONCLUSION: SAC is well-tolerated and may improve dysmenorrhea in women with endometriosis-associated pain. (Trial registration: ClinicalTrials.gov, No. NCT02031523).

[Effects of PRL-3 siRNA on the migration of endometriotic stromal cells].

OBJECTIVE: To evaluate the effects of PRL-3 siRNA (small interfering RNA) on the migration of endometriotic stromal cells. METHODS: Primary endometriotic stromal cells were cultured in vitro. Then PRL-3 (phosphatase of regenerating liver-3) siRNA was transfected to silence the PRL-3 gene. And the PRL-3 protein expression was analyzed by Western blot. The changes of cell migration were detected by cell pseudopod formation, scratch test and transwell cell migration test. RESULTS: The expression of PRL-3 protein significantly decreased in the experimental group versus two other control groups (P < 0.05). The formation of cell pseudopod was much less in experimental group than that in control groups. Within the same time, the number of migration cells was 0.87 ± 0.21 in experimental group. It was less than 1.75 ± 0.28 in blank control group and 1.63 ± 0.39 in negative control group (P < 0.05). CONCLUSION: PRL-3 siRNA can down-regulate the PRL-3 gene and decrease the migratory capacity of endometriotic stromal cells. And PRL-3 may be a promising target in the therapeutics of endometriosis.

Aquaporin 5 Plays a Role in Estrogen-Induced Ectopic Implantation of Endometrial Stromal Cells in Endometriosis.

Aquaporin 5 (AQP5) participates in the migration of endometrial cells. Elucidation of the molecular mechanisms associated with AQP5-mediated, migration of endometrial cells may contribute to a better understanding of endometriosis. Our objectives included identifying the estrogen-response element (ERE) in the promoter region of the AQP5 gene, and, investigating the effects of AQP5 on ectopic implantation of endometrial cells. Luciferase reporter assays and electrophoretic mobility shift assay (EMSA) identified the ERE-like motif in the promoter region of the AQP5 gene. After blocking and up-regulating estradiol (E2) levels, we analysed the expression of AQP5 in endometrial stromal (ES) cells. After blocking E2 /or phosphatidylinositol 3 kinase(PI3K), we analysed the role of AQP5 in signaling pathways. We constructed an AQP5, shRNA, lentiviral vector to knock out the AQP5 gene in ES cells. After knock-out of the AQP5 gene, we studied the role of AQP5 in cell invasion, proliferation, and the formation of ectopic endometrial implants in female mice. We identified an estrogen-response element in the promoter region of the AQP5 gene. Estradiol (E2) increased AQP5 expression in a dose-dependent fashion, that was blocked by ICI182,780(an estrogen receptor inhibitor). E2 activated PI3K /protein kinase B(AKT) pathway (PI3K/AKT), that, in turn, increased AQP5 expression. LY294002(PI3K inhibitor) attenuated estrogen-enhanced, AQP5 expression. Knock-out of the AQP5 gene with AQP5 shRNA lentiviral vector significantly inhibited E2-enhanced invasion, proliferation of ES cells and formation of ectopic implants. Estrogen induces AQP5 expression by activating ERE in the promoter region of the AQP5gene, activates the PI3K/AKT pathway, and, promotes endometrial cell invasion and proliferation. These results provide new insights into some of the mechanisms that may underpin the development of deposits of ectopic endometrium.

Reduced expression of aquaporin 9 in tubal ectopic pregnancy.

Previous studies demonstrate significant roles for passive water channels (aquaporins, AQPs) in maintaining water homeostasis in cell membranes of endometrial cells during decidualisation and embryo implantation. However, there is little information regarding the role of AQPs in the human fallopian tube, specifically their role in human tubal ectopic pregnancy. In this study we took tissue samples from the site of implantation of tubal ectopic pregnancy (group 1, N = 30, mean age 32 years, range 23-42) and the corresponding non-implantation site in women undergoing salpingectomy for tubal pregnancy (group 2). Ampullary fallopian tubes during mid-secretory phase were collected as control group (group 3, N = 17, mean age 37 years, range 30-50). Thin sections were prepared and stained with anti-AQP9, and, for estrogen and progesterone receptors in each group. Immunohistochemical studies showed that AQP9 proteins localize in the cytoplasm of epithelial cells of Fallopian tube. Expression of AQP9 was significantly reduced during tubal pregnancy compared to controls (group 1 vs. group 3, P = 0.036; group 2 vs. group 3, P = 0.029), and, this reduced expression was not related to estrogen receptor or progesterone receptor status (group 2, ER vs. AQP9, Pearson r = 0.173, P = 0.361; PR vs. AQP9, Pearson r = 0.124, P = 0.514, respectively). Similarly, there is no correlation between AQP9 and estrogen receptor or progesterone receptor status in the normal group (group 3, ER vs. AQP9, Pearson r = -0.026, P = 0.923; PR vs. AQP9, Pearson r = -0.292, P = 0.255, respectively). Reduced expression of AQP9 in human fallopian tube may contribute to aspects of pathophysiology of tubal ectopic pregnancy.

Reduced migration of Ishikawa cells associated with downregulation of aquaporin-5.

Aquaporin (AQP)-dependent cell migration has broad implications in angiogenesis, tumor metastasis, wound healing, glial scarring and other events requiring cell movement. There are 13 isoforms of AQP (0-12) that have been identified in mammals. It is unclear whether AQP5 plays a role in the development of endometrial cancer. We recently demonstrated that ovarian steroids may affect the expression of AQP5 in the female genital tract. In this study, we considered whether AQP5 may affect cell migration in Ishikawa cells, an adenocarcinoma cell line derived from the endometrium. The results showed that the downregulation of AQP5 results in reduced Ishikawa cell migration. The estrogen (E2) receptor in the promoter of AQP5 mediated the regulation of AQP5 expression in the normal endometrium and endometrial cancer. By contrast, the upregulation of AQP5 by E2 increased cell migration, invasion and adhesion through increased annexin-2, which is responsible for F-actin remodeling and rearrangement. E2 regulates Ishikawa cell migration by regulating the AQP5 expression.

Plumbagin induces ROS-mediated apoptosis in human promyelocytic leukemia cells in vivo.

Plumbagin, a naphtoquinone from the roots of Plumbago zeylanica is known to possess anticancer and anti-bacterial activity. Based on the former finding of our group in vitro demonstrating its effectiveness in human promyelocytic leukemia cells, NB4, in this study we further revealed the mitochondrial pathway involved in plumbagin-induced apoptosis. We also found that the generation of ROS was a critical mediator in plumbagin-induced apoptosis, which would be abrogated completely by antioxidant, NAC. The anticancer effect of plumbagin was investigated in vivo using NB4 tumor xenograft in NOD/SCID mice. The incidence of formation, growth characteristics, body weight and volume of tumors were observed. The histopathologic examination of tumors and organs were made. The results showed that intraperitoneal injection of plumbagin (2mg/kg body weight) daily for 3 weeks resulted to a 64.49% reduction of tumor volume compared with the control. Furthermore, there was no overt manifestation of toxicity such as weight loss, tissue damage and behavior change which appeared in Doxorubicin-treated mice (1mg/kg thrice a week). These results indicate that plumbagin has potential as a novel therapeutic agent for myeloid leukemia.

Immunohistochemical detection of aquaporin expression in eutopic and ectopic endometria from women with endometriomas.

OBJECTIVE: To investigate the expression of aquaporin (AQP) in eutopic and ectopic endometrial tissues from women with endometriomas. DESIGN: Controlled laboratory research. SETTING: Hospital-based unit for gynecology and obstetrics and research laboratories. PATIENT(S): Premenopausal women undergoing laparoscopy for endometriomas. INTERVENTION(S): Endometrial biopsy samples obtained from 70 women with endometriomas. MAIN OUTCOME MEASURE(S): Semiquantitative analysis by immunohistochemistry. RESULT(S): Aquaporins 2, 5, and 8 were mainly located in luminal and glandular epithelia. The frequency of positive immunostaining for aquaporins 2, 5, and 8 decreased in ectopic compared with eutopic endometria. Aquaporins 2, 5, and 8 were found at a low frequency in the endometria in early proliferative phases but at a higher frequency in late proliferative and secretory phases. There were no significant differences in the menstrual cycle of the proliferative phase and secretory phase in the two groups. CONCLUSION(S): Aquaporins 2, 5, and 8 were expressed with greater frequency in eutopic endometrial cells than inectopic endometrial cells, suggesting that eutopic endometrial cells have stronger migration activity than ectopic endometrial cells in women with endometriosis.

Phosphatase of regenerating liver-3: a novel and promising marker in human endometriosis.

OBJECTIVE: To investigate the expression of phosphatase of regenerating liver-3 (PRL-3) in ectopic, eutopic, and normal endometria and explore its relationship with endometriosis. DESIGN: A clinical retrospective and molecular study. SETTING: Department of obstetrics, gynecology, and reproductive medicine. PATIENT(S): One hundred and five women with histopathologically confirmed endometriosis, and 50 women with histopathologically assessed normal endometria. INTERVENTION(S): Immunohistochemical staining and Western blot analysis. MAIN OUTCOME MEASURE(S): Expression of PRL-3 protein. RESULT(S): As shown by the immunohistochemical analysis, PRL-3 was mainly located in the cytoplasm and membrane. The cells that tested positive for PRL-3 were detected in endometriotic tissues that did not occur in eutopic and normal endometria. Statistical analysis indicated that the expression of PRL-3 was closely associated with the clinical stages and recurrence of endometriosis. CONCLUSION(S): Expression of PRL-3 is related to the clinical stages and recurrence of endometriosis, which provides use with a novel marker and promising target in the treatment of human endometriosis.

Hysteroscopic septum resection of complete septate uterus with cervical duplication, sparing the double cervix in patients with recurrent spontaneous abortions or infertility.

OBJECTIVE: To evaluate the safety and efficacy of hysteroscopic septum resection of the complete septate uterus with cervical duplication in patients with recurrent spontaneous abortions or infertility. DESIGN: Prospective consecutive clinical study. SETTING: University hospital for obstetrics, gynecology, and reproductive medicine. SUBJECT(S): Twenty-five patients with a complete septate uterus, cervical duplication, and history of recurrent spontaneous abortions (13 cases) or infertility (12 cases) were included. INTERVENTION(S): Hysteroscopic septum resection and sparing the double cervix using a bougie served as a means of orientation and blockage of internal cervical os. MAIN OUTCOME MEASURE(S): Intraoperative and postoperative complications, and postoperative anatomic identification of the uterus. RESULT(S): No intraoperative and postoperative complications were encountered. Postoperatively, ultrasound revealed minor fundal septal remnants in 7 (31.8%) of 22 patients receiving the ultrasound detection, and no residual septum in the other 15 cases (68.2%). CONCLUSION(S): By using a bougie technique, hysteroscopic correction of complete septate uterus with cervical duplication and sparing the double cervix can be performed successfully.

Methotrexate therapy for cesarean section scar pregnancy with and without suction curettage.

OBJECTIVE: To compare the clinical effects in women with cesarean scar pregnancy (CSP) who were treated with either methotrexate (MTX) regimen only or MTX regimen followed by dilation and curettage (D&C). DESIGN: Prospective consecutive clinical cohort study. SETTING: University hospital for obstetrics, gynecology, and reproductive medicine. SUBJECT(S): Seventy-one cases of CSP. INTERVENTION(S): The subjects were treated with either MTX only (MTX group, 21 cases) or MTX followed by D&C (combined therapy group, 50 cases). MAIN OUTCOME MEASURE(S): Success rates, hysterectomy rates, and time to resolution of serum beta-hCG and the CSP mass were compared between the two groups. RESULT(S): Compared with the MTX group, the combined therapy group had a shorter time to resolution of the CSP mass and serum beta-hCG. There was no significant difference between the MTX and combined therapy groups regarding success rates (76.2% vs. 90.0%, respectively) and hysterectomy rates (19.0% vs. 8.0%, respectively). CONCLUSION(S): Both therapies could treat the majority of CSP patients successfully, but the combined therapy resulted in a shorter time of therapy and indicated a more favorable effect.

Locally elevated leukemia inhibitory factor in the inflamed fallopian tube resembles that found in tubal pregnancy.

OBJECTIVE: To investigate the association between the expressions of leukemia inhibitory factor and the occurrence of tubal pregnancy. DESIGN: Prospective observational study. SETTING: University-based obstetrics and gynecology hospital. PATIENT(S): Thirty women undergoing salpingectomy for tubal pregnancy and 30 nonpregnant patients with benign uterine or appendix disease. INTERVENTION(S): Oviduct tissues with ectopic gestation were separated into implantation sites and nonimplantation sites. Samples of ampullary fallopian tubes during midsecretory phase were collected as control groups. Immunohistochemical and Western blot analysis were performed. MAIN OUTCOME MEASURE(S): The differences of leukemia inhibitory factor expression between the implantation and nonimplantation sites of oviduct tissues and the normal and chronically inflamed fallopian tubes. RESULT(S): The expression of leukemia inhibitory factor in the implantation group is significantly higher than that in the nonimplantation group or in the normal group. A statistically significant difference was also found for leukemia inhibitory factor between the chronic inflammation group and the normal group by Western blot analysis but no difference between the chronic inflammation group and the implantation group or the nonimplantation group. CONCLUSION(S): Leukemia inhibitory factor might be one of the reasons that cause patients with salpingitis to be more susceptible to tubal pregnancy and might be involved in the implantation process of tubal pregnancy.

Single large cystic adenomyoma of the uterus after cornual pregnancy and curettage.

A case of single large cystic adenomyoma of the uterus (anechoic area 1.6 cm in diameter) was diagnosed by surgery and histopathologic analysis more than 3 years after a transcervical curettage for an early right-cornual pregnancy.