Upregulation of miR-181a-5p and miR-125b-2-3p in the Maternal Circulation of Fetuses with Rh-Negative Hemolytic Disease of the Fetus and Newborn Could Be Related to Dysfunction of Placental Function.

The transplacental transfer of maternal antibodies to the fetus is a critical mechanism for infant protection and perinatal disease. Hemolytic disease of the fetus and newborn (HDFN) is a representative fetal disease caused by transplacental transfer of maternal IgG antibodies. However, it is unclear whether placental-related miRNAs are expressed in Rh-HDFN. Through the investigation of the miR-181a-5p and miR-125b-2-3p levels in maternal plasma using qPCR, we found that both miR-181a-5p and miR-125b-2-3p were highly expressed in maternal plasma of newborns with Rh-HDFN compared with healthy controls, indicating the potential roles of these two miRNAs in Rh-HDFN. To demonstrate whether dysregulation of miR-125b-2-3p and miR-181a-5p contributes to Rh-HDFN development, we analyze the placental miRNA-/mRNA sequencing data (GSE73714) using weighted gene coexpression network analysis (WGCNA), miRNA target predictive databases, and DAVID (Database for Annotation, Visualization, and Integrated Discovery). The results showed that miR-125b-2-3p and miR-181a-5p could regulate several biological processes including cytoplasmic microtubule organization and angiogenesis. Moreover, core promoter sequence-specific DNA binding and protein binding were highly enriched molecular functions, indicating the potential roles of transcriptional regulation. Further pathway enrichment showed that miR-181a-5p and miR-125b-2-3p could regulate several biological pathways that were closely related to placental function, including the FoxO signaling pathway, focal adhesion, mTOR signaling pathway, and central carbon metabolism in cancer. In conclusion, the present results first revealed miRNA expression in the maternal circulation of newborns with Rh-HDFN, which could be caused by dysfunction of the placenta.

Learning curve analysis of transvaginal natural orifice transluminal endoscopic hysterectomy combined under the standard operating procedure.

OBJECTIVE: To investigate the learning curve of transvaginal natural orifice transluminal endoscopic hysterectomy (tVNOTEH) when using a standard operating procedure (SOP). METHODS: Seventy-nine patients were treated with tVNOTEH by a single surgeon. The SOP for tVNOTEH was created after the first eight cases. Patients' perioperative data were retrospectively reviewed. Operative time (OT) was regarded as a replaceable marker for surgical competency. The learning curve was drawn using the cumulative sum method. RESULTS: All patients completed surgeries without switching to other surgical paths. The overall mean OT was 90.23 ± 29.85 min. Four unique phases of the learning curve were identified: phase I (the exploring stage over eight cases), phase II (after adopting the SOP, acquirement of competence over 20 cases), phase III (post-learning of 19 cases, in which more difficult cases were introduced), phase IV (more adept at tVNOTEH), with OT 113.75 ± 43.07 min, 82.50 ± 25.88 min, 101.05 ± 27.83 min, 82.75 ± 25.53 min, respectively. No significant differences were found apart from OT, uterine size, and disease types. CONCLUSION: Our data demonstrated four distinct phases of the learning curve of tVNOTEH. For an experienced surgeon, surgical competence in tVNOTEH can be grasped after eight cases. With SOP, surgical competence could be rapidly acquired.

Irreversible electroporation-mediated shRNA knockdown of the HPV18 E6 gene suppresses cervical cancer growth in vitro and in vivo.

Irreversible electroporation (IRE) is a physical, non-thermal cancer therapy, which leads to cell death via permanent membrane permeability. This differs from reversible electroporation (RE), which is used to transfer macromolecules into target cells via transient membrane permeability. Given the electrical impedance of the electric field, RE co-exists outside the central zone of IRE ablation. In the present study, the feasibility of using IRE at a therapeutic dose to mediate short hairpin RNA (shRNA) knockdown of human papillomavirus (HPV)18 E6 in HeLa cervical cancer cells in vitro and in vivo was investigated. Experimental results indicated that the HeLa cells survived the combined treatment with IRE and shRNA plasmid transfection. Additionally, residual tumor tissue in a nude mouse model demonstrated green fluorescence. Subsequent studies showed that the combined treatment inhibited the growth of HeLa cells and tumors. Western blotting analysis showed marked changes in the growth-associated proteins between the combined treatment group and the control. It was concluded that a therapeutic dose of IRE was able to mediate the transfection of HPV18 E6 shRNA into HeLa cervical cancer cells in vitro and in vivo. This combined treatment strategy has promising implications in cancer treatment for the ablation of tumors, and in eliminating microscopic residual tumor tissue.

Effects of irreversible electroporation on cervical cancer cell lines in vitro.

The effects of irreversible electroporation (IRE) on the proliferation, migration, invasion and adhesion of human cervical cancer cell lines HeLa and SiHa were investigated in the present study. HeLa and SiHa cells were divided into a treatment group and control group. The treatment group cells were exposed to electric pulses at 16 pulses, 1 Hz frequency for 100 µsec with 1,000 V/cm strength. Cellular proliferation was determined 24 h after treatment using a Cell Counting Kit­8 (CCK­8) assay and carboxyfluorescein diacetate­succinimidyl ester (CFDA­SE) labeling assay. The different phases of the cell cycle were detected using flow cytometry. Wound healing, Transwell invasion and Matrigel adhesion assays were performed to evaluate the migration, invasion and adhesion abilities of HeLa and SiHa cells. The expression levels of metastasis­associated proteins were determined by western blot analysis. CCK­8 and CFSE labeling assays indicated that the inhibition of cellular proliferation occurs in cells treated with IRE. Additionally, cell cycle progression was arrested at the G1/S phase. A western blot analysis indicated that the expression levels of p53 and p21 proteins were increased, whilst those of cyclin­dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) proteins were decreased. However, wound healing, invasion and adhesion assays indicated that cellular migration, invasion and adhesion abilities were not significantly altered following exposure to IRE. IRE was not observed to promote the migration, invasion or adhesion capacity of HeLa and SiHa cells. However, IRE may inhibit the capacity of cells to proliferate and their progression through the cell cycle in vitro. Preliminary evidence suggests that the underlying mechanism involves increased expression levels of p53 and p21 and decreased expression levels of CDK2 and PCNA.

Diagnosis and microecological characteristics of aerobic vaginitis in outpatients based on preformed enzymes.

OBJECTIVE: Aerobic vaginitis (AV) is a recently proposed term for genital tract infection in women. The diagnosis of AV is mainly based on descriptive diagnostic criteria proposed by Donders and co-workers. The objective of this study is to report AV prevalence in southwest China using an objective assay kit based on preformed enzymes and also to determine its characteristics. MATERIALS AND METHODS: A total of 1948 outpatients were enrolled and tested by a commercial diagnostic kit to investigate the AV prevalence and characteristics in southwestern China. The study mainly examined the vaginal ecosystem, age distribution, Lactobacillus amount, and changes in pH. Differences within groups were analyzed by Wilcoxon two-sample test. RESULTS: The AV detection rate is 15.40%. The AV patients were usually seen in the sexually active age group of 20-30 years, followed by those in the age group of 30-40 years. The vaginal ecosystems of all the patients studied were absolutely abnormal, and diagnosed to have a combined infection [aerobic vaginitis (AV) + bacterial vaginitis (BV) 61.33%; 184/300]. Aerobic bacteria, especially Staphylococcus aureus and Escherichia coli, were predominantly found in the vaginal samples of these women. CONCLUSION: AV is a common type of genital infection in southwestern China and is characterized by sexually active age and combined infection predominated by the AV and BV type.

Picosecond pulsed electric fields induce apoptosis in a cervical cancer xenograft.

The aim of the present study was to evaluate the efficacy of picosecond pulsed electric fields (psPEF) on a cervical cancer xenograft. Human cervical cancer xenografts were established in nude mice by transplantation of HeLa cells, and the tumors were then treated with psPEF. The histological changes were observed by hematoxylin­eosin staining and transmission electron microscopy. The rate of tumor cell apoptosis was determined using a terminal deoxynucleotidyl­transferase­mediated dUTP nick end labeling assay. The mitochondrial transmembrane potential of the tumor cells was detected by laser scanning confocal microscopy, and the activity of caspase­3, ­8, ­9 and ­12 was determined. The inhibitory rate seven days post­psPEF treatment was also calculated. The results showed that exposure to psPEF led to an increased rate of apoptosis, collapse of mitochondrial transmembrane potential, and activation of caspases. The inhibitory rate was 9.11% at day 7. The results of the present study indicate that psPEF may induce apoptosis in a cervical cancer xenograft through the endoplasmic reticulum stress and caspase­dependent signaling pathways.

Intense picosecond pulsed electric fields inhibit proliferation and induce apoptosis of HeLa cells.

A picosecond pulsed electric field (psPEF) is a localized physical therapy for tumors that has been developed in recent years, and that may in the future be utilized as a targeted non­invasive treatment. However, there are limited studies regarding the biological effects of psPEF on cells. Electric field amplitude and pulse number are the main parameters of psPEF that influence its biological effects. In this study, we exposed HeLa cells to a psPEF with a variety of electric field amplitudes, from 100 to 600 kV/cm, and various pulse numbers, from 1,000 to 3,000. An MTT assay was used to detect the growth inhibition, while flow cytometry was used to determine the occurrence of apoptosis and the cell cycle of the HeLa cells following treatment. The morphological changes during cell apoptosis were observed using transmission electron microscopy (TEM). The results demonstrated that the cell growth inhibition rate gradually increased, in correlation with the increasing electric field amplitude and pulse number, and achieved a plateau of maximum cell inhibition 12 h following the pulses. In addition, typical characteristics of HeLa cell apoptosis in the experimental groups were observed by TEM. The results demonstrated that the rate of apoptosis in the experimental groups was significantly elevated in comparison with the untreated group. In the treatment groups, the rate of apoptosis was greater in the higher amplitude groups than in the lower amplitude groups. The same results were obtained when the variable was the pulse number. Flow cytometric analysis indicated that the cell cycle of the HeLa cells was arrested at the G2/M phase following psPEF treatment. Overall, our results indicated that psPEF inhibited cell proliferation and induced cell apoptosis, and that these effects occurred in a dose-dependent manner. In addition, the results demonstrated that the growth of the HeLa cells was arrested at the G2/M phase following treatment. This study may provide a foundation for further in vivo experiments, and for the potential clinical application of psPEF in the treatment of cervical cancer.

Picosecond pulsed electric fields induce apoptosis in HeLa cells via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

The non-invasive treatment of tumors with preserved fertility holds great promise. The application of pulsed electric field (PEF) is a new biomedical engineering technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely; however, research of the biological effects of psPEF on cells is limited. Electric theory predicts that when the pulse duration decreases to nanoseconds and picoseconds, it will mainly affect organelles and lead to intracellular electromanipulations. Previous studies have shown that psPEF targets the mitochondria and induces apoptosis through a mitochondrial-mediated pathway in HeLa cells. The endoplasmic reticulum is also involved in the intrinsic pathways of apoptosis. In the present study, HeLa cells were exposed to psPEF to investigate the underlying mechanisms of apoptosis. MTT assay demonstrated that psPEF displayed strong growth inhibitory effects on HeLa cells. Treatment with psPEF led to marked cell apoptosis and cell cycle arrest at the G2/M phase. In addition, psPEF affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). These changes were accompanied by the elevation of intracellular Ca2+ concentrations. Furthermore, the activation of caspase-12, -9 and -3, led to the release of cytochrome c, as well as the upregulation of Bax and the downregulation of Bcl-2, as observed in the HeLa cells. Taken together, these data suggest that psPEF is an efficient apoptosis-inducing agent for HeLa cells, which exerts its effects, at least partially, via the endoplasmic reticulum stress and caspase-dependent signaling pathways.

Feasibility and effect of ultrasound microbubble-mediated wild-type p53 gene transfection of HeLa cells.

Gene therapy holds great promise for the treatment of diseases. The key problem of gene therapy is the choice of an effective vector. Ultrasound-mediated microbubble technique (UMMT) has already shown promising applications in numerous types of tumors apart from cervical carcinoma. In the present study, according to the results of an MTT assay, we initially chose an ultrasound intensity of 0.5 W/cm(2), an ultrasound exposure time of 30 sec and a microbubble concentration of 10% as the optimum experimental condition for wtp53 plasmid transfection into HeLa cells. To further investigate the transfection efficiency of ultrasound combined with microbubbles, RT-PCR analysis was used to examine the mRNA level of p53. The transfection efficiency in the plasmid plus microbubbles and ultrasound group was significantly higher than that of the other groups. Following transfection of the wtp53 gene, flow cytometric analysis showed that the cell cycle of HeLa cells was arrested in the G1 phase. The results of the present study suggest that UMMT, a new gene delivery system, increases the transfection efficiency of the wtp53 gene. Moreover, the growth of HeLa cells was arrested by introducing wtp53. This study may afford a new trend for the gene therapy of cervical carcinoma.

Intense picosecond pulsed electric fields induce apoptosis through a mitochondrial-mediated pathway in HeLa cells.

The application of pulsed electric fields (PEF) is emerging as a new technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely, but the research of the biological effects of psPEF on cells is limited. Electric theory predicts that intense psPEF will target mitochondria and lead to changes in transmembrane potential, therefore, it is hypothesized that it can induce mitochondrial-mediated apoptosis. HeLa cells were exposed to psPEF in this study to investigate this hypothesis. MTT assay demonstrated that intense psPEF significantly inhibited the proliferation of HeLa cells in a dose-dependent manner. Typical characteristics of apoptosis in HeLa cells were observed, using transmission electron microscopy. Loss of mitochondrial transmembrane potential was explored using laser scanning confocal microscopy with Rhodamine-123 (Rh123) staining. Furthermore, the mitochondrial apoptotic events were also confirmed by western blot analysis for the release of cytochrome C and apoptosis-inducing factor from mitochondria into the cytosol. In addition, activation of caspase-3, caspase-9, upregulation of Bax, p53 and downregulation of Bcl-2 were observed in HeLa cells also indicating apoptosis. Taken together, these results demonstrate that intense psPEF induce cell apoptosis through a mitochondrial-mediated pathway.

[Evaluation the efficacy and safety of estradiol and drospirenone tablets in the treatment of menopausal symptoms among postmenopausal Chinese healthy women:a randomized, multi-center, double-blind, placebo-controlled clinical study].

OBJECTIVE: To study the efficacy and safety of estradiol and drospirenone tablets (Angeliq) in treatment of menopausal symptoms among postmenopausal Chinese healthy women. METHODS: Total 244 postmenopausal Chinese healthy women who had moderate to severe hot flushes were randomly assigned into estradiol and drospirenone (observation group, n = 183) or placebo group (n = 61) by the ratio of 3:1 for 16 weeks in this randomized multi-center double-blind placebo-controlled study. During the trial, the follow-up visits were conducted at week 4, 8, 12, 16 of treatment and 2 weeks after treatment respectively. Height, weight, vital signs, hot flushes, other relevant menopausal symptoms and vaginal bleeding were observed in each follow-up visit, while the clinical global impression scale was assessed at 16 weeks as well. RESULTS: It showed that hot flushes were reduced significantly more in observation group than that in placebo group (P < 0.01), although both treatments were effective. The absolute values of mean severity index of total hot flushes decreased by -0.6 ± 0.5 in observation group and -0.4 ± 0.4 in placebo group from baseline respectively, which reached significant difference (P < 0.05). However, the absolute values of mean severity index of moderate to severe hot flushes decreased by -0.6 ± 0.8 in observation group and -0.3 ± 0.6 in placebo group from baseline respectively, which had no significant difference (P > 0.05). After 16 weeks treatment, it also showed that estradiol and drospirenone had significant better efficacy than placebo on moderate to severe sweating, vaginal dryness and clinical global impression scale (P < 0.01). During the trial, blood pressure in observation group was stable. The rate of vaginal bleeding in observation group was higher than that in the placebo group, especially during the week 4 to week 8 when 48.9% (87/178) in observation group and 10.7% (6/56) in placebo group of patients bled. Although the cumulative amenorrhea rate of observation group was lower than that of placebo group in each cycle (28 days), it increased gradually along with duration of the treatment. The commonest adverse event in observation group was breast tenderness which accounted for 12.0% (22/183). The level of serum potassium was in the normal range in observation group mostly.Meanwhile, the other adverse events rate was low. Serious adverse events reported in this trial were assessed as not study drug related or as unlikely study drug related. CONCLUSION: Estradiol and drospirenone tablets which could effectively alleviate menopausal symptoms in postmenopausal Chinese healthy women is a novel hormone replacement therapy regimen with high safety and efficacy.

[Recent advances on physical ablation for tumor].

Physical ablation is a new kind of tumor treatment which directly acts on local solid tumors to eradicate or destroy tumor tissues by use of various advanced physical techniques. Physical ablation can be classified by physical characteristics as thermal ablate therapy (TAT), cryoablation and electrical ablation. Recent studies and technical trend of these three physical ablation treatments are reviewed in this paper.

[Efficacy and safety of drospirenone-ethinylestradiol on contraception in healthy Chinese women: a multicenter randomized controlled trial].

OBJECTIVE: To evaluate the contraception efficacy, mode of bleeding, side effects and other positive effects of drospirenone-ethinylestradiol (Yasmin) in healthy Chinese women. METHODS: This was a multicenter, randomized, control study of 768 healthy Chinese women who consulted about contraception. The subjects were randomized into Yasmin group (30 microg ethinylestradiol plus 3 mg drospirenone, 573 cases) or desogestrel group (30 microg ethinylestradiol plus 150 microg desogestrel, 195 cases) with the ratio of 3:1. Each individual was treated for 13 cycles. Further visits were required at cycle 4, cycle 7, cycle 10 and cycle 13 of treatment Weight, height, body mass index were evaluated at each visit. The menstrual distress questionnaire (MDQ) was given to the women at baseline, visit 3 (cycle 7) and visit 5 (after cycle 13). RESULTS: The values of basal features were similar between two groups (P > 0.05). The Pearl index (method failure) of Yasmin was 0. 208/hundred women year which was lower than that of desogestrel (0. 601/hundred women year). The mode of bleeding was similar between two groups after trial without showing any significant difference. According to MDQ subscale, the improvement of water retention and increasing appetite during inter-menstrual period and water retention and general well-being during menstrual period in the Yasmin group (-0.297, -0.057, 0.033, 0.150 respectively) was more obvious than that in the desogestrel group (-0.108, 0.023, 0.231, -0.023 respectively) with a significant difference (P < 0.05). Some other values which improved in both two groups, especially the improvement of breast tenderness and pain and skin abnormality in Yasmin group (18.0%, 89/494; 12.6%, 62/494) was more distinct than that in desogestrel group (11.3%, 19/168; 5.4%, 9/168). The mean weight increased in desogestrel group (0.57 kg) while it decreased in Yasmin group (-0.28 kg) with a significant difference (P < 0.01). CONCLUSIONS: Both Yasmin and desogestrel have good efficacy on contraception and similar modes of menstrual bleeding. Yasmin is better than desogestrel in terms of weight control and premenstrual syndrome of oral contraceptive.

[Research progress of nanosecond pulsed electric field applied to intracellular electromanipulation].

In recent years, many experts have done some researches on experiment and mechanism of intracellular electromanipulation (IEM) under nanosecond pulsed electric field (nsPEF). The experiment results have shown that nsPEF could not induce electroporation of cell membrane, but could induce intracellular effects such as apoptosis, calcium release, enhancement of gene expression, and fragmentation of DNA and chromosome. In order to account for the phenomenon, researchers believe that when the pulse width of the pulsed electric field is larger than the charging time of plasma membrane, the pulsed electric field mainly targets on the outer membrane of cell; and that the effect of the pulsed electric field on nucleus and nuclear membrane increases with the decrease of the pulse width. It is also believed that the effect of electroporation changes from the outer membrane to intracellular electromanipulation when the pulse width decreases to a value being smaller than the charging time of plasma membrane.

[Treatment of nonneoplastic epithelial disorders of skin and mucosa of vulva with focused ultrasound].

OBJECTIVE: To explore the clinical efficacy of focused ultrasound for patients with white lesions of the vulva, as well as its safety and feasibility. METHODS: Clinical data of 941 patients with white lesions of the vulva treated with focused ultrasound from June 2003 to December 2005 were retrospectively reviewed. The mean age of the patients was 40.8 years (18 - 70 years) and the median course of the disease was 6.2 years (3 months - 45 years). Meanwhile, pathological diagnosis was performed in all the patients before treatment, in which 498 cases were squamous hyperplasia, 342 cases were lichen sclerosus and 101 cases were lichen sclerosus with squamous hyperplasia. Patients were followed up and therapeutic effects of focused ultrasound was evaluated at 6 and 12 months after the treatment, respectively. The symptoms of pruritus in the vulva and the changes in the color and elasticity of the vulvar lesions were observed. RESULTS: Of all the patients, 900 were followed up after the treatment, and the ratio of effectiveness was 94.9%. Only 46 patients (5.1%) had no response to the therapy. Of the effective patients, 434 cases were completely cured (48.2%), and 420 cases were improved (46.7%). Pruritus of vulva recurred in 101 patients (11.2%) one year after treatment; however, these patients still had a response to the second or third treatment. CONCLUSIONS: Focused ultrasound therapy is a highly effective instrument in treatment of white lesions of the vulva. It can not only relieve the symptoms of itching, but is also helpful in recovering the color and elasticity of the vulva.