This study proposes an image segmentation method based on bottleneck detection and watershed algorithm to solve the problem of overlapping cervical cell image. First, we use polygon approximation to get all feature points on the cell contour and then use bottleneck detection and ellipse fitting to obtain the correct split point pairs. Therefore, the approximate range of the overlapping region was determined. The watershed algorithm was used to obtain the internal boundary information for the gradient image of the region. Finally, the segmentation results of the overlapped cells were obtained by superimposing with the outer contour. The experimental results show that this algorithm can segment the contour of a single cell from the overlapping cervical cell images with good accuracy and integrity. The segmentation result is close to that of doctors' manual marking, and the segmentation result is better than other existing algorithms.
Algorithms ; Cervix Uteri/cytology* ; Female ; Humans ; Image Processing, Computer-Assisted

BACKGROUNDCervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum (FBS). However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro.

METHODSNormal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity.

RESULTSOur results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%.

CONCLUSIONA novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.

Cell Culture Techniques ; methods ; Cervix Uteri ; cytology ; Epithelial Cells ; cytology ; Female ; Humans ; Keratinocytes ; cytology

OBJECTIVETo study the presence of trophoblast cells in the lower uterus in early pregnancy, identify fetal cells by immunocytochemistry, and determine the best timing for sample collection.

METHODSSamples from healthy gravida in early pregnancy were divided into HE group (I) and immunocytochemistry group (II), and those from healthy nonpregnant women were also divided into HE group (III) and immunocytochemistry group (IV). Four different methods (usage of a cotton swab, aspiration of the cervical mucus, lavage of the endocervical canal, and lavage of the intrauterine cavity) were employed for collecting the samples, which were tested with HE staining or immunocytochemistry, and the presence of fetal cells was observed under optical microscope.

RESULTS AND CONCLUSIONFetal cells were detected in the genital tract of the gravida in early pregnancy. Utilization of the lavage of the endocervical canal or the lavage of the intrauterine cavity allowed greater amount of fetal cell acquisition, and sampling of the fetal cells between 50 days and 79 days of gestational age yielded optimal results. These sampling methods may provide safe and effective means for prenatal diagnosis with minimal invasiveness.

Adult ; Cervix Uteri ; chemistry ; cytology ; Female ; Fetus ; chemistry ; cytology ; Humans ; Immunohistochemistry ; Pregnancy ; Pregnancy Trimester, First ; Prenatal Diagnosis ; Time Factors ; Trophoblasts ; chemistry ; cytology

OBJECTIVETo detect the human papillomavirus (HPV) infectious condition in women with abnormal cytology and evaluate its values in the screening of high grade cervical intraepithelial lesion.

METHOD101 patients who underwent thinprep cell test(TCT) with abnormal cervical cytology were selected to undergo HPV test, all subjects also received tissue biopsy at the same time.

RESULTS(1) Among the 101 patients,the incidence rates of high risk HPV infection of those with ASCUS, LSIL, HSIL and squamous cell carcinoma were 84.2%, 88.6%, 100.0% and 2/2 respectively. (2) Among the patients with abnormal cytology,the number of patients with pathologically confirmed results of CIN I and CIN II or worse were 20 and 81, the incidence rates of high risk HPV infection of those with CIN I and CIN II or worse were 60.0% and 97.5% respectively. (3) In the ASCUS group, the incidence rates of CIN II or worse with high risk HPV infection were 87.5% and the incidence rates of CIN II or worse without high risk HPV infection were 16.7%. (4) The prevalence of high risk HPV types from highest to lowest order were follow: HPV16 (39.6%), 58 (17.8%), 52 (16.8%), 18 (9.9%), 33 (9.9%).

CONCLUSIONSThe infection rate of high risk HPV was positively correlated with the levels of cervical lesions. HPV test is a good triage approach for the patients with ASCUS. HPV16, 58, 52, 18, 33 are the most common in the patients of cervical lesions.

Adult ; Alphapapillomavirus ; genetics ; isolation & purification ; Cervix Uteri ; cytology ; pathology ; virology ; Female ; Humans ; Middle Aged ; Papillomavirus Infections ; diagnosis ; pathology ; virology ; Young Adult

OBJECTIVETo study the molecular biological effects of Guilin Watermelon Frost (GWF) on the mRNA expressions of basic fibroblast growth factor (bFGF) in patients with uterine uterine cervical columnar ectopy.

METHODSOne hundred and sixty patients with uterine cervical columnar ectopy were assigned to two groups by the random digit table. Patients in the treatment group were treated with local spray of GWF, while those in the control group were local applied with bFGF-collagen sponge. The mRNA expressions of bFGF of the uterine tissue were detected in the two groups before and after treatment using RT-PCR.

RESULTSBefore treatment the mRNA expression of bFGF in the uterine cervical columnar ectopy was 0.55 +/- 0.10 in the treatment group and 0.58 +/- 0.13 in the control group, without insignificant difference (P > 0.05). After treatment it significantly increased in the two groups, being 0.82 +/- 0.17 and 0.78 +/- 0.15 respectively, showing statistical difference from before treatment (P < 0.01). But no statistical difference existed between the two groups after treatment (P > 0.05).

CONCLUSIONGWF showed enhancement on the mRNA expressions of bFGF in patients with uterine cervical columnar ectopy.

Adult ; Cervix Uteri ; cytology ; Citrullus ; Drugs, Chinese Herbal ; pharmacology ; Epithelium ; drug effects ; Female ; Fibroblast Growth Factor 2 ; genetics ; metabolism ; Humans ; RNA, Messenger ; genetics

The inactivation of p53 and p105RB by viral proteins or by mutations plays a key role in the oncogenesis of cervical carcinoma. The E6 and E7 proteins of HPV type 16 can bind to p53 and p105RB tumor suppressor gene products, respectively. In the present study, we tested a simple in vivo model that could explain the interactions between HPV E6 oncoprotein and p53 tumor suppressor protein. Our results showed that the life span of normal cervical epithelial cells was increased up to 4.5 times when transfected with expression vector containing E6/E7 ORF of HPV type 16. However, these cells did not divide after second crisis. Therefore, we employed an established human epidermal keratinocytes, RHEK-1. When transfected with an expression vector containing E6 ORF of HPV type 16, RHEK-1 cells showed anchorage independent growth character. When RHEK-E6 cells were transfected with wild type p53 expression vector, the growth rate of the RHEK-E6 cells was diminished. After 48 hours of transfection, many cells showed apoptotic signal but no more apoptotic signal was observed thereafter. These results suggested that the overexpression of the wild type p53 could overcome the dysfunction of the p53 on the cell cycle regulation imposed by E6 protein although not being of physiological condition.
Animal ; Base Sequence ; Cells, Cultured ; Cervix Uteri/*cytology ; Female ; Genes, p53/*physiology ; Human ; Keratinocytes/*cytology ; Mice ; Molecular Sequence Data ; Oncogene Proteins, Viral/genetics/*physiology ; Papillomavirus, Human/*genetics ; Support, Non-U.S. Gov't ; Transfection

BACKGROUNDEndocervical epithelial cells play early roles in the defense of upper female genital tract to pathogens. Toll-like receptors (TLRs) and human defensins (HD) have recently been identified as fundamental components of the innate immune responses to bacterial pathogens. We aimed to use in vitro model of human primary endocervical epithelial cells (HPECs) to investigate their roles in innate immune response of the endocervix.

METHODSTLR4 expression and distribution in HPECs and endocervix were investigated by immunofluorescence (IF). Cultured HPECs were divided into lipopolysaccharide (LPS) group which were treated by LPS for 0, 24 and 48 hours, and control group without treatment. At each time point, the levels of HD5, IL-6 and TNF-alpha in supernants were determined by ELISA. TLR4 and HD5 expressions of cells were detected by Western blotting simultaneously. HD5 expression pattern was also compared between the HeLa cell line and HPECs.

RESULTSEndocervix tissue surface and HPECs expressed TLR4. After incubated with LPS, HPECs expressed significantly higher levels of TLR4 than control group, especially after 24 hours (P < 0.01), however decreased after 48 hours with a similar level of TLR4 expression compared with control group. LPS could upregulate the secretion of HD5, IL-6 and TNF-alpha in a time-dependent manner (24 hours: P < 0.05; 48 hours: P < 0.01, compared with control group). Intracellular HD5 expression levels decreased over time. HD5 expression patterns in HPECs were different from HeLa cell line.

CONCLUSIONSTo respond to LPS stimulation, HPECs may function in the mucosal immune defense through TLR4 activation and HD5 secretion. HPEC is considered a significant model for immunological study.

Blotting, Western ; Cells, Cultured ; Cervix Uteri ; cytology ; Enzyme-Linked Immunospot Assay ; Epithelial Cells ; metabolism ; Female ; Fluorescent Antibody Technique ; HeLa Cells ; Humans ; Interleukin-6 ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; alpha-Defensins ; genetics ; metabolism

OBJECTIVETo study the effect of podophyllotoxin nanostructured lipid carriers (POD-NLC) on immortalized human cervical epithelial cells (H8) infected with HPV in vitro.

METHODSPOD-NLC was prepared by emulsion evaporation method and characterized using transmission electron microscopy, Zetasizer analyzer and high-performance liquid chromatography (HPLC). H8 cells were treated with different concentrations (0.0001-1 µg/ml) of POD-NLC, free POD, or blank nanostructured lipid carriers (NLC), and the cell proliferation was assessed using MTT assay to evaluate the cytotoxic effects. The changes of cell morphology were observed using fluorescence microscopy, and the cell cycle changes and cell apoptosis were analyzed using flow cytometry.

RESULTSPOD-NLC showed a spherical or elliptical shape with good stability in vitro. The average particle size of POD-NLC was 85.6∓10.25 nm, with a Zeta potential of 26.2∓4.1 mV and entrapment efficiency of POD of (88.56∓3.1)%. POD-NLC caused a significant inhibition of H8 cell proliferation in a concentration- and time-dependent manner. At an equivalent concentration, POD-NLC produced a stronger inhibitory effect on cell proliferation than POD. The inhibition rate of H8 cells after a 48-h exposure to POD-NLC and POD reached 95.8% and 65.6%, respectively, and at the highest concentration of 1 µg/ml, the IC(50) of POD-NLC and POD was 0.015 µg/ml and 0.13 µg/ml, respectively. Blank NLC did not obviously affect the proliferation of H8 cells. POD-NLC and POD both caused obvious increases in G(2)/M phase cell percentages and induced typical apoptotic changes of the cells, and their effects were comparable (P>0.05).

CONCLUSIONCompared with POD, POD-NLC has more potent effect in inhibiting H8 cell proliferation and inducing cell apoptosis, suggesting its potential in the treatment of cervical HPV infection.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cervix Uteri ; cytology ; Drug Carriers ; pharmacology ; Epithelial Cells ; drug effects ; Female ; HIV Infections ; pathology ; Humans ; Lipids ; Nanostructures ; Particle Size ; Podophyllotoxin ; chemical synthesis ; pharmacology

OBJECTIVETo observe the effect of Cuichan Zhusheng Decoction (CZD) on cervical maturation factors.

METHODSNinety women with full-term pregnancy and indication for labor inducing were assigned to three groups equally. The treated group was treated by water decoction of CZD, one dose (300 mL) daily, taken orally in the morning 30 min before breakfast, for successive 3 days, the administration would be discontinued if uterine contraction occurred for over 3 times/hour in the course. The control group was treated with pitocin by adding 1 U into 500 mL 5% glucose for intravenous dripping in 6 h, once every day for 3 successive days. The blank group was treated by placebo of CZD, administrated in same way as that in the treated group. The length and width of cervix and diameter of neck tube in all the women were measured on the very day of medication and 72 h later or parturient time by vaginal B-ultrasonography, and the cervical maturation degree was scored referring to the clinical Bishop scale. In the experimental study, the cervical tension of pregnant rats was measured with an in vitro cervical tension-meter, rats' cervical tissues were taken for pathologic examination to observe its morphological change.

RESULTSThe total effective rate for promoting cervical maturation was 96.67% in the treated group and 83.33% in the control group. It was significantly superior in the treated group to that in the control group and the blank group (P<0.05). Moreover, the cervical score in the treated group was higher in comparing with that in the blank group showing statistical significance (P<0.05). Animal experiments displayed that after medication, the cervical tissue of rat loosened with significantly lessened, swollen, convoluted and ruptured collagen fiber, showing sparse disorderly lined-up reticular status. Degradation of collagen fiber, vascular dilatation and congestion with massive amount of inflammatory cells infiltration, increased matrix components, and many leucocyte and fibroblast in the stroma could be seen.

CONCLUSIONCZD can change the morphorlogic structure of cervical tissue, decrease cervical tension, so as to promote the cervical maturation and induce labor.

Adult ; Animals ; Biomechanical Phenomena ; Cervical Ripening ; drug effects ; Cervix Uteri ; cytology ; drug effects ; metabolism ; physiology ; Collagen ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Labor, Induced ; Pregnancy ; Rats ; Young Adult

OBJECTIVETo confirm the relations between the expression of cyclin E, p16ink4, ki67 and HPV16/18 infection using cervical exfoliated cells, and evaluate the usefulness of cyclin E, p16ink4 and ki67 as biomarkers for screening of cervical carcinomas.

METHODSThe expression of cyclin E, p16ink4 oncoproteins and ki67 proliferative activity was evaluated immunohistochemically in 78 cervical exfoliated epithelial specimens. Human papillomavirus type16 and 18 (HPV16/18) infection was assessed by polymerase chain reaction (PCR) using type specific primers.

RESULTSCyclin E, p16ink4 and ki67 were all overexpressed in cervical preneoplasia and neoplasia cells, compared with little expressed in ASCUS (P less than 0.005). Overexpression of cyclin E was observed in CIN, (P less than 0.01), p16ink4 and ki67 overexpressed in invasive carcinoma(100 percent and 90.9 percent respectively). The degree of p16ink4 and ki67 expression correlated well with the degree of cervical neoplasia (P less than 0.005). HPV16 infection was assessed at all stages of cervical neoplasia samples, and a significant relationship with the degree of cervical epithelial lession was observed at the same time. The expression level of p16ink4 and ki67 seemed to be more closely associated with HPV16 infection than cyclin E did (rs=1.0 vs rs=0.4). HPV18 was found positive in only 1 case in CIN1 and in 4 cases in CIN2-3. Therefore no significance was found on statistical analysis (P less than 0.005).

CONCLUSIONCyclin E, p16ink4 and ki67 should be regarded as useful biomarkers of HPV-related cervical neoplasias, and be used for screening patients at high risk for developing cervical carcinomas. Moreover, cyclin E might be a significant cytologic marker for the primary screening of cervical carcinomas.

Adult ; Aged ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; virology ; Cervix Uteri ; cytology ; metabolism ; virology ; Cyclin E ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; DNA, Viral ; genetics ; Female ; Host-Pathogen Interactions ; Human papillomavirus 16 ; genetics ; physiology ; Human papillomavirus 18 ; genetics ; physiology ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; biosynthesis ; Middle Aged ; Papillomavirus Infections ; metabolism ; pathology ; virology ; Polymerase Chain Reaction ; Uterine Cervical Neoplasms ; metabolism ; pathology