Women referred to our institute from September 2002 to May 2006 for investigation of abnormal cervical cytology on the Papanicolaou (PAP) test were enrolled in this study. All patients were submitted to thin-prep PAP test, colposcopy and directed biopsy, and endocervical curettage. HPV test was done in the patients with no evaluating history. For STD screening, vaginal swabs for trichomonas vaginalis and candida species, polymerase chain reaction (PCR) for Chlamydia trachomatis and cervical cultures were performed. Pregnant or menstruating patient at the time of examination were excluded from this study. They were not treated with antibiotics or subject to surgical procedures before examination.
BV is a common, treatable condition resulting from replacement of the normal hydrogen peroxide-producing Lactobacillus-predominant vaginal flora with anaerobic bacteria, e.g. Gardnella vaginalis, Mobiluncus species and Mycoplasma hominis.5
Factors that increase the risk of BV are cigarette smoking, the use of intrauterine devices, frequent douches, multiple sexual partners, and early age at first intercourse. BV is known to be associated with many obstetric and gynecologic complications such as preterm labor and delivery, chorioamnionitis, post-cesarean endometritis, pelvic inflammatory disease (PID), postabortal PID, postoperative cuff infections after hysterectomy, and a possible connection with abnormal cervical cytology and CIN.6-10
BV was diagnosed if three of the following four findings were satisfied: presence of thin, grey vaginal secretions coating the vaginal wall; vaginal pH>4.5; presence of clue cell on microscopic examination of vaginal smear; positive amine or whiff test ().11
Diagnosis of bacterial vaginosis (A) presence of vaginal secretions coating gray and thinly the vaginal wall, (B) vaginal pH>4.5, (C) presence of clue cells on microscopic examination of vaginal smear, (D) positive amine or whiff test.
The result of PAP smears was reported according to the Bethesda III system (2001): atypical squamous cell (ASC), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL). LSIL includes CIN 1 and koilocytotic atypia. HSIL includes CIN 2 and CIN 3. The result of cervical biopsies was reported according to the CIN classification system as mild (CIN 1), moderate (CIN 2), or severe (CIN 3) dysplasia or carcinoma in situ (CIS).12
CIN was subdivided into a low-grade CIN (CIN I) group and a high-grade CIN (CIN II/III) group.
HPV detection and genotyping was performed with HPV DNA Chip, a PCR-based DNA microarray system provided by Microarray Center, Biomedlab Co (Seoul, Korea). HPV DNA Chip contains 22 type-specific probes that consist of 15 high-risk groups (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 69) and 7 low-risk groups (6, 11, 34, 40, 42, 43, and 44). DNA was isolated from swab samples with a DNA isolation kit (Qiagen, Hilden, Germany), and target HPV DNA was amplified by PCR with GPd5+/Gp6d+ primers (GP5d+, 5'-tttkttachgtkgtdgatacyac-3'; GP6d+, 5'-gaaahataaaytgyaadtcataytc-3'; k, g/ t; h, t/a/c; d, a/t/g; y, t/c). β-Globin was PCR amplified with PC03/PC04 primers (PC03, 5'-acacaactgtgttcactagc-3'; PC04, 5'-caacttcatccacgttcacc-3') as an internal control. Amplified DNA was labeled by Cy5-dUTP (NEN; Life Science Products, Inc, Boston, MA, USA). A mixture of 10 HPV-amplified products and 5 β-globin-amplified products were denatured by the addition of 3N sodium hydroxide solution (10% vol/vol), followed by incubation for 5 minutes at room temperature, were neutralized by the addition of 1 mol/L TRIS (tris-[hydroxymethyl]-aminoethane)-hydrochloric acid (pH, 7.2; 5% vol/vol) then 3N hydrochloric acid (10% vol/vol), and finally being cooled for 5 minutes on ice. The samples were mixed with a hybridization solution made of 6_SSPE (saline-sodium phosphate-EDTA buffer; Sigma Chemical Co, St Louis, MO, USA) and 0.2% sodium dodecylsulfate and applied to the DNA chip. The hybridization was performed at 40
for 2 hours and then washed with 3_SSPE for 2 minutes, 1_SSPE for 2 minutes, and air-dried at room temperature. Hybridized HPV DNA was visualized with the use of a DNA Chip Scanner (Scanarray lite; GSI Lumonics, Ottawa, Canada).13
LEEP was performed in women with high grade CIN or with a discrepancy between cytologic and histologic results. Statistical analysis was performed by the student t-test, chi-square test and Fisher's exact test (when n was less than 5), and logistic regression analysis with SPSS ver. 12.0. All statistical tests were two-sided. A level of p<0.05 was accepted as statistically significant.