It is widely accepted that human papillomavirus (HPV) is the primary causative agent of cervical neoplasia (cervical intraepi thelial neoplasia [CIN] and invasive cancer) (zur Hausen, 1991). However, HPV infection of the uterine cervix does not always induce cellular abnormalities (de Villiers et al, 1987), suggesting that other factors play a role in the development of cervical neoplasia. Many epidemiologic factors have been suggested as relevant to the development of cervical neoplasia (Rotkin, 1973;Bornstein et al, 1995). We conducted a case-control study to clarify how epidemiologic factors are involved in the development of CIN in relation to HPV infection.
MATERIALS AND METHODS
Study populationThe study population consisted of women aged 55 or younger who underwent Papanicolaou test screening at nine hospitals between June 1995 and July 1996. For women with abnormal cervical cytology, colposcopically directed biopsies were performed. The cases enrolled in the study were women in whom CIN was first detected during the study period. A total of 167 women who had histological evidence of CIN were included; low-grade CIN (CIN I [n ϭ 94]) and high-grade CIN (CIN II [n ϭ 40] and CIN III [n ϭ 33]). We excluded carcinoma in situ from CIN III. The histological review was performed by one experienced pathologist (KN). Controls matched one-to-one with cases on age (within 5 years) and hospital were selected from subjects who were found to have normal cervical cytology. All CIN cases and controls gave written informed consent.
The questionnaireInformation concerning health issues related to the possible aetiology of CIN was obtained by a self-administered questionnaire. The questionnaire, which included demographic factors, smoking habits, contraceptive and reproductive history, and sexual behaviour, was distributed to each case or control on their second hospital visit. Several participants gave no answers to certain questions.
Detection and typing of HPVCellular DNA was extracted from cervical exfoliated cells by standard procedure. We used the consensus L1 primers, L1C1 (1 µM), L1C2 (0.5 µM) and L1C2M (0.5 µM), for the polymerase chain reaction (PCR) (Yoshikawa et al, 1991). HPV types were identified on the basis of the restriction fragment length polymorphism. The assay can type at least 26 genital HPVs. HPV data were not obtained from nine (5%) of CIN cases and 37 (22%) of controls. HPV types were categorized into three groups of carcinogenic potency according to then risk classification proposed by Lorincz et al, (1992); i.e. high-, intermediate-and low-risk HPVs (Table 1).
Blood sampling and serum antibody detectionA blood sample for serological assays of herpes simplex virus (HSV), cytomegalovirus (CMV) and Chlamydia trachomatis (CT) was collected from each subject. Blood sampling was not available in 9 (5%) CIN cases and 5 (3%) controls. Antibodies were assayed by using commercially available enzyme-linked immunosorbent assay (ELISA) kits; HSV and CMV (Denkaseiken, Niigata, Japan), and CT (Labsystems Oy, Hels...