The original design of the WOLVES study in the pre-vaccination era has been described previously [2]. The ongoing study is a long-term, population-based, prospective, cohort study to measure changes in the prevalence of HPV infections and associated diseases after the introduction of HPV vaccines among the general female population from 2009 to 2020. Voluntary participation in the WOLVES study was open to women irrespective of existing HPV status and all participants gave written informed consent. The study was approved by the local ethics committee (Bo/07/2009).
Four cohorts are reported in this analysis: cohort 1 = participants born in 1983/84 and analyzed in 2009/10; cohort 2 = participants born in 1988/89 and analyzed in 2009/10; cohort 3 = participants born in 1988/89 and analyzed in 2014/15; and cohort 4 = participants born in 1993/94 and analyzed in 2014/15 (Fig. 1). At the time of sample collection, participants were aged 19–22 years in cohorts 2 and 4, and aged 24–27 years in cohorts 1 and 3. Women born in cohort 1 had a single cross-sectional medical examination in 2009/10, whereas those in cohorts 2 and 3 had annual medical examinations from 2009/10 to 2014/15. Participants in cohort 4 were analyzed in 2014/15 and will have annual visits until the end of 2020.
Participants of WOLVES do not differ from the general population of the same age, living in Wolfsburg area city, in terms of education, migration background, or parity. Town registry does not include any medical information, which is the main limitation of this study.
All participants were asked to complete a standardized medical questionnaire and attend regular medical examinations, as described previously [2]. The gynecologist collected data on HPV vaccination status by checking the certificate of vaccination, which includes all vaccinations. For HPV vaccinations, the certificate lists batch numbers and the date of vaccination. A fully vaccinated status, based on number of doses administered according to the recommended schedule, was defined as three doses given at months 0, 2, and 6 (5–13 months).
Women were referred to colposcopy if they had genital warts or an abnormal high-grade Pap smear, or they had an abnormal Pap smear classified as borderline or low-grade and tested positive for high-risk HPV infection. All colposcopy examinations were done at the Klinikum Wolfsburg, the single certified colposcopy unit in the region.
Women were not included if they were not living in the Wolfsburg area, had a diagnosis of cervical or genital cancer, another malignancy, an organ transplant, or were undergoing an immunosuppressive therapy.
Diagnosis of genital warts was based on colposcopy and histological biopsies. The analysis was based on the diagnosis of genital warts and also includes data on untreated genital warts. Genital warts were classified as: (i) typical condylomata acuminata for lesions with typical acuminate morphology, with typical punctuated or cauliflower-like patterns and tend to be pigmentless and are mostly seen on pigmented skin [11]; (ii) flat genital condylomata with a more hyperkeratotic and pigmented surface and flat condylomata with a smooth surface and non-pigmented papules; or (iii) seborrheic wart-like lesions of the cutaneous skin of the external anogenital area. The focus of this paper is on low-risk HPV types and associated disease therefore we do not report on vulvar intraepithelial neoplasia, Bowenoid papulosis, Naevi or Mollusca contagiosa [2]. These patients were excluded from the analysis of genital warts.
HPV DNA testing
HPV testing and genotyping were done using the Hybrid Capture 2 assay (HC2; Qiagen Inc., Hilden Germany) and SPF-10-PCR followed by Reverse Line Probe Assay LiPA Extra, respectively, as described in detail previously [2, 12, 13]. Cervical Pap smear samples were analyzed for the presence of the 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and the five low-risk HPV types (6, 11, 42, 43, and 44). Samples were diagnosed as positive if they attained or exceeded the FDA-approved threshold [2]. The INNO-LiPA Extra test allowed identification of 13 established high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), five known or potential high-risk types (26, 53, 66, 73, and 82), seven low-risk HPV types (6, 11, 40, 43, 44, 54, and 70), additional non-differentiated HPV types, and types with undefined risk (69, 71, and 74).
Study endpoints
The predefined endpoints were the rates of full HPV vaccination coverage (three doses), the prevalence of HPV 6 and 11 infection, and of genital warts (condylomata acuminata). We reported changes in these prevalence rates according to vaccination status and lifestyle factors (sexual history and smoking history) among participants in the four cohorts.
Statistical analysis
Vaccination coverage and prevalence rates of HPV 6/11 infection and genital warts are reported for participants aged 19–22 years (cohorts 2 and 4) and 24–27 years (cohorts 1 and 3) at the time of sample collection. Statistical analyses were done to compare similarly aged participants (cohort 1 versus 3 and cohort 2 versus 4) using 2 × 2 contingency tables for the prevalence of HPV 6/11 infection (positive versus negative) and genital warts (no versus yes). The Röhmel-Mansmann unconditional exact test was used to test for difference, with a two-side alpha of 0.05. Statistical analyses were performed using Testimate V.6.5.14.