This study investigated the age-specific prevalence of overall HR-HPV, HPV16/18/45 and other HR-HPV in 25 to 65-year-old women without cervical precancer and cancer in a prospective study in rural China. It focused on the interpretation for the peak observed in older women. Moreover, we explored the risk factors of HPV incidence and clearance using one-year follow-up data.
The age-standardized prevalence by world population of HR-HPV tested by careHPV at baseline in our study was 14.2%, which was comparable to other studies in mainland China [8–10], and other parts of Asia/Australia (~15%) . However, it was lower than a pooled analysis from China (16.8%) . We reported a prevalence of 2.9% for HPV16 and/or 18 and/or 45 types, which was lower than other studies in China [9, 10]. These differences are likely caused by different HPV DNA tests and different study populations, such as different geographic areas, different age groups, and different distributions of cervical lesions. We found that the prevalence as measured by careHPV was similar to the prevalence as measured by HC2 in our study (p = 0.196), which suggested that the difference between the prevalence in this study and the recent pooled analysis  was primarily due to the differences among study populations.
Previous studies in China had observed a second peak of HPV prevalence in older women, although the peak age varied between studies [8–10, 13]. In our study, HR-HPV prevalence peaked at 55-59 years, but lacked the “first peak” in younger women. It has been well acknowledged that cumulative risks of 40-50% of HPV acquisition happened within 2 to 3 years of sexual debut [29–32]. Since the average age at first sexual intercourse of our study participants was 21.2 years, the theoretical peak of HPV prevalence should appear in women aged 23-24 years. In addition, Chinese women were less likely to report their premarital and extramarital sexual histories. In that case, we hypothesize that the actually age of sexual initiation may be even younger. However, we only enrolled women aged over 25 years, therefore we have no chance to see the “first peak” as observed in other studies . The one-year prospective data demonstrated a larger number of infections that were cleared versus acquired, which indicated that HR-HPV prevalence should decrease with progressing of age, rather than a peak occurred in older women.
The probable explanations for this HR-HPV peak in older Chinese women could be a cohort effect that leads to increased lifetime exposures, increased HPV incidence ,and/or increased viral persistence in older women  versus younger women. We considered each as described below.
Before the one-child policy which had been strictly implemented in year 1979 , most women got married at an early age and gave a large number of births. Risk factor analysis in our study also found an earlier age at first sexual intercourse and a greater number of live births in 49 to 65-year-old women (data not shown), which may have led to a higher cumulative exposure to HR-HPV. And in some high-quality cancer registries in China, a declining trend in cervical cancer incidence from 1970’s to 1990’s has been observed . This may partly reflect a higher HR-HPV burden in older generations (vs. younger generations according to a report from Sharma et al. , which found a positive association between age-standardized HPV prevalence and its square-root age-standardized cervical incidence).
However, one-year follow-up was not sufficient to provide a strong evidence to explain the cohort effect. Long-term follow-up studies with birth cohort analysis are needed to further clarify this effect. With recent changes in sexual norms, we anticipate an upturn in the annual incidence of cervical cancer in China unless widespread secondary prevention through screening, diagnosis, and treatment of precancerous lesions is implemented.
We expected to see a decreased trend in the incidence of HR-HPV infection in older women as reported in other studies [33, 37], but only observed an independent relation. This unexpected higher incidence in older women may be caused by: 1) new HPV infections acquired by changes in sexual behaviors by either the women or their partners; 2) reactivation of latent HPV infection due to immune senescence [38, 39]. No evidence supported the changes in sexual behaviors in older women although that does not rule out that their male partners were having new partners and transmitting HPV to them. However, we noted differences in risk factors associated with HPV infection between women aged 25-40 years and women aged 49-65 years. Number of lifetime sex partners, which may be proxy for cumulative exposure impact , was only found to be associated with HPV infection in older women. Greater early exposure to HR-HPV infection and weakened immune response after menopause  that led to the re-emergence of latent HPV infections could cause a second HPV peak in older women. Gravitt et al.  found that there was a second peak around 50-54 years in women with five or more sex partners but not in those with less than four sex partners. Their findings may support that the second peak could be seen in a more generalized population with a relatively higher cumulative probability of HPV infection.
Another explanation, a greater HPV persistence in older women, was strongly supported by our data (see Additional file 1: Figure S1). We found an obvious trend of decreasing clearance or increasing persistence of HPV with increasing age. The mechanisms could be: 1) decreased ability to clear recent infections with age-related immune senescence; 2) predominance of long-duration prevalent infections in older women from earlier exposures .
Thus, we found that the cause of the second peak in HPV prevalence may be multi-factorial. Despite of the low number of sex partners, the rural Chinese women were usually married at an early age and gave a large number of births. The lifestyles and economic status of the older rural Chinese women were also much different from other populations and younger generations. Their poor nutrition and sanitary condition may cause a poor immune function, which may in turn result in a reduced ability to clear HPV infections and to control/prevent the re-emergence of latent HPV infections.
Our findings agreed with the previous study conducted in Guanacaste, Costa Rica, which also found that HPV infections tended to clear more often than acquire, and persistence increased with age . However, unlike the study in Guanacaste, Costa Rica, we did not observe newly detected infections declining with age.
We also explored the risk factors for HPV prevalence, incidence and clearance using both univariate and multivariate models. Most of our results were similar to the others [42, 43], however, we failed to find the association between the number of sex partners in the past 6 months and HPV prevalence in the multivariate model. This may be explained by the fact that most of the participants reported to have no or one sex partners in the past 6 months. To our knowledge, this may not be entirely accurate as some women may be reluctant to report their actual behaviors.
Impact for cervical cancer screening
The incidence of HPV16,18, and/or 45 was higher and the clearance was lower in 55 to 65-year-old women, they are of the greatest risk of persisting HPV16/18/45 infections, which cause 75% of cervical cancer worldwide . Women aged 55-65 years may be at the highest risk of progression to cervical precancer or cancer. However, the first large-scaled cervical cancer screening program in rural China launched by Chinese government from 2009 to 2011 using VIA or Pap smear does not cover women older than 60 years . If further studies show these infections representing significant cancer risk in older women, expanding screening to older ages should be considered in the future nationwide program in China.
This analysis has some limitations. First of all, convenience sampling was used in recruiting, the participants may not be perfectly representative. Second, we only followed up ~20% of the screen negatives, even with adjustment, the estimated incidence and prevalence at follow-up might differ somewhat from the actual values. Whereas, we conducted a random sampling to get the list of screen-negative women, so the bias was minimized. Third, we did not test for the specific HPV type, so that we could not get the precise status of HPV incidence or clearance. Given that multiple infections typically represent 20-30% of all HR-HPV infections and are often more common in younger women , we likely underestimated both one-year clearance and incidence. However, we granted the same patterns of clearance and incidence for HPV16/18/45 infections as the overall HR-HPV infections, which may reflect a relatively low percentage of women with co-infections of the three types. We only use a pooled DNA test for 14 certain and probable HR-HPV types to generally describe the age-group specific patterns of HR-HPV, which is relevant to cervical cancer screening and the risk factors associated.
We used clinical tests for HPV, which may have missed some lower viral load HPV infections that are not strongly associated with CIN2+. To address this issue, we conducted an additional analysis by defining HPV positive as either careHPV or HC2 tested positive to increase the analytic sensitivity. And we observed similar results.
Another notable limitation was the short follow-up time. As a consequence, some infections persisted for a year but would have later cleared, while some infections that appeared to clear were testing errors and would have tested positive subsequently. However, based on the results from a meta-analysis which found the median duration of HR-HPV detection was 10.9 months in those HPV-positive women with normal cytology , we believe that measuring the pattern over a year was a good surrogate for the longer-term persistence. Previous studies have shown that one-year HPV persistence can strongly predict longer-term persistence  and CIN2+ [47–49].