Human papillomavirus (HPV)–related cervical cancer is a major public health threat to women, with >10,000 new cases diagnosed annually in the United States between 2008 and 2012. Since HPV vaccines can protect against ~80% of HPV-associated cervical cancers, the economic and epidemiological impacts of HPV vaccination have been extensively investigated, particularly at the national level. However, vaccination policies are state-specific, and state-level models are required for state-specific policy decisions. This study adapted an age-structured population model to describe the dynamics of HPV-related cervical cancer in Texas, with model parameters calibrated for Texas. The Year 2000 parameter set was the start point, and the model’s predictions from 2001–2010 were well matched with the real incidence numbers in 23 age groups, suggesting the validity of the model. Application of the model to the Year 2010 parameter set predicted that, over the next 10 decades, incidence would decrease rapidly within the first decade and more slowly thereafter. Sensitivity analysis determined the impact of selected parameters  on future disease incidence. When compared with the US parameter sets, the Texas population was more sensitive to changes in HPV transmission and vaccination . The incidence of cervical cancer in Texas is among the highest in the country, 8.7 cases per 100,000 women in 2013, compared with a national rate of 7.2 cases per 100,000 women2. About 1,300 new cases of cervical cancer are expected in Texas in 20171.Cancer of the uterine cervix is one of the most common cancers affecting women in the United States (US), ranking third in terms of incidence and mortality among all gynecologic cancers. In the US, 12,820 new cases of cervical cancer and 4,210 cervical cancer–related deaths are expected in 20171. Declines of cervical cancer incidence and mortality slowed down in recent decades: during the decade 2005–2014, incidence did not change significantly and mortality declined by an average of 0.8%1.Cervical cancer was once one of the leading causes of death among women in the US. However, with the introduction of the Pap test and the promotion of cancer screening and prevention programs in the 1950s, the incidence and mortality of invasive cervical cancer declined by more than 60% between 1955 and 19923. The first effective HPV vaccine became available in the US in 2006, and a national HPV vaccination program was implemented thereafter. The HPV vaccination program is expected to significantly reduce the incidence of HPV-related cervical cancers, but because of the latency period between HPV exposure and occurrence of cervical cancer, this decline is not expected for two to three decades.Nearly all cases of cervical cancers are associated with human papillomavirus (HPV) infection. HPV-16 and HPV-18 infection are highly oncogenic and account for the majority of HPV-related cervical cancer cases4. However, currently only 49.5% of females and 36.5% of males have been fully vaccinated, falling short of the national goal of 80% vaccination5. Currently vaccination-related legislation is state-specific7. therefore, understanding the effect of vaccination on a state level  is required to justify policy-based changes6. In Texas, the vaccination rate is lower than the national average, especially in males. The discrepancy between national and state vaccination rates as well as the differences between individual states necessitate further investigations on the state level8.HPV vaccination is recommended for all adolescents12) frequently call for the use of mathematical models, given the complexity of the long natural history of HPV-related cancers. In general, such mathematical models will include HPV transmission dynamics, progression and regression of cervical disease from premalignant to malignant states, screening strategies for early detection, and vaccination strategies for prevention13. Also, once established, a persistent HPV infection may progress to cervical intraepithelial neoplasia (CIN), carcinoma in situ (CIS), and finally invasive cervical cancer, so such models should allow for regression of disease and early detection and treatment through screening14. Considering the substantial heterogeneity in different age groups , it is suitable to employ age-structured population models, which are a system of ordinary differential equations (ODEs) generated by discretizing a first-order partial differential equation for population dynamics according to consecutive age groups16. In one of the many pioneering modeling efforts, Elbasha et al.17 built a comprehensive age-structured model with all the aforementioned components and factors considered. While the natural history of cervical cancer is the best understood of all HPV-related cancers, new evidence is continually emerging that can be incorporated into existing models. For instance, Campos et al.18 proposed an updated natural history model of cervical cancer by combining the CIN1 stage with the HPV-infected stage. Of course, age-structured ODE models are not the only possible model structure for this study; however, the recent work of Brisson et al.19 selected and compared 16 transmission-dynamic models that differ in structure and data for parameter calibration, finding that the population-level prediction results of these models were concordant with each other.Studies on the economic and/or epidemiological impacts of HPV vaccination  or knowledge of HPV and cervical cancer. Note that the Texas population is very different from the overall US population . Therefore, the exact same model structures are used for both Texas and US, but two sets of parameter values and initial conditions are derived and employed for Texas and US, respectively.To build a model that would help us understand the impact of HPV transmission and vaccination on the incidence of HPV-related cervical cancer in Texas and compare Texas outcomes with the national-level results, we developed an age-structured mathematical model consisting of demographic, HPV epidemiological, and cervical cancer components. Briefly, the demographic component depicted the age- and sex-specific population dynamics of Texas and the US; the epidemiological component described HPV transmission, progression, and vaccination; and the cervical cancer component sketched the natural history of the disease. The resulting model is a large system of ODEs  similar to the previous work of Elbasha and Dasbach21 and statistical inference22 techniques for a large-scale nonlinear ODE system like our model, it is infeasible to perform model fitting here. However, Fig. 23 for model validation, and they also reported mismatches between real data and model predictions for the older age groups. In comparison with Elbasha and Dasbach’s work, our prediction results better matched the real data in the older age groups, possibly suggesting an improvement in model structure refinement and parameter value calibration.The proposed model was validated by comparing model prediction results with real cervical cancer incidence numbers from Year 2001 to Year 2010, with the Year 2000 parameter values and initial conditions , under the assumption that model parameter values remain the same as Year 2010 Texts  and S5. 17, include vaccine coverage rate, vaccination covered age group, rate of sexual partner change, degree of assortative mixing between age and sexual activity groups, heterogeneity in sexual partner acquisition rates, and the impact of vaccinate efficacy. We found that if the progression rates from CIN to CIS  were sufficiently small, model prediction results were not notably affected by the changes in these parameters. Therefore, the following sensitivity analyses were based on π2 = 2.1 (% per year), π3 = 6.45 (%per year), and π5 = 0.62 (%per year) and other Year 2010 parameter values. All the predicted incidence rates monotonically decreased from Year 2010 to 2110. Also, all the predicted incidence rates dropped rapidly in the first 10 years and then slowed down. The incidence was higher in Texas at the beginning of the time period but decreased faster than that of the US.Sensitivity analyses were conducted to investigate the impacts of changes in several selected factors on the future incidence of HPV-related cervical cancer. These factors, which are considered to be important contributors to HPV-related cervical cancer and thus are often considered as parameters in predictive models for this diseaseAs shown in Fig. The starting age of vaccination affects the incidence rate. To understand the effects of age-specific vaccination, we made predictions based on the assumption that people can receive vaccination two age groups earlier than existing vaccination coverage of 23 age groups . We then compared those results with the results of a similar analysis in which vaccination started two age groups later  depicts the level of assortative mixing between different age and sexual activity groups. As shown in Fig. 1 or ε2 values indicate a greater degree of assortative mixing. ε3 is for persons older than 60 years. The difference in the incidence rate for Texas and for the US increased slightly  at the end of prediction period (Year 2110).The degree of assortative mixing parameters  pcl = 1, pai = 1, et al.17; all the other parameters were fixed at default. Here pcl and pai are the relative partner acquisition rates for sexual activity group l and for age group i, respectively; To assess the impact of heterogeneity in sexual partner acquisition rates , we consider three scenarios (1) heterogeneity in age groups only, (2) heterogeneity in sexual activity groups only, and (3) no heterogeneity, and we set (1) ψv1 and ψv2. The baseline values of ψv1 and ψv2 are 0.91 and 0.99, respectively; and the baseline values of ψv1 and ψv2 at 0.5; (2) we decreased the ψv1 and ψv2 at 0.75. As shown in Fig. To study the impact of HPV vaccine efficacy on the incidence of HPV-related cervical cancer, sensitivity analyses were conducted by changing the values of parameters 25. More importantly, both the prediction and sensitivity analysis results suggested a clear difference between the Texas and the US populations in response to disease dynamics or policy changes.In this study, an age-structured HPV infectious disease model was adapted from the literature, updated, and validated for understanding the effects of HPV vaccination on cervical cancer progression in Texas and comparing them with those for the US overall. Model parameter values were taken from the literature or calibrated by using epidemiological data from Years 2000 to 2010. Model prediction results were validated against cervical cancer incidence for Years 2001–2010, matching the actual data well. The model shows a rapid initial decline of predicted incidence for the first 10 years and then a slower decrease after that. This predicted decreasing trend in incidence is consistent with the real trend observed over the past 7 years (2010–2017). As our study only considered the effect on HPV-16 and -18, the subsequent slower rate of decrease in cervical cancer incidence may represent incident cases due to other HPV types. Previous studies have demonstrated the lack of type-replacement following vaccination, supporting this trend. The model was then used to predict cervical cancer incidence rates for Texas and the US until Year 2110. Interestingly, when the progression rates from CIN to CIS were small, the five parameters used for the sensitivity analysis, parameters that are frequently chosen for this type of analysis, did not have a significant impact on the predicted cancer incidenceReaders should be aware of several restrictions in this study. First, model fitting was not performed because of the lack of feasible regression techniques for large-scale ODE systems; therefore, parameter values were from the literature or estimated using available data or based on certain assumptions. A better match between model outputs and real data is expected if model fitting becomes feasible in the future. Second, a constant vaccination rate was assumed and thus might not truthfully capture the real evolution of vaccination strategy over time. Therefore, our prediction results may be conservative. Third, the age-structure model is of a very large scale, comprising more than 6,000 equations and a large number of parameters. Thorough interrogation of every aspect of this model  was not feasible in one study. Thus, one may consider reducing model complexity by dropping certain parameters or variables if their roles are trivial. Fourth, the proposed model was validated only by comparing the predicted cervical cancer incidence with the actual data from Years 2000 to 2010. When more data become available, further assessment of model validity is necessary to meet our goal of better assessing the health impacts of different vaccination strategies as well as their cost-effectiveness. Finally, solving a large system of ODEs repeatedly is computationally expensive. High performance computing techniques or alternative modeling approaches  may be considered in future work.In summary, this study quantitatively investigated the HPV-related cervical cancer incidence in both Texas and the US using an age-structured model. The differences revealed via computer simulation and prediction in the Texas and US populations and disease dynamics suggest the necessity of further research work on the state level. These results provide a basis for future modeling of HPV-related cervical cancer incidence as well as the investigation of economic impacts of HPV vaccination related to prevention of cervical cancer.20. This modeling framework comprises three major coupled components: the demographic, the epidemiological, and the cervical cancer natural history models for different sexes as well as different age groups. The demographic model describes the population dynamics of 23 consecutive age groups (from age 0–1 to age ≥85 years); the HPV epidemiology model describes HPV transmission, persistence, and vaccination; and the cancer natural history model describes the incidence and progression of cervical cancer in women.Building a mathematical model from scratch to depict population growth, HPV transmission, vaccination, and natural history of HPV-related cervical cancer demands tremendous effort and time. Therefore, we adapted the previously compared and validated nonlinear age-structured model framework proposed by Elbasha and colleaguesIn the demographic model, individuals of one age group transfer to the next successive age group, except for the oldest (age ≥85 years) group, at an age- and sex-specific rate. For example, the calibrated parameters suggested that around 13% of 8-year-old females from the second age group (age 1–8 years) move to the third age group (age 9–10 years) every year for both Texas and the US. The population size of an age group of specific sex depends on the proportions transferring from a younger group or into an older group and on cancer-related and non–cancer-related death. The population growth of the youngest age group is calculated from the birth rate because no younger population exists. In the epidemiological model, HPV transmission among subpopulations is specific to sex, age, and sexual activity. The female populations of various ages and sexual activity levels were further dichotomized by cervical cancer screen status (never or routine). Important subpopulations in this transmission model include susceptible individuals, infected individuals, recovered individuals, persistently infected individuals, vaccinated individuals, infectious vaccinated individuals, persistently infected vaccinated individuals, and recovered vaccinated individuals. In the cancer natural history model, the pre-cancer and post-cancer stages consist of undetected CIN, treated CIN, infected treated CIN, benign hysterectomy, undetected cervical cancer, detected cervical cancer, and cervical cancer survivors.et al.20 to reflect the current understanding of HPV transmission and cervical cancer progression. First, the updated natural history model of cervical cancer from Campos et al.18 was incorporated into Elbasha et al.’s model, where CIN1 was combined with the HPV-infected state and CIN2 and CIN3 were treated as non-sequential states18. Second, Elbasha et al. made a distinction between two subtypes of undetected CIS; however, since the two CIS subtypes are not clinically distinguishable, CIS was not divided into two stages in our model. Third, the three-dose HPV vaccination strategy has recently been replaced by a two-dose strategy26 therefore, the populations who received two or more doses were combined into one population in our model. Such a change affects many model structure details. For instance, the force of HPV infection (λ) was determined bycklaij is the number of sexual partners, rk is the relative infectivity of vaccine breakthrough cases, ρklaij is the probability of someone being in group k, l, a, i and j. Here, k denotes sex, l denotes sexual activity group, i denotes age class, a denotes the sexual activity group that an opposite-sex partner was from, and j denotes the age class for the partner; Y and U denote infected and persistently infected persons, respectively; C includes undetected cervical cancer and detected cervical cancer populations; WS denotes infectious vaccinated persons. In a deviation from the model of Elbasha et al., here P consists of P1 and P2 only, which denote the persistently infected vaccinated persons who receive one or two doses, respectively. Finally, the complete model equations are given in Supplementary Text Several changes were made to the model of Elbasha 28 and screening characteristics (two levels), also categorized by age29. For previously infected or vaccinated individuals, we assumed that there exists no protection against future infection if no seroconversion was observed in these individuals. For a vaccinated individual with seroconversion, protection against future infection was assumed to be 100% because of the demonstrated efficacy of HPV vaccination31. Because Harper et al. showed no persistent infections following vaccination30, we assumed that no individuals with previous vaccination would develop premalignant cervical lesions if an infection occurred following vaccination.Model parameter notations, definitions, and sources of parameter values are presented in Table 32. Patients with detected CIN3 or CIS were assumed to have received treatment according to standard practice. Since no data were available to estimate the number of undetected cervical cancer cases , the undetected cancer cases were ignored in our model.The occurrence rate of undetected cervical premalignant disease was estimated from the sensitivity and specificity of the conventional Pap smearThe model was implemented in MATLAB software  and solved using the ode15s solver. All the simulation studies were conducted for both the Texas and the US populations. Specifically, the entire population was divided into two sex groups and 23 age groups . The population was further stratified into three sexual activity groups according to the number of partners . The cervical cancer screening behavior for females was dichotomized into two categories (never or routine).https://seer.cancer.gov/ and https://www.dshs.texas.gov/tcr/). The Year 2000 data were used as initial conditions for the model to predict the Years 2001–2010 outcomes, and the Years 2001–2010 data were compared with the model prediction results for validation purposes. Similarly, using the Year 2010 data and parameter values in the model, the cervical cancer incidence rates from Year 2011 to 2110 were predicted for both Texas and the US. All parameter values and initial conditions for Texas and the US are listed in Supplementary Texts The US and Texas annual cancer incidence data by age group and cancer site were available from Year 2000 to 2010 in the SEER and TCR databases (Supplementary InformationSupplementary dataset


Until the 1960s, only leading cyclists originating from the province of West- and East-Flanders were considered as flandriens. After 1960, the media extended the use of this term to Belgian cyclists in general and even to international cyclists. The present study examined whether Flemish citizens agree with this generalization considering that the term flandrien still plays a highly symbolical role in the public discourse on Flemish identity. First, the main aim was to investigate whether having an ethno-cultural identity representation of the Flemish identity is positively related to perceived regional exclusivity of the term flandrien. Second, this study explored whether Flemish identification moderates this relation (i.e. this relation is only expected for high identifiers) and also predicts Flemings’ regional exclusivity of the term. Results revealed that the more Flemings endorse an ethno-cultural identity representation the more they consider a flandrien as an exclusively Flemish cyclist, and the less they will include international cyclists in their consideration of a flandrien. Flemish identification did not moderate this relation but did predict the consideration of a flandrien as a Flemish cyclist. These findings indicate that the current interpretation of the historical cycling term flandrien is influenced by cultural conceptualizations of Flemish identity.In Flanders the term  Just one week later, he sped over the cobbles of Paris-Roubaix to his second one-day classic victory in seven days. Mainly because of this exceptional double victory, Cancellara was honored with the ‘International Flandrien Award of 2010’ during the annual ‘gala of the flandrien was reserved in the media for Flemish cyclists that were physically powerful, had a visibly high sportive work ethic and exhibited great perseverance while racing , which has become the biggest Flemish party during the elections in 2014.In Belgium, cycling has historically played a crucial role in the construction of group identities at different levels  . This smflandrien, which started in West- and East Flanders (i.e. the Western part of Dutch-speaking Flanders), served to convey Flemish identity and pride during the interbellum, because the Dutch-speaking Flemings strived to become more autonomous from the French-speaking Walloons.Being the first modern sport that was already adopted by the lower classes around 1900 , cyclingflandrien had a rather negative connotation as shorthand description for the rude West- and East-Flemish seasonal workers in Wallonia and the Northern part of France . The French-speaking press described these flandriens as savages, displaying unfair and aggressive behaviour. From this moment, the term flandrien became synonymous with the straightforward style of Flemish cyclists. The latter characteristics were further confirmed by finishing first, second and third at the ‘Six hours of Paris’ in 1913, again using their aggressive, perseverant style. Nevertheless, Van Wijnendaele consistently casted flandriens in a positive and even heroic light. He described ‘his’ flandriens as personifications of a supposed ‘essence’ of the Flemish people: poor, but strong and showing a lot of willpower. They lacked tactics, but won through strength and perseverance  of a cyclist became less important to be considered as a flandrien. The use of the term expanded and in the 1980s and 1990s also non-West- or East Flemish (but still Flemish) cyclists, such as the ever-attacking Ludo Dierckxsens, were labelled as flandriens.After 1960, even though Schotte was still considered as the prototype of the flandrien by the Flemish media. This was illustrated by the recent documentary The Flandriens , in which the legendary Eddy Merckx, who grew up in Brussels, a non-Flemish region of Belgium were the majority speaks French, was presented as a flandrien. An even more poignant illustration of this trend was the fact that Philippe Gilbert won the trophy ‘flandrien of the year 2011’, despite his Walloon origin. More recently, even non-Belgian cyclists are labelled and honoured as flandriens. This is further illustrated by the fact that in 2003, 2006 and 2007, the trophy ‘flandrien of the year’ was awarded to Italian cyclist Paolo Bettini.As the new millennium approached, even the Flemish origin became less important in order to be considered as a flandrien, the present study will question if and to what extent Flemish citizens agree with this labelling of foreign cyclists as flandriens. Nowadays, flandriens are still considered as important symbols of a Flemish identity , and the descriptions of heroic performances of previous flandriens in the Flemish media. This creates a feeling of ‘us (i.e. the Flemings) versus them ’ and symbolizes Flemish identity  disagreed with this statement. Importantly, this means that a large group of Flemings was not inclined to consider international cyclists as flandriens, even if these cyclists exhibited the same characteristics as a prototypical flandrien like Briek Schotte. These findings suggest that there are interindividual differences in the interpretation of a flandrien in terms of regional exclusivity, and that a large proportion of Flemings do not share the most recent media depiction of a flandrien.It should be noted that the conclusions of the previous section were based on historical and sociological studies, which used contemporary primary sources  and secondary literature. By contrast, in a recent social-psychological study Knuts et al.  intervieN = 203) and did not test whether the proportion of Flemings who have a regionally exclusive view on a flandrien (71%) significantly differed from the proportion of Flemings who are less restrictive and do not limit their definition of a flandrien in terms of regional exclusivity (29%). Furthermore, this study did not include variables that allowed the authors to directly probe into the possible underlying reasons for Flemings’ interpretation of a flandrien.However, this study only included a limited sample size .A first aim of the present study was to replicate Knuts et al.’s  observeds et al.  and the flandrien. More specifically we wanted to investigate the relation between the national identity representation of Flemings and their interpretation of this historical cycling term as an important symbol of Flemish identity. Because a flandrien is an important, historically grounded cultural symbol for the Flemish identity, we hypothesized that the more strongly Flemings endorse an ethno-cultural identity representation of Flanders, the more they would be inclined to protect the meaning of a flandrien against change and would therefore show more regional exclusivity in their interpretation (Hypothesis 2). More specifically, the more Flemings endorse an ethno-cultural identity representation, the more they consider a flandrien as an exclusively Flemish cyclist (Hypothesis 2a) and the less they would include international cyclists in their consideration of a flandrien (Hypothesis 2b).The second and main aim was to address the absence of an underlying explanation of Flemings’ interpretation of a flandrien. First, it is known that ingroup identification positively predicts ingroup favouritism . It should be noted that previous research did not show a straightforward relation between ingroup identification and outgroup derogation . As mentioned before, identification with a group is necessary in order to attach value to the norms and values of its prototype. Moreover, Pehrson, Vignoles, and Brown  and the less they will include international cyclists in their consideration of a flandrien (Hypothesis 4b).Second, it can also be expected that Flemish identification moderates the relation between an ethno-cultural identity representation and the interpretation of a nd Brown  reportedflandrien. A link referring to the web-based questionnaire was included. Participants were informed that completing the questionnaire would take 20 to 25 minutes and they were asked to give their honest, personal opinion.Participants were recruited by means of a standardized message. This message stated that the KU Leuven was investigating how cycling fans interpret the term De Zondag , printed each Sunday on more than 600,000 copies. Fourth, social media  were used to distribute the message. Fifth, the message was printed on 500 flyers, which were distributed in various places: in Mechelen on March 24, 2012, in Oudenaarde on March 31, 2012 (during the Tour of Flanders for leisure cyclists) and in Schoten, Lille and Herentals on April 4, 2012 (during the cycling race ‘Scheldeprijs’).This message was spread through different channels. First, this message was sent via e-mail to 285 cycling clubs. The selected cycling clubs were either located in a cycling-specific region  or in a cycling-nonspecific region . Second, staff members of cycling events, administrators of cycling-related and tourism websites  were contacted. Third, the message was published in the free newspaper In total, 2010 participants completed the web-based questionnaire, which was online from February 14, 2012 until April 16, 2012. Participation was voluntarily and participants could end the questionnaire at every moment. Anonymity was ensured and an e-mail address of one of the researchers was provided in case participants had any questions or remarks.Table Demographic variables. The questionnaire assessed respondents’ age (in years), gender and education. With respect to education, respondents had to indicate whether they had a degree in lower education, lower secondary education, higher secondary education, higher, non-university education or university education.Cycling fandom. To assess respondents’ level of cycling fandom, they had to indicate the extent to which they agree with the statement that they consider themselves as cycling fan on a 5-point Likert scale, ranging from 1  to 5 .Flemish identification. Identification with the Flemish in-group was assessed through three items  on a 5-point Likert scale, ranging from 1  to 5 . This scale was based on a six-item affective in-group identification scale from Boen et al.  to 5 . Two items that assessed the desire for more autonomy for Flanders at the expense of the Belgian state . The other six items were part of the citizenship representation scales from Reijerse et al.  to 5 .Regional exclusivity of the term flandrien: inclusion of international cyclists as flandriens. Two measurements were constructed to assess whether respondents also included international cyclists in their consideration of a flandrien. First, respondents had to rate the item ‘A flandrien can also be a foreign cyclist’ on a 5-point likert scale, ranging from 1  to 5 . Second, the questionnaire also contained eight international cyclists. Respondents had to indicate the extent to which they considered these cyclists as a flandrien on a 5-point likert scale ranging from 1  to 5 . Four international cyclists were selected  because they were expected to be best known amongst Flemings and contain prototypical flandrien-features such as a straightforward, fierce cycling style, winning on willpower and demonstrating a lot of perseverance. Therefore, for each participant, the average score for these four international cyclists was calculated. This combined score served as another, less abstract measurement of the inclusion of international cyclists as flandriens. This measurement demonstrated a good internal consistency (α = .83).flandrien can also be a foreign cyclist’. Respondents who gave a rating of 1 or 2, were assigned to the category ‘disagree’, while respondents who rated this item 4 or 5 were assigned to the category ‘agree’. Participants who gave a rating of 3 were considered as ‘non-deciders’. This procedure resulted into three categories that all contained a proportion of the study sample: a ‘disagree’ category (N = 731), an ‘agree’ category (N = 972) and a ‘non-decider’ category (N = 298). To test Hypothesis 1, we compared the number of respondents who agreed with the statement that a flandrien can also be a foreign cyclist with the number of respondents who disagreed with this statement. In line with Hypothesis 1, a Chi-Square statistic indicated that the non-agreement category contained significantly more respondents than the agreement category  = 34.11, p < .001). That is, there are more Flemings who adhere a regionally exclusive definition of a flandrien than Flemings who opine that a flandrien can also be an international cyclist.First, in order to test Hypothesis 1 and to compare our results with those from Knuts et al. , it was flandrien can also be an international cyclist). Results demonstrated that the proportion of disagreeing respondents was significantly lower in the present study as compared to the study from Knuts et al.  = 14.36, p < .001). To summarize, although these results confirm Hypothesis 1, we did not fully replicate the exact distributions from Knuts et al. . As opposed to the previous analysis of Hypothesis 1, all following analyses used the full range of likert-scale scores.The multiple hierarchical regression analysis was conducted for both operationalizations of regional exclusivity of the term flandrien as a Flemish cyclist and the inclusion of international cyclists in the consideration of a flandrien. In step 3, the interaction term between ethno-cultural identity representation and Flemish identification was entered. Ethno-cultural identity representation and Flemish identification were mean centered, following the recommended procedures for testing interactions in multiple regressions  versus no higher education group (0). In step 2, ethno-cultural identity representation and Flemish identification were included to test their contribution to the consideration of a ressions . The resflandrien as a Flemish cyclist  = 28.61, p < .001). Step 2 demonstrated that, when the background variables were statistically controlled for, both ethno-cultural identity representation and Flemish identification contributed significantly to the consideration of a flandrien as a Flemish cyclist  = 62,56 p < .001). The results of step 3 revealed that the interaction term between ethno-cultural identity representation and Flemish identification did not contribute significantly to the consideration of a flandrien as a Flemish cyclist  = 0.03, p = .87), which contradicts Hypothesis 4a. Therefore, only the regression coefficients after step 2 were interpreted.The results of the first step showed that the background variables explained 6% of variance in the consideration of a p = .041), cycling fandom , education  and age  were revealed as significant predictors. In particular, men consider a flandrien less as a Flemish cyclist than women, and older and higher educated individuals consider a flandrien more as a Flemish cyclist than younger and lower educated individuals respectively. Moreover, the more strongly respondents claim to be cycling fans, the less they consider a flandrien as a Flemish cyclist.First, gender . In other words, the more strongly Flemings endorse an ethno-cultural identity representation, the more they consider a flandrien as a Flemish cyclist. Third, the same relation was found regarding Flemish identification (β = .08 p = .002). That is, the more Flemings identify themselves as Flemish, the more regionally exclusive their interpretation of a flandrien in terms of considering a flandrien as a Flemish cyclist, which confirmed Hypothesis 3a.Second, in line with Hypothesis 2a, ethno-cultural identity representation was a significant positive predictor of the consideration of a flandrien can also be a foreign cyclist’  = 40.17, p < .001). The results of step 2 revealed that, when statistically accounting for the background variables, including ethno-cultural identity representation and Flemish identification contributed significantly to the prediction of the inclusion of international cyclists in the consideration of a flandrien  = 26.06, p < .001). The results of step 3 showed that the interaction term did not contribute significantly to the inclusion of international cyclists as flandriens  = 0.44, p = .51). This result contradicts Hypothesis 4b. Therefore, the predictors of the third step were not included for the interpretation of the regression coefficients.In the first step the background variables accounted for 8.3% of variance in the item ‘A p = .006), cycling fandom , age  and education  were significant predictors. This means that men include international cyclists more in their consideration of a flandrien than women, and older and higher-educated Flemings include international cyclists less in their consideration of a flandrien than younger and lower educated Flemings, respectively. Furthermore, the more strongly respondents claim to be cycling fans, the more they include international cyclists in their consideration of a flandrien.First, gender . That is, the more Flemings endorse an ethno-cultural identity representation, the less they will include international cyclists in their consideration of a flandrien. This finding is in line with Hypothesis 2b. In line with Hypothesis 3b, Flemish identification was no significant predictor in this model .As expected, ethno-cultural identity representation emerged as a significant, negative predictor  = 74.50, p < .001). The results of step 2 revealed that, when the background variables were statistically controlled for, the addition of ethno-cultural identity representation and Flemish identification contributed significantly to the inclusion of international cyclists as flandriens  = 3.19, p = .041). The results of step 3 showed that, when controlling for the background variables and ethno-cultural identity representation, the interaction between ethno-cultural identity representation and Flemish identification did not contribute significantly to the inclusion of international cyclists as flandriens  = 2.39, p = .092). This result contradicts Hypothesis 4b. Therefore, we further only discuss the regression coefficients for the predictors after the second step.Step 1 revealed that the background variables accounted for 14.8% of variance in the average score given to the four international cyclists (p = .002), cycling fandom , age  and education  emerged as significant predictors. This means that men and older respondents include international cyclists more in their consideration of a flandrien than women and younger respondents, respectively, and that higher educated respondents include international cyclists less in their consideration of a flandrien than lower educated respondents. Furthermore, the more strongly respondents are cycling fans, the more they include international cyclists as flandriens.First, gender . That is, the more Flemings endorse an ethno-cultural identity representation, the less they include international cyclists in their consideration of a flandrien. This finding is in line with Hypothesis 2b. Again, Flemish identification was no significant predictor in this model , which confirmed Hypothesis 3b.As expected, ethno-cultural identity representation was a significant, albeit modest, negative predictor  attitudes and norms in sports contexts.An important finding was that the more Flemings endorse an ethno-cultural identity representation, the more they protect the meaning of a flandrien as a Flemish cyclist, thereby confirming Hypothesis 3a. Considering that a flandrien is generally regarded as a positively valued term, Flemings who strongly identify themselves as Flemish want to ascribe the term to their ingroup, thereby aiming to verify the positive view they (want to) have on their group membership. This finding is in line with previous findings regarding the positive relation between ingroup identification and ingroup favouritism  as cycling fans than as Flemish persons, because they were addressed as cycling fans to fill in the questionnaire. As a consequence, their social identity of ‘cycling fans’ was more salient to them than their social identity of a Flemish person.flandrien than women. However, additional analyses showed that on average, men (M = 4.47) were stronger cycling fans than women (M = 3.73) (t(2006) = –13.44, p < .001). For this reason, the aforementioned explanation with respect to the relation between cycling fandom and regional exclusivity can also account for the finding that men are less regionally exclusive than women.A second interesting finding is that men are less regionally exclusive in the use of the term flandrien in symbolizing a Flemish identity. By using regression analyses on a large-scale internet-survey data, the present study extends previous studies, which only used qualitative material  to study this topic. This approach, including the large sample size, elaborates previous qualitative findings that simply describe the existence of differences in perception of a flandrien  can be considered to be more abstract than the concrete list of cyclists. The fact that we found similar relations between these outcomes on the one hand and Flemish identification and ethno-cultural identity representation on the other hand testifies to the validity of our findings.One of the major strengths of the present study is that we adopted a quantitative approach to investigate the importance of a landrien , by inclflandrien in terms of regional exclusivity, this effect was not particularly pronounced. In line with the findings regarding the other predictors, this indicates that other factors, such as cycling fandom, have a stronger influence on the perceived regional exclusivity of a flandrien.A first limitation of this study addresses the relatively small effect sizes (0.3%–6%) of our focal predictors: ethno-cultural identity presentation and Flemish identification. This means that although endorsing an ethno-cultural identity representation and Flemish identification did predict the interpretation of a flandrien. That is, respondents had to rate to which they considered four international cyclists as a flandrien. We chose only these four cyclists because we expected them to be largely known amongst Flemings and demonstrate the typical ‘Briek Schotte-characteristics’. This enables to really capture the regional aspect of these cyclists and limits the possible confounding influence of these characteristics although we acknowledge that this choice is subjective, but still based on the opinion of cycling experts.A second limitation regards one of this study’s operationalizations of the inclusion of international cyclists as A third limitation of this study is that an ethno-cultural identity representation, as a combination of an ethnic and cultural identity representation, has never been studied as an identity representation in itself. Traditionally, the most occurring distinction is between a civic and ethnic identity representations. More recently, Kymlicka  argued fflandrien in terms of regional exclusivity is not shared by all Flemings and is influenced by their identity representation. On the one hand, the results of this study reveal that a flandrien, a historically contested cycling term, is still considered as an important symbol for the Flemish identity. On the other hand, adhering to an ethno-cultural identity representation seems to influence Flemings’ perception of this identity-relevant symbol in terms of changeability. In spite of his heroic performances and match with behavioural characteristics, Flemings with an ethno-cultural identity representation will less likely consider the cycling champion Cancellara as a flandrien, simply because he is not a Fleming.To conclude, this study demonstrates that the expansion of the term The additional file for this article can be found as follows:pb-58-1-358-s1.savSupplementary file 1 Dataset. DOI: https://doi.org/10.5334/pb.358.s1


British Journal of Cancer1 and contributing to the evaluation of the potential risk of HPV-related second cancer in patients with a history of a primary HPV-related cancer in the same or an independent mucosal site.We read with a great interest the meta-analysis of Gilbert D.C. et al. recently published in 2 and it is now well established that HPV infection is involved in the oncogenesis of genital  or oropharyngeal carcinomas. Patients with primary HPV squamous cell carcinoma may have increased risk to develop HPV-related second malignancies, in the same or in another mucosal site.8 However, the vast majority of available data regarding the risk of developing secondary cancers or high-grade intraepithelial lesions for a patient who has presented an index HPV-induced cancer originate from retrospective studies essentially focused on two mucosal sites1 and as it was discussed by Gilbert et al., all of these retrospective studies are very heterogenous.Oncogenic high-risk HPV (HR-HPV) are involved in about 5% of overall cancersEven if the tendency towards an increased risk was observed in all studies and across each of the mucosal sites, it is still difficult to clearly recommend a systematic follow-up for these patients and to define how this monitoring should be carried out. Indeed, the studies are very heterogenous and present clear limitation due to their retrospective design, especially for the ones which reported rates of a second independent cancer after an initial diagnosis of primary HPV-associated cancer.The European Georges Pompidou Hospital  takes advantage of a large recruitment of patients suffering from HPV-related cancer , as well as HPV biological and clinical experts. Diagnosing and treating HPV-induced cancers represents a unique opportunity to prevent subsequent HPV-associated cancers with a multidisciplinary follow-up. We have created in 2014, for the first time to our knowledge, the so-called CoMPap (Consultation Multidisciplinaire Papillomavirus) HPV multidisciplinary consultation based on an annual prospective monitoring of patients with a history of HPV-related primary tumour. To be enroled, the patients have to sign a dedicated informed consent approved by the ethical committee.Included patients are annually examined by clinicians  to detect macroscopic mucosal lesion in all HPV-concerned mucosal site. In parallel, pap-smears are collected for cytological and virological evaluation. Different infection sexually transmitted serological status are checked and a psychologist can also be requested for patient care if indicated. Finally, the CoMPap consultation allows an innovative medical care of the HPV cancers allying supervision, prevention, psychological support and biobanking.n = 41; 69.5%) essentially enroled after in situ or infiltrative genital carcinoma diagnosis. Eighteen men were also included and monitored.Until now, 59 patients (median age 55 years) have been enroled in the CoMPap. The cohort includes a majority of women  to closely monitor HPV-related cancer patients in prospective cohorts to prevent or diagnose at an earlier curable stage of these potential secondary malignancies.


Background: We aimed to investigate whether infection with high-risk (HR) types of human papilloma virus (HPV) or HPV-associated cervical disease were associated with preterm birth (<37 weeks gestation). In a sub-group of younger women who were eligible for the HPV vaccine, we aimed to determine whether prior vaccination against the specific HPV-types, HPV-16 and -18 modified preterm birth risk.Methods: This was a data-linkage study, which linked HPV-associated viral and pathological information (from the Scottish HPV Archive) from women aged 16-45 years to routinely collected NHS maternity- and hospital-admission records from 1999–2015. Pregnancy outcomes from 5,598 women with term live birth , preterm birth  or early miscarriage . Of these, data from HPV vaccine-eligible women  were available, of whom 588 had been vaccinated. HPV-associated disease status was defined as: HR HPV-positive no disease, low-grade abnormalities or high-grade disease.Results: High-grade HPV-associated cervical disease was associated with preterm birth  in adjusted binary logistic regression analysis, in all women, but there were no associations with HR HPV-infection alone or with low-grade abnormalities. No associations between any HPV parameter and preterm birth were seen in vaccine-eligible women, nor was there any effect of prior vaccination.Conclusions: HPV-associated high-grade cervical disease was associated with preterm birth, but there were no associations with HR HPV-infection or low-grade cervical disease. Thus HPV-infection alone  does not appear to be an independent risk factor for preterm birth. For women who have undergone treatment for CIN and become pregnant, these results demonstrate the need to monitor for signs of preterm birth. Persistent HPV infection causes pre-cancerous cervical intraepithelial neoplasia (CIN); left untreated, CIN can develop into cervical cancer2. Over 200 HPV types exist, of which 14 are oncogenic or high-risk (HR). Two such HR types, HPV-16 and -18, are responsible for 70–80% of invasive cervical cancers3. Treatment of CIN and cervical cancer is by removal of the abnormal cervical cells4. Human papillomavirus (HPV) is a sexually transmissible virus that infects cervical cells. Normally, the virus is cleared by the immune system, becoming undetectable within two years5. HPV-infection may alter cervical function, possibly increasing the risk of intrauterine infections6 and subsequent complications such as preterm birth7 or miscarriage8.The cervix is important for maintenance of pregnancy, with barrier and immune mechanisms that protect the growing foetus10, whereas others have suggested that HPV-infection, cervical disease and/or its treatment are associated with adverse pregnancy outcomes, such as miscarriage11, premature preterm rupture of membranes (PPROM)12 or spontaneous preterm birth14.The relationships between HPV infection and pregnancy outcomes are unclear. Some studies have shown no associationWe performed a data-linkage cohort study, linking women with HPV viral and pathological data with their own pregnancy records. The aims for this study were to determine whether HR HPV-infection, specific HR HPV types, or the presence of HPV-associated cervical disease were associated with preterm birth (<37 weeks gestation), early miscarriage (<13 weeks gestation) or stillbirth (pregnancy loss at ≥24 weeks gestation) and whether previous vaccination against HPV-16/18 affected these outcomes.This study was a data-linkage study, linking data from women with HPV viral and pathological data contained within the Scottish HPV Archive (see below) with their own pregnancy records. After data-cleaning, the retrospective data from the women in the resulting cohort were analysed for associations between the HPV viral/pathology parameters and adverse pregnancy outcomes: preterm birth (<37 weeks gestation), early miscarriage (<13 weeks gestation) and stillbirth (≥24 weeks gestation). The study population for whom data were obtained and linked were women who had samples and data within the Scottish HPV Archive and who had had details of pregnancy outcomes.1 and women are advised to delay their next scheduled cervical smear until at least 12 weeks postnatally4, women who had a pregnancy up to 1 year prior to the cytology date were also included. In women who had had more than one pregnancy, the pregnancy outcome nearest the cytology date was included.Women whose HPV-infection status was known at the time of birth were included. As HPV is usually cleared within 2 yearsExclusion criteria for the study were: Multiple (twin) pregnancies, as these babies are more often routinely delivered preterm; therapeutic terminations, ectopic pregnancies and trophoblastic disease were also excluded.Term live births were women who had a live birth at term (≥37 weeks gestation);All preterm births were women with births before 37 weeks’ gestation including miscarriages of ≥13 weeks gestation;Spontaneous preterm births were women with births prior to 37 weeks’ of gestation, including those with PPROM and miscarriages of ≥13 weeks gestation, but excluding iatrogenic preterm births (those delivered by elective caesarean section or those which were induced);Early Miscarriage was any spontaneous pregnancy loss before 13 weeks’ gestation;Stillbirth was defined as any pregnancy loss after 24 weeks’ gestation.The definitions for pregnancy outcomes were:Scottish HPV Archive. Scotland has a cervical screening programme with population coverage of around 70%15. Until June 2016, women aged 20–60 years were screened; thereafter the age-range was 25–65 years. In September 2008, a routine, school-based HPV vaccination programme for girls aged 11–13 years was introduced, with a “catch-up” immunisation programme for girls aged 13–17 years16. In 2009, the Scottish HPV Archive was established as a biobank for HPV-associated research. It is a collection of collections which includes residual cervical samples from women attending routine cervical screening and colposcopy clinics, from the year 2000 to the present. Many samples within the Archive have a patient identifier  and associated clinical, cytology and histology results, obtained from the Scottish Cervical Cytology Recall System (SCCRS). SCCRS is the national IT system that supports the Scottish cervical screening program and contains a woman’s entire screening record4. A number of samples in the Archive are annotated with HPV status as a consequence of immunisation surveillance and specific research projects18, using different HPV assays depending on the nature and objectives of the research projects (rojects . At the Scottish Morbidity Record (SMR)02. SMR02 is the Maternity Inpatient and Day Case Dataset19, held by the Information Services Division (ISD) of the National Health Service (NHS) National Services Scotland (NSS) and covers maternity admissions in Scotland. SMR02 includes over 98% of deliveries, as less than 2% of deliveries occur at home. A quality assurance audit of SMR02 showed that accuracy of the records exceeded 90%, when compared with corresponding written medical records20. SMR01. SMR01 is the General/Acute Inpatient and Day Case Dataset, held by NHS NSS ISD, and covers hospital admissions in Scotland19. Audit of SMR01 found that accuracy of clinical data was more than 89%, when compared with written medical records22. Miscarriage data were obtained from both SMR01 and SMR02. If an SMR01 record had no corresponding SMR02 record, we assumed that the miscarriage occurred before 13 weeks’ gestation, as it is usual practice for pregnancies to be registered on SMR02 before 13 weeks gestation23. Scottish birth statistics from the Scottish population, obtained from SMR02 and SMR01, are available from the NHS NSS ISD website, Births in Scottish Hospitals23.24; such cases would not generate a hospital or maternity record. Women admitted to a hospital ward via an emergency or gynaecology department or early pregnancy unit generate an SMR01 record; women admitted to a maternity unit generate an SMR02 record. Between 1998 and 2016, the population rate of miscarriages managed in Scottish hospitals (data from SMR01 and SMR02) fell from 7.0 to 4.4 per 1000 women aged 15–44 per year23. From these numbers, the percentage of miscarriages in the Scottish birth data for each year was calculated as:Miscarriage may occur before the mother is aware she is pregnant, and is often managed in an outpatient or general practice settingFrom this calculation, for the years 1998–2016 the percentage of miscarriages approximated to 8.0- 12.0% of all births per year.National Records of Scotland (NRS). This resource hold the records of stillbirths and infant deaths in Scotland25.26. The Safe Haven is a remote server through which the researcher accesses the health data and services to enable research while protecting the confidentiality of the data. Data remains under the control of the NHS and complies with legislative and NHS policies. The linked dataset is archived within the Safe Haven and is available by application to NHS Scotland via the electronic Data Research and Innovation Service (eDRIS)27, within the Farr Institute for Health Informatics Research.Approval for use of NHS NSS and NRS data was obtained from the NHS Privacy Advisory Committee (PAC 74/14). Approval for linkage of previous unlinked datasets was given by the NHS Scotland National Caldicott Guardian Forum (Ref 2015-19). The linked dataset was analysed within the NHS National Safe Haven, provided by NHS Research ScotlandThe data-linkage and -cleaning processes are summarised in23 to determine how representative our cohort was of the Scottish population.Pregnancy admission records from SMR02, SMR01 and NRS records for women in the Scottish HPV Archive were received from January 1981 to August 2015. Many of the older women within the Scottish HPV Archive had their pregnancies before their HPV-infection status was known and so were excluded. Records in the Scottish HPV Archive started in 2000, so pregnancy admissions before 1999 were removed; therefore, the time-period for this study was 1999–2015. Duplicate records were removed. Information from multiple admissions per pregnancy outcome were condensed into one record. The variables obtained from SMR01, SMR02, NRS records and the Scottish HPV Archive are presented inHR HPV-positive if there was a positive result for at least one HR HPV type ; low-grade abnormalities  or high-grade disease .HPV vaccination status. Some women had received the bivalent HPV vaccine17 (1–3 doses) against HPV-16 and -18. Vaccine-eligible women were classified as: those who had been vaccinated, those eligible (by age) but unvaccinated, or those eligible but whose vaccination status was unavailable; women born before 1990 were too old to have been eligible for vaccination.4, but data for the specific treatments used were not available.Scottish National guidelines require over 90% of women with HPV-associated high-grade cervical disease to have undergone excisional treatment28, as HPV results are not recorded as part of routine cervical screening. Pathology data (low- and high-grade disease) were compared with data from ISD29.To determine how representative our cohort was of the Scottish population, the proportions of women with specific HPV viral/pathology parameters within our final cohort, were compared with a recent HPV prevalence study in women attending screening in ScotlandPotential confounders for pregnancy outcomes were obtained from SMR02 and SMR01: ethnicity, parity, Scottish Index of Multiple Deprivation (SIMD), smoking history and during current pregnancy, diabetes  and hypertension (any). Coding and definitions for these variables are shown in30. For the logistic regression analyses, pooled results from five imputations are presented in the results. The HPV viral/pathology parameters  were not imputed, as these were the primary predictive factors of interest for the analyses.All statistical analyses were carried out using SPSS v22 . Missing data analysis  of the cThe proportions of maternal characteristics according to pregnancy outcomes or according to HPV viral/pathology parameters were compared by chi squared or Fisher’s exact test analysis and are shown inPregnancy admission records  were received for women who had been pregnant and had HPV viral/pathology data in the Scottish HPV Archive . After data-cleaning and exclusions, we had obtained 7,512 pregnancy outcomes from 5,626 women . Of thes23, the age distribution of the cohort was much younger than that of the Scottish childbearing population. There were more pregnancy outcomes in women of <25 years (62.9%) than in the Scottish population23 (26.5%) and a smaller proportion of mothers aged 25–34 years (28.8% vs. 53%) or ≥35 years (8.3% vs. 20.5%) in our cohort, respectively. The percentage of all women who had a preterm birth in our cohort (6.9%) was similar to that of the Scottish population23, which ranged between 5.8 and 6.7% (between 1999 and 2015). The percentage of women having hospital admission for early miscarriage in our population was much lower (4.8%) than that in the Scottish birth data23. Due to this low rate of miscarriage, the miscarriage data were deemed to be problematic due to uncertainty in case ascertainment and was thought to not be representative of the Scottish child-bearing population. Therefore, the results for early miscarriage are not presented.Women had had term live births ; all preterm birth  and early miscarriage . On comparison with Scottish birth statistics28, probably due to the nature of ‘selective’ sampling in the Scottish HPV Archive. Similarly, the percentage of women with HPV-16/18 was higher than that in the routine prevalence in the same population . For HPV-associated disease, some women were HR HPV-negative or of unknown genotype but showed evidence of HPV-associated disease. The proportion of women with low-grade disease was higher in our cohort than in the national screening data  and our cohort had a higher percentage of women with high-grade cervical disease .The characteristics of the women according to the HPV viral/pathology parameters are shown inThe numbers and percentages for the maternal factors were compared by pregnancy outcomes  or low-grade HPV-associated cervical disease and all or spontaneous preterm birth.14. In this study, such associations were not seen in the younger, vaccine-eligible subgroup of women, suggesting that the older women were infected for longer and had time to develop high-grade cervical disease31. Previous studies showing associations between HPV-associated cervical disease and preterm birth have been ambiguous regarding whether these were due to HPV-infection itself, development of associated CIN lesions or the excision of these lesions33. Low-grade cervical lesions are more likely to be associated with productive HPV-infections, where the viral life-cycle is completed, compared with infections associated with severe disease where the viral life-cycle is poorly supported34. Productive infections are also associated with a peak/burst of viral load over a narrower time-frame than infections associated with high-grade disease35. Therefore, in the absence of any associations with HR HPV-infection alone or low-grade cervical disease, and given that over 90% of those who had high-grade disease would have been treated4, we suggest that the association with high-grade cervical disease, in our cohort, is likely to be due to the treatment of HPV-associated cervical disease rather than a direct effect of HPV-infection itself. The clinical implication is that women who have undergone treatment for HPV-associated cervical disease should be closely monitored in subsequent pregnancies for any signs of preterm birth.Associations between high-grade cervical disease and all preterm birth or spontaneous preterm birth have been suggested in previous studies36. Diabetes and hypertension were also independently associated with all preterm birth, but this was not surprising as these are likely to be the indicative reasons for early delivery of the baby. A recent study suggested that HPV infection in the placenta was associated with a number of pregnancy complications37, including gestational diabetes. However, our data did not find an increased association between HPV viral/pathology data and diabetes and so do not support such an association.Although only HPV-associated high-grade cervical disease was associated with all preterm birth and spontaneous preterm birth, current smoking was consistently associated with spontaneous preterm birth. Smoking is a previously recognised risk factor for spontaneous preterm birthThere are strengths and weaknesses for this study. The strengths of this study are that this was a data linkage study starting with women for whom HPV-infection status was known. The use of the Scottish HPV Archive meant that the HPV viral/pathology parameters for these women had been robustly characterised and the HPV information was not available from any other source. The use of routinely collected data should also reduce bias in the recording of clinical information. Primary HPV screening in Scotland will soon include results from HPV testing. Such information may facilitate a future population-wide analysis of the effects of HPV-infection and pregnancy outcomes in the Scottish population.23, and therefore, it is hard to determine how representative these results are of the wider population. The study cohort was drawn from the general population by the fact that they underwent cervical screening, but were considerably younger at pregnancy outcome and had higher proportions of women with HPV-infection and HPV-associated cervical disease, due to the selective nature of the samples within the Scottish HPV Archive. The preterm birth rates were similar to that in the Scottish population. The early miscarriage data were obtained from maternity/hospital admission records, but would not include miscarriages managed at home, through outpatient and emergency departments or by General Practitioners. The risk of miscarriage increases with age, particularly after the age of 40 years, although a slightly increased risk of miscarriage exists in women aged under 20 years38. The miscarriage rate in our population was much lower than the that reported for miscarriages managed in hospitals23. While this lower rate may particularly reflect the youth of our cohort, the differences are hard to explain, and so these data were not presented.A weakness of the study is that we did not have details of any treatments for cervical disease, so we have had to rely on an assumption that women with high-grade disease received excisional treatment as per national guidelines. The age-distribution of the women did not reflect that of the Scottish childbearing population13.In summary, our data linkage study has found no evidence that HPV infection per se, or low-grade HPV-associated cervical disease was associated with preterm birth. High-grade disease, the majority of which is treated with excisional treatments, was associated with spontaneous preterm birth. This is consistent with previous evidence linking excisional cervical treatment with increased risk of spontaneous preterm birthhttps://www.isdscotland.org/Products-and-Services/eDRIS/).The linked dataset was analysed within the NHS National Safe Haven, provided by NHS Research Scotland. The Safe Haven is a remote server through which the researcher accesses the health data and services to enable research while protecting the confidentiality of the data. Data remains under the control of the NHS and complies with legislative and NHS policies. The linked dataset is archived within the Safe Haven and is available by application to NHS Scotland via the electronic Data Research and Innovation Service (eDRIS), within NHS National Services Scotland  is an important area of research because it is the major cause of neonatal mortality and morbidity worldwide, and infection is a major cause, particularly at low gestational ages.Scientific merit: I am not qualified to comment on the use of registry data, the statistical methods in general or the use of imputation techniques in particular. Otherwise I have no problem with the conduct or methodology of the study.CommentsBonde U, et al. The potential role of HPV vaccination in the prevention of infectious complications of pregnancy1.Bearing in mind the paucity of research in his area, the authors should consider citing the review of adverse outcomes of pregnancy in association with HPV . The authors should consider alluding to the safety or otherwise of HPV vaccination in pregnancy (3). This should be mentioned.The study by Bruinsma (cited as Reference 14) had significant flaws and these were highlighted in the subsequent correspondence-pyrene-dio-epoxide (BPDE), the metabolite of a chemical carcinogen in cigarette smoke, can be found in the vaginal secretions of women who smoke. With increasing concentrations of BPDE, there is an increase in the frequency of phage released. Cigarette smoke chemicals promote phage induction in vaginal lactobacilli, and these phages lyse other vaginalLactobacillus strains. Tobacco products can be found in the cervical secretions of women with CIN and in the semen of smokers. Bacterial vaginosis is more common in women who smoke and both BV and smoking are linked by common sequelae such as PTB. It is therefore possible that female smokers secrete, or their partners who smoke, may introduce, by sexual transmission, tobacco products like BPDE into the vagina, which puts them at greater risk of BV by inducing endogenous or sexually acquired Lactobacillus phages to become lytic and an increased the risk of PTB. The authors record that current smoking was independently associated with an increased risk of PTB, but did not speculate on the link. One possibility is through temporate phage viruses being provoked to become lytic by cigarette smoke carcinogens in cervicovaginal secretions. Diet acquired phage viruses, may be induced to become lytic by factors related to sexual activity, and per se. This did not become clear to me until the last sentence of the discussion. Could this be made clearer earlier in the manuscript.The clear message to me was, that PTB in association with HPV and CIN, was more likely to be due to the treatment of HR-HPV with HGCIN, than the presence of HR-HPV and HGCIN I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This study is an important and timely contribution to the evidence base around the influence of oncogenic HPV subtypes and pre-term labour. It is excellently written. I am not an expert in statistics but the methodology looks fine. Previous reports by Bruinsma cited by the author have suggested that the presence of dysplasia of all types potentially increases the risk of preterm labour whilst there is clear evidence that excisions treatment significantly increases risk particularly if excisions are of excessive volume/depth. This linkage study from robust databases within the Scottish health care system relating HPV status and pregnancy outcomes is very timely as it clearly demonstrates that having HPV infection alone without treatment does not influence pregnancy outcomes . There is growing evidence that observational management for low and intermediate grade cervical precursors leads to high rate of spontaneous regression and therefore excisional treatment can be avoided. Women can have the re-assurance that their risk of adverse pregnancy outcomes is not increased by HPV transient disease.  Furthermore, with the advent of primary HPV screening, when significant proportion of women are going to found to be High risk HPV positive but disease free, can also be re-assured by this data that this will not increase the risk of pre-term delivery etc. I think this study is an important contribution and should be shared with individuals designing information resources for women regarding HPV testing etc.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Saccharomyces cerevisiae that covers all nine types, raising the need for microbial production of broad-spectrum HPV vaccines. Here, we investigated whether N-terminal truncations of the major HPV capsid proteins L1, improve their soluble expression in Escherichia coli. We found that N-terminal truncations promoted the soluble expression of HPV 33 (truncated by 10 amino acids [aa]), 52 (15 aa), and 58 (10 aa). The resultant HPV L1 proteins were purified in pentamer form and extensively characterized with biochemical, biophysical, and immunochemical methods. The pentamers self-assembled into virus-like particles (VLPs) in vitro, and 3D cryo-EM reconstructions revealed that all formed T = 7 icosahedral particles having 50–60-nm diameters. Moreover, we formulated a nine-valent HPV vaccine candidate with aluminum adjuvant and L1 VLPs from four genotypes used in this study and five from previous work. Immunogenicity assays in mice and non-human primates indicated that this HPV nine-valent vaccine candidate elicits neutralizing antibody titers comparable to those induced by Gardasil 9. Our study provides a method for producing a nine-valent HPV vaccine in E. coli and may inform strategies for the soluble expression of other vaccine candidates.Human papillomavirus (HPV) is the causative agent in genital warts and nearly all cervical, anogenital, and oropharyngeal cancers. Nine HPV types  are associated with about 90% of cervical cancers and 90% of genital warts. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent HPV-associated diseases. However, there is only one commercially available HPV vaccine, Gardasil 9, produced from  E. coli and their self-assembly of T = 7 icosahedral particle in vitro• N-terminal truncations promote the soluble expression of HPV L1 proteins in E. coli-derived HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58 VLPs, and conferred comparable immunogenicity with Gardasil 9• An HPV 9-valent vaccine candidate was formulated with  Nearly 200 HPV types have been identified to date, with over 15 high-risk types closely linked to cervical cancer and other epithelial tumors4. Cervical cancer is one of the most common types of female cancers, of which 88% are caused by HPV types 16, 18, 31, 33, 45, 52, and 586. Specifically, HPV 33, 45, 52, and 58 account for 15% of cervical cancers worldwide8. HPV 6 and HPV 11, which are considered as low-risk HPV subtypes, are usually not connected with cancer but are responsible for more than 90% of genital warts9. There are currently three prophylactic HPV vaccines on the market: Gardasil , a quadrivalent vaccine containing HPV 6, 11, 16, and 18 antigens10; Gardasil 9 (Merck Sharp and Dohme), a 9-valent vaccine covering HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58 antigens11; and Cervarix , a bivalent vaccine against HPV 16 and 18 antigens12.Human Papillomavirus (HPV) is a small, non-enveloped epitheliotropic DNA virus associated with multiple human diseases, such as benign verrucae vulgares (common wart), condylomata acuminata , and malignancies of the cervix, vulva, anus, and penisT = 7 icosahedral particle containing two capsid proteins, L1 and L214. The major capsid protein, L1, can be generated by recombinant expression in insect cells (using baculovirus vectors), yeast cells, plant cells, and E. coli cells, and self-assembles into virus-like particles (VLPs) that closely resemble the native papillomavirus virion16. HPV L1 VLPs are highly immunogenic antigens and can induce the production of high-titer neutralizing antibodies, conferring protection against HPV infection18. The market-available HPV prophylactic vaccines are all designed based on L1 VLPs and were generated from Saccharomyces cerevisiae or insect cells. Other expression systems have also been used to produce HPV L1 proteins, such as Escherichia coli (E. coli), Bacillus subtilis, Salmonella typhimurium, silkworm larvae, and plant mammalian cells21. Among these, E. coli has been shown to be an efficient and versatile tool for producing recombinant proteins, offering the advantages of rapid growth rate, inexpensive media for growth, and ease of purification24. E. coli has also been used to express L1 proteins of HPV using GST or β-galactosidase fusion, which helps to alleviate the difficulty associated with the formation of insoluble inclusion bodies30.Cryo-EM structures of virus capsids have shown that the HPV virion assumes a T = 1 small particles, and this suggested that the N-terminal region of the protein was involved in the VLP assembly process32. Further research found that the 5′-end of the HPV L1 gene contained an element that could negatively affect the expression of L1 proteins in human epithelial cells33, and showed that point mutations or deletions in the inhibitory element to inactivate the effector could improve the production of L1 proteins24. However, the exact location of this regulatory region might vary with different HPV types, with maximum protein expression levels achieved with different N-terminal truncations for HPV 6, 11, 16, 18, and 3136. Therefore, it is necessary to investigate the effects of the N-terminal region on L1 expression for different HPV types for the successful and efficient manufacturing of an E. coli-based HPV vaccine.Previous studies on HPV particle assembly have shown that an N-terminally truncated L1 protein lacking over 10 aa could assemble into E. coli36, among which the HPV 16/18 bivalent vaccine and HPV 6/11 bivalent vaccine have been in phase 3 and phase 2 clinical trials, respectively. The L1 proteins were produced through non-fusion expression, and harbored N-terminal deletions (except for HPV 31) that rendered them still capable of self-assembling into T = 7 particles. Here, we successfully generated L1 VLPs for another four HPV types, HPV 33, 45, 52, and 58, using E. coli expression, and found that the non-fusion soluble expression of recombinant HPV 33, 52, and 58 L1 genes could be increased markedly with different N-terminal truncations. We then employed a combination of biochemical and biophysical methods to characterize the structural and antigenic properties of these four VLPs. Combined with our previous L1 VLPs, here we present an E. coli-based HPV 9-valent vaccine capable of inducing high and durable neutralizing antibody titers against HPV 6,11,16,18,31,33,45,52,58 infection. Overall, our results introduce a feasible method to optimize the soluble expression of proteins in a bacterial expression system. Furthermore, our 9-valent VLPs generated from E. coli offer another highly potent, second-generation, prophylactic HPV vaccine that could benefit the costs associated with worldwide vaccine production and distribution.In our previous work, we successfully produced high-potency VLP vaccine candidates of HPV 6, 11, 16, 18, and 31 using 37, suggesting that deletion of these residues might not affect the formation of T = 7 particles .The previously solved structure of the HPV 16 pseudovirus (PDB no:5KEP) highlighted that the initial few amino acids of HPV 16 L1 proteins were not involved in inter-pentameric contactsles Fig. . In our E. coli. , HPV 45 (aa 1–513), HPV 52 (aa 15–503), and HPV 58 (aa 10–498)—were then selected for our 9-valent vaccine. To this end, we scaled up the protein production of truncated HPV 33, 45, 52, and 58 L1 proteins to 5 L-scale fermentation. The purified L1 proteins of HPV 33, 45, 52, and 58 were confirmed for consistency by SDS-PAGE and WB , HPV 11 (aa 5–501), HPV 16 aa 5–503), HPV 18 (aa 5–507), HPV 31 (aa 1–504), HPV 33  and differential scanning calorimetry (DSC) to test the primary, secondary structures and thermal properties of HPV 33, 45, 52, and 58 L1 proteins. MALDI-TOF MS was employed to determine the intact molecular weight (MW) of HPV 33, 45, 52, and 58 L1 proteins Fig. . These Hely Fig. .Fig. 2Mo36. Overall, the L1 VLPs of nine HPV types qualified for inclusion in the manufactured HPV vaccine.Purified HPV L1 proteins maintained their pentamer form (about ~8 nm in diameter) under reducing conditions Fig. S, and couE. coli-generated L1 VLPs for the 5 HPV types determined previously 36, particles for HPV 33, 45, 52, and 58 VLPs appeared to be irregularly spherical and heterogeneous in size 41 revealed structural differences in the diameters of the E. coli-generated HPV VLPs    might be another possibility to explain the resolution variance.The morphology of HPV 33, 45, 52, and 58 VLPs was determined by negative staining TEM. Similar to the ers Fig. , lower; nm) Fig. ; this maer) Fig. , which m5 neutralization titer. We then assessed the antibody binding activity of HPV VLPs using indirect ELISA. We found that HPV 33, 45, 52, and 58 L1 VLPs were capable of binding efficiently (>105 ELISA titer) to their corresponding mAbs  calculations36. Mice were separated into groups and vaccinated with the HPV 9-valent vaccine . Sera were collected after 5 weeks to analyze neutralizing antibody titers, and analyzed according to the Reed and Muench statistics43. The ED50 values for all nine HPV types were lower than 10 ng  at 0, 2, and 4 weeks with a 20-, 200- or 2000-fold diluted dosage of our 9-valent vaccine and commercial Gardasil 944. Sera were drawn at 2 weeks after the third vaccination to detect the neutralizing antibody titers. We found that mice receiving as low as 0.135 μg of E. coli-generated 9-valent vaccine were capable of inducing about 102.36, 102.70, 102.36, 102.60, 102.60, 102.88, and 102.74 anti-HPV 6, 11, 31, 33, 45, 52, and 58 neutralization titers, and 103.75 and 104.02 against the rest two types, HPV 16 and -18  after the last inoculation, with neutralization titers of 104.2, 103.6, 104.1, 103.9, 103.8, 104.2, 104.1, 104.2, and 104.5, respectively   might have also affected the resolution.The SDS-PAGE and MALDI-TOF experiments found similar molecular weights of the HPVs for the nine L1s. All of the L1 proteins in the 9-valent vaccine had the desired characteristics, with similar secondary structure composition, robust thermal stability, and the ability to self-assemble into a VLPs in vitro. Purified HPV 33, 45, 52, and 58 L1 VLPs showed variable particle size but with a mean of ~55 nm, similar to that previously reporteder) Fig. , and thiE. coli-based 9-valent vaccine confers comparable neutralization titers against HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58 infection to that induced by Gardasil 9 .The full-length gene fragments of HPV 33, 45, 52, and 58 L1  were synthesized by GLS . DNA sequences coding for HPV 52 and 58 L1 proteins were from the second initiation codon. The synthesized gene fragments were used as template for PCR amplification, then a series of N-terminally truncated HPV L1 genes were subsequently cloned into 35. In brief, cells were grown at 37 °C overnight in LB medium and the L1 protein expression was induced by the addition of isopropyl-β-d-thiogalactoside  upon reaching an OD600 of 0.6, and then further incubated at 25 °C for 8 h. Cells were harvested by centrifugation and resuspended with cell lysis solution . HPV 33, 45, 52, and 58 L1 proteins were released from the cells by sonication and separated from the cell debris by centrifugation. The supernatant was then combined with 20 mM DTT and further purified by SP sepharose  and CHT-II resin . Samples eluted in the 800 mM NaCl fraction. After purification, the proteins were analyzed by SDS-PAGE and dialyzed into neutral buffer (PBS) without a reducing agent to allow VLP self-assembly. The concentration of HPV L1 VLPs was determined using the BCA method.N-terminally truncated HPV 33, 45, 52, and 58 L1 proteins were produced as previously describedAluminum hydroxide adjuvant was used to absorb HPV 6, 11, 16, 16, 18, 31, 33, 45, 52, and 58 L1 VLPs, respectively. The VLPs were then mixed together. The final product of the HPV 9-valent vaccine contained 30 μg HPV 6 L1 VLPs, 40 μg HPV 11 L1 VLPs, 60 μg HPV 16 L1 VLPs, 40 μg HPV 18 L1 VLPs, 20 μg HPV 31 L1 VLPs, 20 μg HPV 33 L1 VLPs, 20 μg HPV 45 L1 VLPs, 20 μg HPV 52 L1 VLPs, and 20 μg HPV 58 L1 VLPs with 0.42 mg aluminum hydroxide adjuvant suspended in 1.0 mL phosphate-buffered saline (PBS). Gardasil 9 HPV vaccine (Lot no. N023354) was purchased from Hong Kong SAR China.50. BALB/c mice were immunized subcutaneously three times at 2-week intervals  with nine HPV monovalent vaccines, respectively. The anti-HPV L1 mAbs were produced from mouse ascites fluid and were screened by HPV L1 VLP-based ELISA and a pseudovirion-based neutralization assay. Protein A affinity chromatography was used to purify the anti-HPV mAbs IgGs. The purified mAbs were diluted to 1 mg/mL and stored in PBS at −20 °C.HPV L1-specific mouse mAbs were produced using the hybridoma technology previously described51.HPV L1 proteins were analyzed by SDS-PAGE using 10% performed gels. For western blotting, electrophoresed samples transferred to nitrocellulose membranes, were probed with HPV L1 mice antibodies and alkaline phosphatase-conjugated secondary antibody, and scanned as described previously38, HPV 33, 45, 52, and 58 VLPs were treated using reducing and alkylating conditions before tryptic digestion. The peptide fragments and their amino acid compositions were analyzed using the Triple TOF 5600 System fitted with a Nanospray III source  and a pulled quartz tip as the emitter . The mass spectra of HPV L1 peptide mixtures were gained using a Reflex III MALDI-TOF mass spectrometer  with α-cyano-4-hydroxy cinnamic acid as a matrix before the MS analysis.According to previously described38 HPV 33, 45, 52, and 58 VLPs were placed into a 1.0-mm path-length cuvette for far-UV measurements.CD spectra were acquired using a Jasco J-810 spectropolarimeter , as previously described by Zhang et al.38The thermostability of HPV 33, 45, 52, and 58 VLPs was determined using a MicroCal VP-DSC instrument , as previously described by Zhang et al.35.Particle size is an essential parameter of a VLP-based vaccine. The distribution of molecular sizes, homogeneity and sedimentation velocity, hydrodynamic size distributions and overall morphology distribution of purified HPV 33, 45, 52, and 58 VLPs were investigated using size-exclusion chromatography (HPSEC), analytical ultracentrifugation (AUC), dynamic light scattering (DLS), and transmission electron micrograph (TEM), respectively, as previously described41, purified HPV 33, 45, 52, and 58 VLPs, at a concentration of ~3 mg/mL, were vitrified on Quantifoil holey carbon grids in an FEI Vitrobot. Images were recorded on an FEI Falcon II direct detector camera at a nominal ×93,000 magnification in an FEI TF30 FEG microscope at 300 kV, with underfocus settings estimated to be between 1 and 3 μm, and with an electron dose of 25 e/Å2. The T = 7 particle images were manually boxed and extracted with the program Robem52. RELION, AUTO3DEM, and EMAN2 programs were used for image processing and three-dimensional (3D) reconstructions54.As described by Li et al.35 HPV L1 VLPs were coated into the wells of 96-well microplates (100 ng/well). The wells were blocked and then incubated with 100 μL of twofold serially diluted anti-HPV 33, 45, 52, and 58 L1 mAbs. HRP-conjugated goat anti-mouse Ig antibody  was used as a secondary antibody. The absorbance  was recorded using an automated ELISA reader . The cut-off value was set at an absorbance of 450 (Δ620) nm = 0.1 to define the positive titer.The antigenicity of HPV 33, 45, 52, and 58 VLPs was determined by indirect binding ELISA, as described by Pan et al.50 calculation assays, the final HPV 9-valent vaccine was diluted 200-fold, 600-fold, 1,800-fold, 5,400-fold, and 16,200-fold with aluminum hydroxide adjuvant. Fifty special pathogen-free (SPF) BALB/c mice (n = 10 per group) were vaccinated intra-muscularly with a single dose of HPV 9-valent vaccine. Serum samples were taken 5 weeks after immunization to determine the seroconversion of anti-HPV 6, 11, 16, 16, 18, 31, 33, 45, 52, or 58 neutralizing antibodies.For EDE. coli-based HPV 9-valent vaccine with Gardasil 9 vaccines, two groups (n = 4) monkeys were immunized three times with a 1:4 dilution of HPV 9-valent vaccine or Gardasil 9 vaccine at weeks 0 and 4. The serum samples were collected at 6-week post first immunization to detect anti-HPV L1 neutralizing antibodies. To assess the kinetics of virus-neutralizing antibody induced by our E. coli-based HPV 9-valent vaccine, the other two groups monkeys (n = 6) were immunized three times at weeks 0, 4, and 27 with an intramuscular injection of 270 μg HPV 9-valent vaccine; the placebo group received aluminum hydroxide adjuvant only. Serum samples were collected weekly after the first immunization to identify anti-HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58 neutralizing antibodies in a pseudovirus-binding neutralizing assay.The immunogenicity of the HPV 9-valent vaccine was tested in mice and adult Cynomolgus Macaques. For mice immunizations, 4- to 6-week-old female BLAB/c mice were immunized three times at 0, 2, and 4 weeks with intraperitoneal injections of a 1:20, 1:200, or 1:2,000 dilution of HPV 9-valent vaccine, respectively, and Gardasil 9 vaccine served as controls. The sera were harvested at week 6 and subjected to neutralization assay. For the non-human primate study, a total of eight monkeys were used during screening. These monkeys showed no detectable anti-HPV L1 IgG in serum at 1:10 dilution using ELISA testing. To compare the immunogenicity of our 57. HPV 6,11,16,18 L1/L2 expression vector and pN31-EGFP used in the experiment were kindly provided by Dr. J. T. Schiller58. HPV 31, 33, 45, 52, and 58 L1/L2 genes were chemically synthesized by GLS . In briefly, 293FT cell were harvested 72 h after transfection, lysed with cell lysis buffer containing 0.5% Brij58 , 0.2% Benzonase , and 0.2% PlasmidSafe ATP-Dependent DNase  DPBS-Mg solution, and incubated at 37 °C for 24 h. Samples were then combined with a 5 M NaCl solution to extract the cell lysates. The TCID50 (tissue culture infective dose) of the supernatants was then measured to determine the titers of the PsVs, calculated according to the classical Reed–Muench method59.HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58 pseudoviruses were produced according to previous studies57. Briefly, 293FT cells were incubated at 37 °C in the wells of a 96-well plate at a density of 1.5 × 104 cells per well for 6 h. The sera of mouse and monkeys were 2-fold diluted, and PsVs were diluted to 3 × 105 TCID50/µL. Equal volumes (60 µL) of the PsV diluent and the serially diluted sera were mixed and incubated at 4 °C for 1 h. The negative control was prepared by mixing equal volumes (60 µL) of the PsV diluent and culture medium. Then, 100 µL of these mixtures were added to designated wells and incubated at 37 °C for 72 h. After 72 h, the cells were trypsinized and analyzed by flow cytometry. The endpoint titers were calculated as the log10 of the highest sera or antibody dilution with a percent infection inhibition higher than 50%. Every sample was detected at least three times, and the values presented here are calculated as the mean value of all repeats.A pseudovirus-binding neutralizing assay was used to determine anti-HPV L1 neutralizing antibodies within the samples, as previously describedThe EM density map for HPV 33, 45, 52, and 58 VLPs have been deposited in the Electron Microscopy DataBank .Supplementary information