Both vaccines enclose viral antigenic proteins, but differ as to the biological systems of culture and the adjuvant components. Recently, a collection of symptoms, indicating nervous system dysfunction, has been described after HPV vaccination. We retrospectively described a case series including 18 girls (aged 12–24 years) referred to our “Second Opinion Medical Network” for the evaluation of “neuropathy with autonomic dysfunction” after HPV vaccination. All girls complained of long-lasting and invalidating somatoform symptoms  that have developed 1–5 days (n = 11), 5–15 days (n = 5) and 15–20 days (n = 2) after the vaccination. These cases can be included in the recently described immune dysfunction named autoimmune/inflammatory syndrome induced by adjuvants (ASIA). HPV vaccine, through its adjuvant component, is speculated to induce an abnormal activation of the immune system, involving glia cells in the nervous system too. Further researches should aim at defining the pathological and clinical aspects of these post-vaccination diseases and identifying a genetic background predisposing to these adverse reactions.Human papilloma virus (HPV) is recognized as a major cause for cervical cancer among women worldwide. Two HPV vaccines are currently available: Gardasil Human papilloma virus (HPV) infection has been recognized to have a significant role in the occurrence and progression of several tumours, such as cervical cancer, representing one of the most common malignancies among women worldwide .Following this discovery, some vaccines have been produced to prevent such a sexually transmitted infection, against the most frequently involved serotypes, namely HPV-6, HPV-11, HPV-16 and HPV-18. Particularly, the latter two have been estimated to account for 70 % of cases of cervical cancer .®  was licensed in 2006 and Cervarix®  in 2009. Both vaccines enclose self-assembled antigenic pentamers of L1 proteins, produced by DNA recombinant technology, but differ as to the biological system of culture and the adjuvant components, as summarized in Table ® is produced by Saccharomyces cerevisiae cells, and viral antigens are pooled with the adjuvant aluminium hydroxyphosphate sulphate (225 μg). Cervarix® is obtained through a L1-recombinant baculovirus-infected insect , and the adjuvant component is represented by ASO4 complex that is made of aluminium hydroxide and Salmonella minnesota lipopolysaccharide extract. The intramuscular (IM) administration protocol encompasses three bimonthly injections that elicit a strong persisting antibody titre against HPV serotypes over six months and a good control of its related infection [Currently, two HPV vaccines are available on the market: Gardasilnfection –5.Table So far, more than 100 million HPV vaccine doses have been administered worldwide and it has been shown to provide protective and long-lasting antibody titres against the virus. The largest study investigating the safety of HPV vaccine included almost 300,000 young girls administered with nearly 700,000 doses of the quadrivalent vaccine: no increases in the number of cases of autoimmune and/or neurological diseases were detected. Moreover, a pooled analysis of five clinical trials involving 11,778 quadrivalent HPV vaccinated people and 9686 placebo-treated controls, aged 9–26 years, showed that 0.2 % of both vaccine and placebo recipients had dropped out because of adverse drug reactions (ADRs). Most dropouts occurred after the first dose and the most severe cases were due to autoimmunity; however, those events affected equally the vaccine and placebo groups .The temporal relationship between the vaccination and the development of immune-mediated diseases raised the issue of the possible aetiological or co-causative role of such an immunization procedure, which may occur in patients with a genetic predisposition. Moreover, long-standing neurological and/or psychiatric symptoms after HPV vaccine being refractory to any treatment elicited further concerns –21.Recently, these ADRs have been pooled in a heterogeneous group of medical conditions  named autoimmune/inflammatory syndrome induced by adjuvants (ASIA). The common feature of different ASIA clinical conditions is considered to be the previous exposure to an external stimulus (including vaccines) triggering an undefined immune-mediated response elicited by its adjuvant properties –25.Here an incidental finding of a small cluster of young females with severe and persistent neurological and/or psychiatric symptoms arising after HPV vaccination led us to review critically this issue.® (n = 9) or Cervarix® (n = 9). Patients’ and immunization features are reported in Table In 2015, through our “Second Opinion Medical Network”, we have been asked for evaluating a girl with cervical lymphadenitis, headache, severe persistent fibromyalgia-like pain, paresthaesia, chronic fatigue, insomnia, cognitive impairment, abnormal menstrual cycles and fingers/toes vascular abnormalities (changes in skin colour and temperature perception). Such a clinical picture was defined  as “neuropathy with autonomic dysfunction”. Later, some other families from Northern and Central Italy, whose daughters had been complaining of similar symptoms after HPV vaccination, were referred to our centre through the mother of this first patient. That fact allowed us to visit and analyse a case series including 18 young girls aged 12–24 years who developed post-vaccination phenomena after receiving Gardasiln = 4), scarlet fever (n = 2), fifth disease (n = 2) or CMV-related eruption (n = 1). During childhood, eight patients received the MMR  vaccine, in addition to the regular immunization schedule administered at that time, including at least DTP, poliomyelitis and HBV. Nine girls were vaccinated against HPV via the quadrivalent vaccine (Gardasil®), between January 2008 and December 2015, and nine girls received HPV-16/18 vaccine (Cervarix®) between January 2008 and December 2015 (Cervarix®). The mean age at HPV immunization was 11 and 12 years  have been included in this case series. As concerns their past personal history, all of them reported chickenpox during childhood, and some had mononucleosis (n = 11), (2) girls whose symptoms appeared 5–15 days after the vaccination (n = 5) and (3) girls whose symptoms occurred 15–20 days post-vaccination (n = 2).The ADRs described in Table n = 5), involuntary movement at the injection arm, especially hand tremor (n = 1), swelling (n = 2).Local ADRs were also reported: pain at the injection site (n = 9), headache (n = 12), recurrent syncope (n = 7), persistent convulsive hunger (n = 1), irritability (n = 3), epileptic seizures (n = 1), transient speech loss (n = 1), lower limbs paresthaesia and paresis (n = 1), hot flushes (n = 1), severe stomach pain (n = 4), insomnia (n = 5), hypersensitivity reactions  (n = 1), muscle pain (n = 5), severe gait impairment and orthostatic intolerance (n = 1), excessive sweating (n = 1), vomiting (n = 1).The systemic reactions observed 1–5 days after the vaccination consisted in low-grade fever (n = 8), excessive thirst (n = 3), severe hands and feet itching (n = 1), optic neuritis (n = 1). Finally, other patients complained of symptoms after 15–20 days after vaccination: amenorrhoea (n = 1), skin rashes (n = 2), tachycardia (n = 2), difficult breathing (n = 1), weight loss (n = 3).Clinical manifestations occurring 5–15 days after vaccination were asthenia (n = 17), muscle pain (n = 13), finger and toes vascular abnormalities with skin colour and temperature changes (n = 13), headache (n = 12), asthenia (n = 9), dizziness (n = 6), abdominal pain (n = 4), low-grade fever (n = 3), sinus tachycardia (n = 2), skin rashes (n = 1).Among the most common and persistent symptoms Table , the patImmunization practice definitely modified the epidemiology of human infectious diseases since its introduction some centuries ago: indeed, a dramatic reduction of the morbidity and/or mortality in the populations recruited by mass vaccine campaigns was obtained , 27. DesHowever, available studies reported no statistical association between vaccines (including those against HPV) and the incidence of autoimmune diseases, except for a few and well-defined situations , 30. In Moreover, both the use of standardized protocols and the coordinated surveillance by the regulatory drug agencies have currently erased vaccine-related diseases due to malpractice . Obviously, despite the best medical practice, vaccines as any other drug, can display unforeseen side effects: these intrinsic risks are relatively rare and are considered acceptable in front of the health benefits produced in terms of life quality and expectancy. Moreover, the warnings to avoid vaccinations in people with allergies to some components, with history of previous ADRs and with transient (e.g. during pregnancy) or permanent (e.g. severe primary and secondary immunodeficiency) impairment of the immune system, substantially reduced the incidence of complications . UnfortuThe adjuvant is devoted to enhance the immune response to the sensitizing molecules (antigens or haptens); yet they may contain some pathogen-associated molecular patterns (PAMPs) and/or create a mild tissue injury exposing some damage-associated molecular patterns (DAMPs), which are recognized by the innate immune system through specific pattern recognition receptors (PRRs) , 36–38.If autoimmune or immune-mediated phenomena were related to a vaccination, the cross-reactive adaptive immune response to both antigenic and adjuvant components of vaccine might be considered. Therefore, at the site of injection, at afferent lymph nodes or also at distant organs, autoreactive immune processes may be activated by the vaccine adjuvant and not only by the antigenic part of the vaccine. The central and peripheral nervous system has been supposed to be one of the targets of this autoimmune reaction, leading to neurological and/or psychiatric symptoms, diagnosed as being ASIA, in some  predisposed individuals –40.Our case series included a wide spectrum of post-vaccination general, neurological and psychiatric symptoms during and/or early after the scheduled HPV immunization. Most patients showed an “acute phase” characterized by a variable combination of clinical manifestations, including low-grade fever, skin rashes, muscle pain, headache and sensorial disturbances. Its onset occurred at a variable time after HPV immunization, ranging from hours to days. Then, this acute phase subsided in some weeks, but the girls developed some chronic and/or recurrent symptoms resembling chronic fatigue, fibromyalgia or other functional somatic syndromes. The clinical pictures displayed by each patient were actually polymorphous, except for asthenia, concentration problems, sensorial impairment , recurrent or persisting muscle pain, paresis and headache. Previously, all these girls declared excellent fitness and wellness, including sport practice. No significant co-morbidities or previous relevant diseases were reported before the HPV immunization.Currently, the diagnosis of ASIA is based upon the criteria proposed by Shoenfeld , 41, as The description of our case series aimed at analysing clinical phenomena, being putatively immune mediated and arising after HPV immunization. The latency of the onset of symptoms is quite variable, but it can be consistent with the occurrence of cell-mediated and/or antibody response against some neurological/neuromuscular structures.The majority of post-vaccination ADRs were consistent with severe fibromyalgia and chronic pain syndrome, according to the literature criteria.Because of these symptoms, at least 10 of these eighteen young girls developed a long-standing social impairment .Certainly, the diagnostic criteria for ASIA need to be improved and this medical category requires a better clinical, immunological and, possibly, genetic definition. Indeed, in addition to the aforementioned diagnostic criteria, the diagnosis is currently supported by a strict temporal association with the onset of symptoms and, of course, by the exclusion of other well-defined infectious, immune-mediated and oncologic diseases. ASIA is thought to represent an infrequent side effect of vaccinations due to an adjuvant or adjuvant-like stimulus acting on a predisposed, but not well-defined, genetic background . BecauseAmong commercialized vaccines, those against HPV have received a lot of descriptions of medical conditions being consistent with the diagnosis of ASIA, regardless the type of vaccine. Recently, Pellegrino et al. carried out an interesting analysis of HPV vaccine-related ASIA epidemiology in the vaccine adverse event reporting system (VAERS), which is a large database collecting almost 30,000 post-vaccination ADRs per year. Pooling the data until the end of 2013, the authors reported more than 26,000 ADRs after HPV vaccine: of these, about 8 % have been labelled as being probable or possible ASIA, according to the Shoenfeld and Agmon-Levin’s guideline for the diagnosis. A strong causality evaluation between HPV vaccination and ASIA has been supposed in this study . UnfortuA recent observational analysis promoted by UK Medicines and Healthcare products Regulatory Agency (MHRA) investigated the association between bivalent HPV vaccine and chronic fatigue syndrome (CFS) that is considered one of the main expressions or evolution of ASIA. No increase of CFS incidence was noticed in girls aged 12–20 years old after the introduction of such an immunization .Moreover, in 2015, the European Medicine Agency (EMA) revised the safety profile of HPV vaccines as regards the occurrence of complex regional pain syndrome (CRPS) and postural orthostatic tachycardia syndrome (POTS). Such a revision was carried out through the Pharmacovigilance Risk Assessment Committee (PRAC) and the Global Advisory Committee on Vaccine Safety (GACVS), which were not able to find enough scientific evidence so far to support a clear causal link between HPV vaccine and the aforementioned syndromes and, thus, to alter the current recommendations for HPV immunization programme .These studies were prompted by the burden of reports regarding syncope, asthenia and pain syndromes occurring after HPV immunization , 47. RecThis same author speculated that post-vaccination fibromyalgia, POTS and CRPS might be due to a dysautonomia, precisely a sympathetic nervous system dysfunction, because of a small fibre neuropathy triggered by the HPV immunization in susceptible people. Actually, such a hypothesis has been endorsed by Kinoshita et al. , who desThe aforementioned EMA investigation had been prompted mainly by a retrospective analysis (carried out in Denmark) on 53 patients developing orthostatic intolerance and other symptoms consistent with autonomic dysfunction within two months after the quadrivalent HPV vaccination . The autAlthough no cause/effect evidence between HPV immunization and non-organic symptoms has been detected so far, the strong burden of concerns expressed by the parents’ associations from several European countries should not be underscored at all.® between 2008 and 2013 in France, who showed similar symptoms as our case series , was published by an independent parents’ association [A database of 35 young girls immunized against HPV through Gardasilociation .In Spain, the Asociacion de Afectadas pro la Vacuna del Papiloma (AAVP) collected 42 cases from 2008 to 2014: in addition to few cases of demyelinating/autoimmune diseases, most patients developed somatoform/functional disorders or not well-defined patterns of symptoms without a final diagnosis .Again, in the UK, another parents’ association, named Association of HPV Vaccine Injured Daughters (AHVID), currently counts 265 members: through a questionnaire which has been responded to by almost 100 families so far, variable patterns of neuropathic disorders, being labelled as POTS, CRPS, CFS or fibromyalgia, were reported in most cases .All these alerting reports are not institutional and cannot be reputed as systematic epidemiologic investigations; however, in our opinion, they have to be taken into adequate account to reach definite conclusions upon the relationship between HPV vaccines and ASIA/functional somatoform diseases, through further studies. This will be the only way to clarify the real risk of HPV vaccines, as regards these specific health issues: that will help to retract all potential and/or unfounded concerns related to HPV immunization, and, thus, will reinforce the perception of safety and effectiveness of HPV vaccines among people all over the world. Conversely, the identification of a genetically and/or immunologically cohort of young girls being potentially exposed to these kinds of diseases might lead to the selective exclusion from this immunization practice.® since the introduction of the National Immunization Programme in 2009, according to the data analysis updated at December 2015: 231 reports were about long-lasting AEFI, whose duration was 2 months or more, and most related complaints were persistent/recurrent fatigue, headache, dizziness, musculoskeletal discomfort and syncope [® and the reported long-lasting AEFI could not be concluded, but neither excluded, based upon the available data. Previously, in 2013, the same institution edited an overview of the reports on long-lasting fatigue following immunization with Cervarix®. As many others, the authors observed that the age of vaccinated girls had to be taken into account as potential confounding factor, but they concluded that additional epidemiological research was needed anyway, in order to confirm or reject a causal link [Indeed, like the UK Medicines and Healthcare products Regulatory Agency (MHRA) and other European Pharmacovigilance, other systems have been notified about this kind of side effects regarding HPV vaccines. For instance, the Netherland Pharmacovigilance Centre Lareb received 1271 reports of Adverse Events Following Immunization (AEFI) with Cervarix syncope . Interessal link . It is wA plan of prospective and specific epidemiological studies, among active surveillance of post-vaccination ADRs, addressed to face the HPV immunization major concerns, was attempted in Italy through the National Centre of Epidemiology, Surveillance and Health Promotion. The second report on post-marketing surveillance of vaccines in Italy described the side effects in a cohort of 12,990 female subject aged 9–16 years who received free immunization through the Local Health System Institutions in the period going from June 2009 to December 2011. The main complaints observed were local reactions and symptoms like headache, muscles and joint pains. However, this surveillance was limited to symptoms arising within 15 days after the vaccination. Moreover, this analysis included only girls who voluntarily accepted to participate to the study, moreover, it was not possible to ascertain the effective surveillance coverage in each recruited vaccination centre . It mighConclusively, our study highlighted some important post-vaccination phenomena temporally linked to HPV immunization, which needs further epidemiological analysis and biological investigations in order to establish or exclude a causal relation.The exact incidence of these post-vaccination syndromes in the general population has not been known yet and a definite cause/effect link between HPV vaccine-related immune activation and symptoms onset has to be established. However, the studies on fibromyalgia, chronic pain with dysautonomia and CFS considered that a previous subclinical and spontaneous reaction to viral agents could be often involved in the start of pain and functional impairment. Therefore, a link between HPV vaccination and some neuromuscular and systemic impairment might be possible, considering also the immunization properties displayed by HPV vaccines, according to several studies .Moreover, the HPV vaccine formula, containing also high polysorbate 80 (50 mcg) concentration, might also induce a greater meningeal permeability leading to a facilitated entrance of many substances to the central nervous system. Based upon these observations, it might be speculated that this vaccine—and not others—could induce some abnormal activation of immune competent cells in the central nervous system, such as the glia , 63. Fin


The incidence of oropharyngeal cancer (OPC) in New Zealand (NZ) has more than doubled over the last 14 years with 126 cases in 2010. Overseas studies have shown that human papillomavirus (HPV) plays a significant role in the development of these cancers. However, the role of HPV in OPC and the burden on the NZ health system is unclear.The aim of the study was to determine the prevalence and the genotypes of HPV associated with OPC in New Zealand.In this study, 621 OPC were identified from cancer registry data from 1996–98, 2003–05, and 2010–12. Biopsies of 267 cases were then retrieved from laboratories throughout New Zealand. p16 immunohistochemistry and a human beta globin PCR were performed on all specimens. HPV genotyping was performed on all beta globin positive specimens using real-time PCR with melt analysis.Using a p16/PCR algorithm, 77.9% (95% CI: 71.1–83.5%) of cases were attributable to HPV. Of these, 98.5% were HPV 16 positive. There was also one case each of HPV 33 and 35. The percentage of HPV positive cases increased from 61.9% (95% CI: 40.9%– 79.2%) in 1996–98 to 87.5% (95% CI: 79.8%– 92.5%) in 2010–12. Results from the multivariable model, adjusted for sex and ethnicity found statistically significant associations between HPV positivity and timeframe , and between HPV positivity and patient age .® 9.This data is consistent with data from other developed countries showing an increase in cases of HPV positive OPC in New Zealand, and the majority of cases being attributable to HPV 16. These results support the recent inclusion of males into the nationally funded immunization schedule for Gardasil Head and neck squamous cell carcinomas (HNSCC) comprise cancers of the oral cavity, larynx, hypopharynx, and oropharynx . The incIn the past, the majority of HNSCC was seen in the larynx and oral cavity and was associated with smoking and heavy alcohol consumption . The incThe number of cases of OPC caused by HPV in New Zealand is unknown, however, when looking at anatomical site alone there has been an approximately threefold increase in the number of OPC cases from 35 cases in 1996 to 126 cases in 2010 . In contrast, the incidence of oral cavity cancers has remained steady during the same time period . CurrentEthics approval was obtained from the Health and Disabilities Ethics Committee (reference: 14/STH/128). Participant privacy was preserved by de-identification of samples.Cases were identified by a New Zealand cancer registry search of all cases of oropharyngeal cancer of ICD-10 codes C01, C05.1–5.2, C09.0–9.9, and C10.0–10.9 from 1996–98, 2003–05, and 2010–12. The New Zealand cancer registry is a population based register of all primary malignant diseases diagnosed in New Zealand. The information available from the registry comprises: patient demographics  and tumour specific information ). Data was cross checked with National Health Index (NHI) data to identify patients that were deceased. Study information sheets and consent forms were sent to all living patients whose samples were stored in laboratories with five or greater specimens in storage as per the laboratory code from the cancer registry data, and with current and complete address details. In addition the whānau (extended family) of deceased participants identified as Māori from the Auckland region were requested to give consent for these specimens to be used, as per local Iwi (tribe) wishes. All participants provided written informed consent prior to specimens being requested from storage. After specimen requests were made, laboratories sent the specimens (n = 267), a copy of the pathology report, and any existing p16 slides (n = 57) to Massey University.Samples with a historic p16 slide had the existing slide used in this study. Samples were recoded and read blinded. All other samples had p16 IHC performed at a diagnostic medical laboratory . Formalin fixed paraffin embedded (FFPE) tissue was used for IHC. Sections were cut at four microns and baked onto Superfrost Plus slides at 60°C for 60 minutes. Staining was performed on the Ventana Benchmark Ultra , with antigen retrieval performed on board, using Ventana CC1 buffer for 32 minutes at 100°C. A 1:150 dilution of the p16 (INK4a) antibody  , was incubated on the slide for eight minutes at 36°C. The detection of p16 was then visualised using the Ventana Optiview DAB kit . A multi-tumour block positive control containing a serous ovarian carcinoma was included with each run of samples. All slides were read independently by two pathologists (BL and UV), and discordant cases reviewed to reach a consensus. Samples showing strong diffuse staining in the nucleus and cytoplasm of greater than 75% of tumour cells were considered positive. Samples showing staining in 10% to 75% of tumour cells were considered focally positive, and samples with staining in less than 10% of tumour cells were considered negative.® Blood and Tissue kit  as per manufacturer’s instructions for tissue. A control extraction was included with each set of samples. The blank extractions were then used as negative controls in the beta globin and HPV 16 qPCRs. A pre-treatment for paraffin embedded samples was included, and a final elution volume of 100 μL was used. The quality of extracted DNA was assessed using a Nanodrop™ Spectrophotometer .A 10 μm slice of representative FFPE tumour block from each case with sufficient material (n = 224) was cut for DNA extraction using a new blade between each sample to avoid cross contamination of samples. DNA extractions were performed using the DNeasyTm of 81.0°C (±1.0°C). Samples showing low amplification on the qPCR were visualised using gel electrophoresis through a 1.0% agarose gel containing 1X SYBR® Safe (Thermo Fisher Scientific) to confirm product amplification.The presence of amplifiable DNA was assessed on all cases with extracted DNA by qPCR targeting the human beta-globin gene using the PC03 and PC04 primers . All priTm of 77.6°C (±1.0°C).The presence of HPV 16 was assessed on all cases with a positive beta globin qPCR result. A qPCR with melt curve analysis was developed using previously described primers . Each re®-T Easy kit with JM109 High Efficiency Competent Cells , according to the manufacturer's instructions with blue/white selection. The cloned plasmid contained an approximately 1.8 kb fragment of the HPV 16 genome that included the E6 and E7 gene sequence amplified using the primers detailed in A positive control was made from cloned DNA of a HPV 16 positive clinical sample provided by MedLab Central Ltd . Cloning was performed using a pGEM7 copies of HPV 16 were used to determine the limit of detection of the HPV 16 qPCR.Duplicate standard curves from eight, 10-fold serial dilutions starting from 9.56 x 102, 1X PCR buffer, 0.3 mM each dNTP, 1 unit Platinum™Taq , 1.5 μM Syto® 9 (Invitrogen). Up to 50 ng extracted DNA was used as template. Cycling conditions were 95°C for 10 minutes, followed by 40 cycles of: 95°C for 15 seconds, 49°C for 30 seconds and 72°C for 30 seconds. PCR was followed by a melt curve from 70–85°C with 0.2°C increments and a five second hold. Known HPV 16, 18, 33, and 52 positive samples were included in each run. Samples with a Tm in the range of 76.0–81.0°C were considered positive for HPV DNA. The GP5+/6+ PCR could not reliably discriminate HPV genotypes therefore, the product of samples positive for the GP5+/6+ PCR were purified using the PureLink® PCR purification kit . The HPV type present was then confirmed by sequencing as previously described.Samples that were p16 positive / focally positive, and HPV 16 negative were subject to qPCR using the GP5+/6+ primers . Each reThe continuous variable age was categorised into a binary variable: aged 60 years or younger, and 61 years or older. Ethnicity was classified into NZ European, NZ Māori, and other.To investigate the association between HPV positive OPC and putative risk or confounding factors , each factor was tested individually for significance at p < 0.2 in a logistic regression model using the software package R version 3.2.0 . A multivariable model was built by a stepwise selection process, retaining all variables significant at p < 0.05 and any confounding variables. Once a main effects model was built two-way interaction terms were introduced to the model and retained if significant at p < 0.05.The national dataset from the cancer registry comprised a total of 621 cases from the 1996–98 (n = 113), 2003–05 (n = 185), and 2010–12 (n = 323) time periods. Diagnosis was based on histology in 94.8% (n = 589) of cases, cytology in 4.6% (n = 29) of cases and other non-specified tests in 0.6% (n = 3) of cases. The proportion of cases diagnosed on histology versus cytology was consistent over all time frames. The mean age of an OPC patient was 59.2 years. NZ Europeans made up 68.8% of the national cases, and NZ Māori a further 10.5%. There was an approximate 4:1 ratio of males to females.Of the 344 living patients identified from the national dataset, 303 were able to be contacted, of which 52% (157/303) gave consent. Of the 157 cases, 109 specimens could be retrieved. There were 277 deceased patients identified in the national dataset. Of the 277 cases, 158 specimens were retrieved for use in the study.Thus, the total study population comprised 267 cases with retrievable specimens, and represented all major centres. Between four and 61 cases were retrieved from each of the 15 laboratories involved. The demographic factors of the national dataset and study population were comparable as shown by the high p-values for age, sex, ethnicity and timeframe . Therefop16 results were categorised as positive, focally positive, or negative . ConsensThere were 57 cases with a historic p16 slide. When comparing study p16 results to original results from cases with historic p16 slides, concordance was seen in 49/49 positives, 1/2 focally positives, and 5/5 negatives. The discordant focally positive case was identified as negative by our study. The remaining case had a slide sent for the study but no p16 result in the original pathology report.DNA could be extracted from 224 samples. Following the beta-globin qPCR  a furtheResults of the GP5+/6+ qPCR (n = 17) were 82% (14/17) negative, and 18% (3/17) positive . Of the For the purpose of further analysis, a sample is considered positive if positive by p16, and positive for either the HPV 16 qPCR, or a high risk HPV type was confirmed by sequencing. A negative sample is negative by p16, and HPV 16 qPCR negative. This approach resulted in an overall study prevalence of HPV positive OPC of 77.9% (95% CI: 71.1–83.5%). HPV 16 accounted for 98.5% of HPV positive OPC.The proportion of cases attributable to HPV increased from 61.9% (95% CI: 40.9%– 79.2%) in 1996–98 to 63.8% (95% CI: 49.5%– 76.0%) in 2003–05, and finally to 87.5% (95% CI: 79.8%– 92.5%) in 2010–12.The mean age of an HPV negative patient was 66.2 years (95% CI: 65.8–66.4), compared to 56.8 years (95% CI: 56.6–56.9) for an HPV positive patient. The univariate analysis of putative risk factors showed age (p < 0.01) and timeframe (p < 0.01) to be significant . In the The increase in the proportion of HPV positive cases between 1996–98 and 2010–12 seen in this study augments Ministry of Health data showing over a three-fold increase in OPC case numbers, regardless of HPV status between 1996 and 2010. Overall, the results of this study showed that the majority of OPC cases in New Zealand are caused by HPV. An HPV positive patient is more likely to be under 60. The younger age for HPV positive patients and increase in prevalence from this study are consistent with international data ,12,21. OThere was a higher proportion of deceased patients in our population than the national dataset (59% versus 45%) and this is likely due to the different consenting requirements. Due to the poorer prognosis of HPV negative tumours , the incWe requested 389 specimens from laboratories throughout New Zealand to capture our final study population of 267 cases. There were several reasons why cases were not retrieved, such as 1) there were logistical difficulties identifying cases , 2) cases were diagnosed on cytology only and had no biopsy / cell block, 3), specimens had been destroyed in the 2011 Christchurch Earthquake, and 4) specimens had been removed from storage and could not be located. Given the many reasons for samples not being included and the final comparisons of our study population to the national dataset, there was no reason to suspect any systematic bias in our sample.In New Zealand, laboratories are required by law to report any new diagnosis of cancer to the cancer registry. This generates complete national incidence data. Our study compared only risk factors with data available from the cancer registry . It is important to note these are not the only risk factors for HPV positive OPC and other significant factors such as alcohol consumption, tobacco smoking and sexual behaviours ,11,12 coIt should be noted, that our final criteria for a positive result was both p16 positive and either HPV 16 qPCR positive, or a high risk HPV type confirmed by sequencing. These criteria resulted in 172 results from 267 cases for analysis. This approach excluded focally positive cases and those cases that were p16 negative but HPV 16 positive by PCR. The exclusion of focally positive cases, and use of the p16 positive cut-off at 75% of tumour cells stained, aligned our definition of a p16 positive result with that used in many other studies ,12,21,25Although p16 is routinely used alone in the diagnostic setting , the use2 and 1.34 x 107 copies per 50 ng of DNA [et al [et al [The viral load in tonsillar cancer has been reported to be between 1.54 x 10g of DNA . Our HPVg of DNA ,28. JunoA [et al  reportedet al [et al [et al [While multi-levelled result categories or scoring systems for p16 have been reported –34, moreet al  and Lewil [et al ,34. Apprl [et al ,34. It il [et al , howeveret al, with HPV 6 the most commonly detected with a prevalence of 3.1% [Worldwide HPV is responsible for over 90% of HPV positive OPC . Co-infe of 3.1% . It is aet al, focused on tumour site alone without assessing HPV status, the results support the apparent increase seen in our study.Based on our results, a HPV positive patient is more likely to be under 60, and diagnosed recently (2010–12). The prevalence of HPV positive OPC in females appears to be increasing at a faster rate than in males based on our study data. However, interpretation of this apparent trend is limited due to the low number of cases in females in this study. There were no HPV positive cases in females in 1996–98, although there was one case that was p16 positive that could not have DNA extracted. This is compared to 20% of p16/HPV DNA positive being in females in the 2010–12 time period. A statistically significant increase in New Zealand OPC cases in females of 2.1% per year between 1982 and 2010 has been previously reported [et al [®9 in New Zealand. HPV types 16, 18, 6, 11, 31, 33, 45, 52, and 58 are included in Gardasil®9 [Chaturvedi et al  estimaterdasil®9 . TherefoS1 File(XLSX)Click here for additional data file.


Head and neck cancer (HNC) is a heterogeneous group of squamous cell cancers that affect the oral cavity, pharynx, and larynx. Worldwide, it is the sixth most common cancer but in parts of Southern and South-East Asia, HNC is one of the most common cancers. A significant proportion of HNC is driven by human papillomavirus (HPV) infection, whereas HPV-independent HNC is associated with alcohol, smoking, and smokeless tobacco consumption. Here, we review the past and present experience of targeting HNC with vaccination focusing on HPV-derived antigens as well as non-viral antigens for HPV-negative HNC. Novel therapeutic approaches for HNC will focus not only on effective vaccine platforms but will also target the stroma-rich immunosuppressive microenvironment found in those tumours. Head and neck cancer (HNC) is a heterogeneous group of squamous cell cancers that affect the oral cavity, pharynx, and larynx. Overall, it is the sixth most common cancer worldwide with an annual estimated incidence of 550,000 cases and around 300,000 deaths –3. In paCombinations of surgery, chemotherapy, and radiotherapy form the standard current first-line treatment regimens for HNC. But despite improvements, these are associated with significant morbidity and a relatively static 5-year survival rate of around 40–50% . HPV-posThe incidence of HNC has risen dramatically since the later 1970s and this has been linked to HPV infection. Around 25–50% of HNCs are HPV-driven with a higher percentage in developed countries –14. Thisvia micro abrasions which expose the deeper basal epithelial cells  have been found in the HPV-negative HNC subset. Further alterations in the genes associated with smoking such as those involved in oxidative stress CUL3, KEAP1, and NFE2L2 are also associated with the HPV-negative subset –28.® and more recently Gardasil®9 have been approved by the FDA to protect from HPV infection as well as HPV-associated diseases such as genital warts and cancer –32® and in vitro (These vaccines are based on virus-like particles (VLPs) consisting of different HPV capsid proteins L1 . For exain vitro –36. Supein vitro . Prophylin vitro . The vacFor HPV-positive cancers, the expressed viral antigens are available. For HPV-negative cancers, other antigens have to be considered. Cancer antigens can be broadly classed into two categories: tumour-specific antigens (TSAs) and tumour-associated antigens (TAAs). Here, we describe TSAs as proteins only expressed in cancer cells and mutated self-proteins (neoepitopes), and TAAs as unmutated self-proteins such as glycosylated proteins MUC1 and CEA or cancer testis antigens (CTAs) . TSAs ofClinicalTrials.gov database utilising “Head and Neck Cancer” or “Oropharyngeal Cancer” or “Oral Cancer” as the disease, and “Vaccine” as the search string, yielding 55 studies. Terminated, withdrawn, suspended trials as well as trials with unknown recruitment statuses were excluded and the results are summarised in Table ClinicalTrials.gov database, but registered on the Japanese UMIN-Clinical Trials Registry .To cover antigens which have been targeted to date, current clinical trials for HNC were queried at the NIH Viral proteins are considered to be good targets since they are foreign, and hence, the available T-cell repertoire has not been subjected to central tolerance. Most vaccination strategies for HNC target the HPV-positive subset where HPV antigens can be used. HPV E6 and E7 play a critical role in carcinogenesis of HNCs, similar to ano-genital cancers. During malignant transformation when HPV frequently integrates into the host genome, E6 and E7 are thought to be the only proteins expressed and hence have been targeted by many types of vaccines. Vaccines against these antigens have demonstrated efficacy in HPV-induced cervical dysplasia –46 and aOther potential targets are E2 and E5. E2 has been successfully targeted in ano-genital intraepithelial lesions . In HNC,HPV-negative HNC lacks the immunogenic HPV viral proteins of HPV-associated HNC and appears less responsive to current treatments . A viralDespite earlier reports that HPV-negative HNC had a greater mutation rate than HPV-positive , more revia vaccination. However, as each case exhibits its own unique mutanome 1 cells must also be co-induced (via dendritic cells (DC)-licensing by binding to 12-15mer or longer peptides presented in the context of MHC II on DCs which leads to DC activation . CTLs are powerful effector cells which directly kill target cells -induced . These ptivation , 90. Thetivation –94. The tivation ]. Tetanu vaccine . A recenbenefits .For effective cancer attack, Th1 cells that play not only a helper role to CTLs but a more direct role in anti-tumour immunity are important –99. ThosDelivering antigenic epitopes in an immunogenic context which leads to the induction of a durable T-cell response is the goal. The available vaccine platforms are illustrated in Figure in vivo electroporation . For ingets DCs . The effgets DCs . T-cell + epitopes predicted for a particular HLA allele or include long single or overlapping peptides which often contain both CD8 and CD4 epitopes. The latter approach circumvents HLA restriction issues. Unlike DNA and RNA vaccines peptides do not carry “inbuilt” adjuvants. The efforts to enhance their immunogenicity have focused on combining with appropriate adjuvants. MF59®, emulsion of squalene oil approved in both Europe and USA, has been used in earlier trials , for HPV16-positive tumours and premalignant lesions has also been initiated . These e models –126, ince models , 128.The first clinical trial of a recombinant vaccinia virus targeting HPV was conducted more than 20 years ago. The TA-HPV vaccine was based on a live vaccinia virus and was engineered to express E6 and E7 proteins from HPV16 and 18. Two patients with advanced cervical cancer remained tumour free 15 and 21 months after vaccination, in one of them an HPV-specific T-cell response was also induced . Two morEfficacy of an MVA-based vaccine encoding modified HPV16 E6 and E7 together with IL-2 TG4001 (Transgene) was demonstrated in patients with HPV16-related CIN2/3. In 7 out of 10 patients who were evaluated as clinical responders, cytological and colposcopic regression together with HPV16 mRNA clearance was observed . A phaseListeria monocytogenes (Lm) incorporating a non-toxic version of the listerionlysin O (LLO) as adjuvant has been utilised to target HPV16 E7 oncoprotein  [(A well-characterized attenuated facultative intracellular bacterium 11-001) [; for rev11-001) . Severa11-001) [.Using autologous tumour cells as vaccines ensures that patients are vaccinated with cells containing the same tumour antigens that their tumour expresses saving time and effort needed to identify TSAs. Irradiated cells are used but undesired immune responses are still a potential safety concern. To enhance immunogenicity cells genetically modified to express costimulatory molecules, TLR ligands or cytokines have been utilised . Phase ISeveral immunosuppressive immune subsets have been found in HNC including tumour-associated macrophages, myeloid-derived suppressor cells, and regulatory B and T cells. The role of these have been reviewed elsewhere , 144, buIncidence of both HPV-positive and -negative HNC is on the increase. The trend is unlikely to change at least in the near future, particular for HPV-independent HNC, especially OSCC, where the habitual and cultural causes are unlikely to disappear.Human papillomavirus targets E6 and E7 are considered to be less challenging because of their foreign nature. Their targeting by DNA, peptides, and other vaccines has already demonstrated clinical efficacy in HPV-driven dysplasia. These vaccines are now in clinical trials for HPV-driven cancers including HNC. On the contrary, HPV-independent HNC has received less attention largely because such targets have not been available. A number of interesting antigenic targets has started coming through; these include personalised mutanome-derived neoepitopes but also novel TAAs . The mutin vivo delivery are promising. However, these will still need to be developed within a strategy that overcomes a suppressive tumour microenvironment and further work is required to develop these therapeutic approaches.More affordable vaccine modalities such as DNA vaccines combined with a simple CW, JD, RS, and NS wrote sections on target cancer antigens, immune mechanisms, and vaccine platforms. CP and GT wrote sections on HNC and tumour microenvironment. All authors contributed to discussion of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heart failure (HF) is a complex clinical syndrome defined by the inability of the heart to pump enough blood to meet the body’s metabolic demands. Major causes of HF are cardiomyopathies , among which the most common form is dilated cardiomyopathy (DCM). DCM is defined by ventricular chamber enlargement and systolic dysfunction with normal left ventricular wall thickness, which leads to progressive HF. Over 60 genes are linked to the etiology of DCM. Titin (TTN) is the largest known protein in biology, spanning half the cardiac sarcomere and, as such, is a basic structural and functional unit of striated muscles. It is essential for heart development as well as mechanical and regulatory functions of the sarcomere. Next-generation sequencing (NGS) in clinical DCM cohorts implicated truncating variants in titin (TTNtv) as major disease alleles, accounting for more than 25% of familial DCM cases, but these variants have also been identified in 2–3% of the general population, where these TTNtv blur diagnostic and clinical utility. Taking into account the published TTNtv and their association to DCM, it becomes clear that TTNtv harm the heart with position-dependent occurrence, being more harmful when present in the A-band TTN, presumably with dominant negative/gain-of-function mechanisms. However, these insights are challenged by the depiction of position-independent toxicity of TTNtv acting via haploinsufficient alleles, which are sufficient to induce cardiac pathology upon stress. In the current review, we provide an overview of TTN and discuss studies investigating various TTN mutations. We also present an overview of different mechanisms postulated or experimentally validated in the pathogenicity of TTNtv. DCM-causing genes are also discussed with respect to non-truncating mutations in the etiology of DCM. One way of understanding pathogenic variants is probably to understand the context in which they may or may not affect protein–protein interactions, changes in cell signaling, and substrate specificity. In this regard, we also provide a brief overview of TTN interactions in situ. Quantitative models in the risk assessment of TTNtv are also discussed. In summary, we highlight the importance of gene–environment interactions in the etiology of DCM and further mechanistic studies used to delineate the pathways which could be targeted in the management of DCM. Heart failure (HF) is a complex clinical syndrome which concerns the impaired ability of the heart to pump and/or fill with blood, resulting in inadequate cardiac output to meet metabolic demands or, more commonly, adequate cardiac output but only due to compensatory neurohormonal activation  domains, interspersed with Z-insertion sequences. Z-disk TTN ligands are shown in Fig. I-band TTN is a highly interactive structure with great potential for alternative splicing. This results in isoforms contributing to cardiac and skeletal muscle specific phenotypes. The meta-transcript  of I-band TTN is mainly composed of Ig domains, cardiac N2B region, and skeletal N2A region (which contains non-repetitive sequences and Ig domains), followed by the Pro-Glu-Val-Lys (PEVK) TTN domain, all of which contribute to TTN-related elasticity  motifs. FN-III motifs are exclusive to A-band TTN. Ig and FN-III are further arranged into two super-repeats, in which FN-III domains are bisected by Ig domains. In contrast to I-band TTN, A-band TTN is inextensible, as it provides binding sites for myosin, and, hence, functions as a stable anchor. TTN binding to myosin S1 fragments also plays a role in regulating Ca+2-calmodulin regulated mechanochemical signal transduction pathways -Nbr1-p62 and MURFs in the M-band region. MURFs work in concert with other transcription factors in order to suppress the hypertrophic gene expression program, whereas the TK-Nbr1-p62 axis activates pro-hypertrophic gene expression (Granzier and Labeit TTN is a unique protein with highly extensible (I-band TTN) and inextensible regions . In addition, it contains regions undergoing extensive regional alternative splicing  and regions which are constitutive . Alternative splicing in the A-band TTN results in the cardiac-specific N2B isoform with interspersed unique sequence (N2B-Us), N2BA, which contains the N2B and N2A sequence, and skeletal isoform N2A (without N2B sequence). All these isoforms contain the extensible PEVK region. Cardiac isoforms contain three extensible regions: tandem Ig segments, PEVK segments, and cardiac-specific N2B-Us. Upon increasing their length, Ig segments stretch first, followed by PEVK and, finally, N2B-Us segments, which stretch at the upper limit of physiological sarcomeric length , routine analysis of the TTN gene had been extremely challenging because of its size and complexity. Thus, only a few TTN mutations had previously been reported, and the general incidence and spectrum of titinopathies was significantly underestimated. Early work pointed towards the importance of TTN mutations in HCM  in TTN, are all factors that have generated considerable challenges in demystifying the functional effects of TTNtv in disease penetrance, allele dosage, and by posing challenges to clinicians in genetic risk assessment for cases identified positive for TTNtv with or without overt cardiac phenotypes  also described TTNtv high enrichment in DCM compared to the control 1000 Genomes cohort , HGMD (http://www.hgmd.cf.ac.uk/ac/index.php), and/or LOVD (http://www.LOVD.nl/TTN). We will discuss individual cohorts in their respective studies, and we will then present the re-evaluation of the pooled cohorts analyzed by Akinrinade et al. , and that the required additional stress for the DCM to emerge means that TTN insufficiency alone is unlikely to be the sole cause of DCM. Another conclusion drawn from TTNtv epidemiology studies is the differential sensitivity of TTN mutations along its length towards the DCM phenotype. It appears that I-band TTNtv are less prevalent and perhaps more tolerable compared to A-band TTNtv. As has been stated earlier in the TTN signaling section, I-band TTN is mainly a substrate for signaling and metabolic activity as compared to A-band TTN, which provides structural and functional interactions with major sarcomeric contractile protein, such as myosin heavy chain and myosin binding protein C. With A-band TTNtv, such interactions are severely compromised in DCM patients and/or are at risk from defective functions relating to stress in genotype-positive asymptomatic individuals. I-band haploinsufficiency or I-band dominant negative TTN might still contribute towards signaling events occurring at I-band TTN on rest or stress conditions without severely impairing the cardiac conditions. Further, A-band TTN exons were constituted by high PSI values compared to I-band TTN, indicating that any mutation in A-band will affect most or all of the splice variants and, hence, be more deleterious, whereas mutations in I-band variants could spare splice variants and, hence, have milder outcome. The Schafer et al.  study alhttp://www.world-heart-federation.org) published a new classification scheme for cardiomyopathies, called MOGE(S) , which accounts for the environmental triggers in the quantitative model in the background of genetic susceptibility to DCM. Implementation of MOGE(S) classification in a pilot study improved the stratification of patients with DCM, most likely by using the combination of genetic evaluation and non-genetic, environmental factors  have also been described in sarcomeric genes, Z-disk genes, cytoskeletal genes, ion channels genes, nuclear envelope genes, desmosomal genes, transcription factors genes, gamma secretase activity genes, sarcoplasmic reticulum genes Fig. . Z-disk In this context, a study by Walsh et al.  re-assesn = 176) included in the study compared to 187 missense variants found in 37.2% (n = 187) of the control population (1000 Genome Europeans) , it is more feasible to introduce truncating variants  in tissue-restricted manners as to substantially aid in understanding the pathogenic mechanisms of these variants. In this regard, Deo  introducIn summary, our current understanding on truncating and non-truncating variants in DCM and other cardiac phenotypes needs to be explored beyond simple genetic events and on the functional level. The pathogenesis of DCM needs to be addressed, in particular, beyond the explanation of haploinsufficient and dominant negative lesions. Disease-causing mutations in DCM might also be addressed in the epigenetic context. Do truncating variants in different TTN domains and/or bands dictate specific epigenetic lesions? Is there a differential epigenetic sensitivity towards haploinsufficiency or dominant negative TTNtv? How do environmental stressors shape the DCM genome and epigenome in patients with genotype-positive/phenotype-negative individuals and vice versa. The limited number of genotype-positive DCM patients presenting with the spectrum of specific cardiac phenotypes and even more limited patients with one particular mutation are the major obstacles to confidently assigning the risk carried by each variant. On the other hand, it is possible that patients with variable titinopathies could present with converging patterns of epigenetic lesions, which might aid in the development and implementation of epigenotype-specific treatment regimens  in the management of DCM.Another important issue is to identify to which extent TTNtv contribute to the phenotype in the presence of other disease-causing mutations. Therefore, in order to establish the pathogenicity of rare variants, further studies should focus on prospective longitudinal population cohorts without apparent overt cardiac phenotypes needing to be established if the occurrence of cardiac events (if any) is significant in pre-symptomatic genotype-positive individuals. This can help the geneticist in counseling such individuals, or in the design of screening programs which can help to identify further family members at risk, and, finally, develop prophylactic managements . However, considerable challenges lie ahead, including the integration of masses of genetic information and their interpretation in the biological context. This will also comprise building standard algorithms for comprehensive diagnostic workups in the evaluation and management of inherited cardiac disease.Given the gigantic size of TTN, it is expected that the TTNtv landscape of DCM will increase in much higher numbers than currently known. As discussed, truncating changes are likely to be pathogenic; however, the mechanism of pathogenicity is probably less well understood. Recent functional studies highlighting the position-independent toxicity of TTNtv raise further questions on dominant negative modes of action than previously envisaged. If evidence is generalized to broader populations of TTNtv, then the preponderance of TTNtv in the A-band requires further explanation.


Forty-one pregnancies, 11 in the control group and 30 in the vaccine group, were recorded  and resulted into 4 deliveries and 7 miscarriages (control group) and 29 deliveries and one miscarriage . HPV detection on sperms was predictive of negative pregnancy outcome. Adjuvant vaccination associated with enhanced HPV healing in semen cells and increased rate of natural pregnancies and live births.In this study we aimed to evaluate the effect on reproductive outcome of HPV vaccination in male subjects of infertile couples with HPV semen infection. In this single-center study, we retrospectively enrolled 151 infertile couples with detection of HPV in semen, attending our Hospital Unit of Andrology between January 2013 and June 2015, counseled to receive adjuvant HPV vaccination. Seventy-nine accepted vaccination (vaccine group) whilst 72 did not (control group). Our protocol of follow-up, aimed to evaluate HPV viral clearance, consisted in semen analysis, INNO-LiPA and FISH for HPV in semen cells after 6 and 12 months from basal evaluation. Spontaneous pregnancies, miscarriages and live births were recorded. Progressive sperm motility and anti-sperm antibodies were improved in the vaccine group at both time points (p < 0,05  In this context, despite genital human papillomavirus (HPV) is acknowledged as the most common sexually-transmitted viral infection worldwide, very few studies have investigated the effect of HPV on human reproduction. Data on the actual rate of spontaneous abortions, major birth defects and pregnancy complications during natural conceptions in couples exposed to HPV appear scarce and controversial4. Dealing with assisted reproduction, only a clinical study performed on women undergoing in-vitro fertilization (IVF) reported a significant reduction of pregnancies in the presence of cervical HPV detection5.Sexually-transmittable diseases are among the primary causes of infertility7. Even in male patients with accessory gland infection the prevalence of HPV detection in semen is 2 to 3 to folds higher than healthy subjects, regardless of the mere inflammatory or the microbial form of the disease8. Furthermore, prevalent sperm motility impairment and detection of anti-sperm antibodies (ASA) have been described in male subjects with detection of HPV-DNA in semen11.New recent insights on the role of HPV in human reproduction derived from studies on infertile couples with viral infection detected in semen. Indeed, the prevalence of HPV-DNA detection in semen from infertile males is nearly 3 to 4 folds higher than fertile controls12. As a consequence, a possible role of sperm cells as vectors for HPV transfer into the oocytes could be suggested12. To this regard, the possible consequences of embryo exposure to HPV are not well defined. However in vitro experiments have shown that trophoblast cells transfected with HPV-DNA have an increased rate of stage-specific maturation arrest, apoptosis and a reduced placental invasion into the uterine wall, compared with controls13. These data are largely in agreement with the few available clinical evidence showing an association between the detection of HPV-DNA in spermatozoa and reduced pregnancy rate or recurrent miscarriage18.From a mechanistic point of view, the effect exerted by HPV infection in semen during the fertilization process is currently under investigation. To this regard, data from our group showed that sperm cells, either transfected with HPV E6/E7 genes or exposed to HPV L1 capsid protein, were able to penetrate the oocyte and to transfer the virus DNA into oocytes where viral genes were then activated and transcribed26 together with anal pre-cancerous lesions, external genitalia lesions and pharyngeal pathologies in males29.The prophylactic vaccination anti-HPV is demonstrated to be highly effective in preventing HPV-related pathologies in both sexes, such as cervical lesions, vulvo-vaginal lesions, condylomas in females30.However, despite the overall genital HPV infection is highly prevalent among all age groups of men, the HPV vaccination coverage in eligible males is less than 11%31. This evidence arises questions about the possible application of prophylactic vaccination in male patients whose couple infertility is likely related to the tresence of HPV in semen33. On these bases, here we aimed to evaluate the effect of HPV vaccination on reproductive outcome, in terms of natural pregnancy, delivery and abortion rate. To this aim, we retrospectively evaluated the pregnancy outcome in the 12 months-follow up of infertile couples where the male partner showed HPV semen infection and received the HPV prophylactic vaccination. A group of infertile couples with equal HPV-detection in semen that did not receive vaccination served as control group. We also evaluated the effect of HPV vaccination on viral persistence and semen parameters, particularly in terms of motility and presence of ASA in both groups.We recently described that HPV vaccination in males with HPV detection in semen, led to a significant reduction of the prevalence of HPV semen infection together with amelioration of sperm parameters, such as motility and anti-sperm antibodies (ASA), through the likely stimulation of humoral immunity12. No significant difference was observed between control and vaccine groups in terms of clinical characteristics. The cumulative frequency of each HPV genotype, detected by INNO-LiPA, in the whole cohort of patients at baseline is reported in supplementary Figure As summarized in Fig. vs corresponding time point of the control arm). This change was present at T1  and was still present at T2 . No significant modification was observed in total sperm count and normal morphology.Figure Table In Fig. vs control group). Among couples of the former group, 4 deliveries and 7 miscarriages were recorded whereas, among couples of the vaccine group, we recorded 29 deliveries and one miscarriage. The control group also showed a higher miscarriage rate than the vaccine group  recent evidence reported a possible role of HPV sperm detection in assisted reproduction failure and adverse pregnancy outcome (reviewed in15 and35). However, the actual role of HPV semen infection in couples seeking natural fertility receive less interest.The state of the art concerning HPV sperm infection and infertility, indicates that: i) there is a higher prevalence of HPV semen infection in men affected by idiopathic infertility compared with the general population, independently from the genotype of HPV detected; ii) in infected infertile patients, HPV is mainly localized on the sperm surface, in particular along the equatorial region; iii) there is a significant relationship between asthenozoospermia and HPV sperm infection; iv) the detection of HPV-DNA on sperm surface is more frequently associated with the detection of ASA and this condition is characterized by a further reduction of sperm motility; v) 29. However, healing effects of neutralizing antibodies against recombinant self-assembled virus-like particles were early suggested in animal models38. In humans, a recent systematic review showed that when HPV vaccination is used as an adjuvant treatment for active clinical disease, a decreased disease burden is observed39. Accordingly, we recently reported that HPV prophylactic vaccination improves viral healing and sperm parameters by involving humoral immunity31. To this regard, a recent study from our group on infertile couples eligible for assisted reproduction showed a reduced pregnancy rate and a significantly higher abortion rate when the male partner had positive FISH analysis on sperm16. Based on these evidence, we suggested that HPV vaccination may represent a valuable tool to improve viral healing in infertile couples, aimed to improve reproductive outcome. It should also be noted that HPV vaccination itself does not increase the risk of miscarriage in pregnancy, as a long-term observational study has recently confirmed40. As in previous studies, we observed that patients with HPV-DNA on sperm at FISH analysis showed a positive staining in a relatively small percentage of cells. Major concerns may then derive from the fact that patients, despite this pattern of infection, have worse pregnancy rates and fail to achieve live births. To this regard, we hypothesize that the lack of HPV detection in the remaining sperm population is likely related to the small viral DNA quantity, lower than method’s sensitivity41.The available HPV prophylactic vaccine demonstrated high efficiency in preventing viral infection and HPV-related diseases11 and, accordingly, our patients showed a wide variety of types from low to intermediate and high risk. However, it is recognized a degree of cross-protection provided by quadrivalent vaccine41. In addition, as reported for general population, in infertile patients, and in those here described, HPV infection is mainly ascribed to both the types covered by the vaccine and those defined by cross-protection31.The main limitation of this study is represented by the relatively small sample size in relation to the wide range of HPV genotypes detected and the use of a vaccine whose specificity is restricted to only four genotypes. Indeed, the effect of HPV in male infertility was shown to be largely independent from viral genotype33.Considering the wide prevalence of HPV infection in infertile couples, these results may directly impact on their diagnostic work-up, being supportive of a screening for HVP even at the very beginning of this process. In case of viral DNA detection in semen, FISH analysis for HPV would address the diagnostic accuracy of infection and possibly the convenience of a therapeutic approach with vaccinationIn conclusion, HPV screening in infertile male patients is strongly recommended and we suggest to perform FISH analysis in infected subjects, in order to ascertain the pattern of the viral infection. Since prophylactic vaccination enhances healing from HPV infection in semen cells, we propose HPV vaccination as a suitable strategy to improve assisted and even natural fertility.We conducted a retrospective study on infertile couples who attended the Unit of Andrology and Reproductive Medicine between January 2013 and September 2016. At recruitment, all couples reported the seek for fertility since at least one year and gave written informed consent for the access to their clinical information and the use of their data according to the Italian Privacy Law. The study was approved by the Ethical Committee for Clinical Experimentation of the Padova University Hospital, Protocol number 2336 P and subsequent amendments.42. Patients who agreed HPV vaccination, received off-label prescription of the quadrivalent vaccine Gardasil . Vaccine was administered as 3 injections over 6 months, the second dose received 2 months after the first dose, and the third dose received 6 months after the first dose respectively. The procedure was performed at the Unit of Hygiene and Public Health of Padova Hospital after acceptance of written informed consent.Among infertile couples, those eligible for recruitment had idiopathic infertility and detection of HPV-DNA in semen of the male partner. At the time of diagnosis, infected patients received medical counseling, including HPV vaccination aimed to accelerate viral clearance, as previously describedSemen analysis, ASA detection, INNO-LiPA for HPV detection/genotyping and FISH for HPV-DNA on sperm and exfoliated cells (see below) were evaluated in all male patients throughout an overall follow-up of 1 year. Analyses were performed at baseline (T0), at the end of HPV vaccination  and after further 6 months from the end of HPV vaccination . During the study period, clinical pregnancy rates (by positive serum β-hCG test), ongoing pregnancy rates , miscarriages and healthy born babies were also recorded. The prevalence of HPV infection in semen and pregnancy outcomes during the whole study period, were retrospectively evaluated in patients that received vaccination (the “vaccine group”) and in non-vaccinated patients (the “control group”).6 cells per ejaculate, independently from the other sperm parameters evaluated according to World Health Organization guidelines 201043. Exclusion criteria were: concomitant infection of Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoae or other sperm infections, together with seropositivity toward human immunodeficiency virus type 1 or 2, human T-cell lymphotropic virus type 1 or 2, hepatitis B or C virus and Treponema pallidum. Patients with karyotype abnormalities or CFTR mutations were also excluded. Screening and genotyping for HPV-DNA together with analysis of localization of HPV on spermatozoa and/or exfoliated cells were performed on whole semen respectively by INNO-LiPA Genotyping and Fluorescent in situ hybridization (FISH) as previously described31. Multiple HPV infections at recruitment were defined as the detection of ≥2 different HPV types in whole semen by the genotyping assay.As male partners eligible for the study were considered subjects aged 25 to 40 years with HPV semen infection and total sperm count ≥40 × 1044 idiopathic/unexplained infertility, age between 25 and 35 years, BMI between 18 and 30, negative pap smear and genital swab for the presence of Chlamydia trachomatis, Neisseria gonorrhoae, Trichomonas vaginalis, bacterial vaginosis. Patients with previous ovarian/tubal surgery, patients with a previous history of cervical dysplasia, positive history for endometriosis, pelvic inflammatory disease, tubal occlusion and polycystic-ovarian-syndrome were exluded. In addition, patients treated for benign endouterine disease  in the previous 6 months were excluded. Patients with history of smoking, karyotype abnormalities, mutations of the cystic fibrosis gene, major systemic disease  previous neoplasia, previous chemo and/or radio therapy, untreated uterine diseases  were also excluded.Dealing with the female partner, were considered as eligible normo-ovulatory women with the following characteristics: normo-responder according to Bologna Criteria43. Samples were assessed for semen volume, pH, sperm concentration, viability, motility, and normal morphology. SpermMar Test kit was also applied to detect the presence of anti-sperm antibodies of the IgG and IgA class according to the manufacturer’s protocol 46. Complete coverage of sperm cells by latex particles was considered as positive result for the test whilst the presence of uncovered and moving sperms was considered as negative.Semen samples were collected from all the study partecipants by masturbation. Patients were required to attend the visit with 3 days of sexual abstinence. Semen samples were let liquefy at room temperature and evaluated according to the World Health Organization guidelines for semen analysis47. Briefly, total DNA isolated form semen underwent PCR amplification using the INNO-LiPA HPV Genotyping Extra assay reagents. Biotinylated PCR products were genotyped by hybridization to HPV type-specific oligonucleotide probes, bound to nitrocellulose membrane, and detected by colorimetric reaction using an Auto-LiPA 48 instrument in accordance with the manufacturer’s recommendations. All results were confirmed by visual inspection by trained personnel.HPV-DNA screening-genotyping was performed with INNO-LiPA HPV Genotyping Extra assay  according to the manufacturers’ protocols as previously reported by Barzon and collegues6 smeared sperm, fixed with a methanol-acetic acid solution, were used for FISH analysis for HPV as previously described48. Briefly, permeabilized samples were pre-treated with pepsin and incubated with hybridization solution  containing biotin-labeled HPV DNA probe. After denaturation of target DNA, hybridization with HPV-DNA probe was performed by incubating the samples at 37 °C overnight in a humidified chamber. The biotin-labeled HPV probe was detected by incubation with streptavidin Texas Red (Vector Laboratories) at the proper dilution. Slides were mounted with an anti-fade buffer solution containing DAPI as counterstainer . Samples were analyzed using a fluorescence microscope  equipped with a triple band-pass filter set . For each slide, at least 200 spermatozoa and 200 exfoliated cells (E.c.) were analyzed by three independent investigators and results were reported as the percentage of sperm or E.c. showing a positive staining as described. Supplementary Figure Glass slides containing at least 2 × 10No patients were involved in setting the research question or the outcome measures, nor were they involved in developing plans for design or implementation of the study. No patients were asked to advise on interpretation or writing up of results.p value was reported. The proportion of HPV-positive patients during the study was compared with non-parametric Cochran’s test. Post-hoc pairwise comparisons were performed with McNemar’s test. Differences between groups in categorical variables at each time point were compare with Pearson’s Chi-square test, or Fisher’s exact test when expected frequency was five or less. p values < 0.05 were considered as statistically significant.Statistical analysis of the data were conducted with SPSS 21.0 for Windows . The results are expressed as means ± standard deviation (SD) and categorical variables are expressed as a percentage. The Kolmogorov–Smirnov test was used to check for normality of distribution. Variables not showing normal distribution were log-transformed. Characteristics of subjects between the control and vaccine arm at each time point were compared by using unpaired Student’s t test. Repeated-measures ANOVA was performed to test for differences in continuous variables during the study at three time points . Age was included as a covariate and post-hoc analysis with Bonferroni-Holm correction for multiple comparisons was performed to test differences between time-points. The Levene’s test was used to test the homogeneity of variance among groups. If homogeneity of variance assumption was violated, Welch test was performed and the respective Supplemental Information


Cervical cancer is a major problem in women and it is important to find a suitable and acceptable screening method, especially among young in low-resource areas for future human papillomavirus (HPV) vaccine follow-up investigations. The study sought to test the acceptability of self-sampling as well as the suitability of the specimen collecting devices.Ninety-eight young women from rural KwaZulu-Natal were enrolled between March and July 2014. Collected genital specimens were transferred to colour indicator cards for HPV detection. Participants answered a questionnaire where they described their experiences with self-sampling. Samples were tested for high-risk HPV using GP5/6+ PCR.Of the enrolled participants, 91 answered questionnaires and indicated that self-sampling was preferred by 51/91 (56%) women while 40/91 (44%) indicated preference for sampling by a doctor (p = 0.023). The majority, 64% were comfortable using a swab, 22% preferred a brush while 11% were comfortable with both devices. Of the 98 self-sampled specimens 61 were negative for HPV in both specimens while 37 were HPV-positive in either brush or swab. Of the 37, 26 (70%) were HPV-positive in both brush and swab (kappa = 0.743) and 11 (30%) were discordant.Self-sampling was acceptable to the majority of participants in this rural area. The Dacron swab was the preferred device, and can be used in combination with colour indicator cards for comfortable self-sampling, easy storage and transport of specimens plus detection. The global burden of cervical cancer is estimated at 528,000 new cases and causes 266,000 annual deaths . Eighty-A variety of sampling devices can be used for HPV detection for example Dacron swabs, cotton wool tipped swabs, cytobrushes and vaginal washings (lavage), either by self-collection or collected by a health worker. Studies in America, showed an 80% agreement between physician-directed swabs and self-collected vaginal tampons . In a meSelf-collected samples for HPV testing might be a better approach in areas where cultural and programmatic barriers may limit the use of routine gynecologic procedures . This seThe main objectives of the study were to (i) assess the acceptability of self-sampling among young rural women, (ii) to evaluate the optimal sampling device, and (iii) investigate the use of Flinders Technology Associates (FTA) cartridge indicator cards for safe storage and transport of DNA for hr-HPV detection.The study had a cross-sectional design and was nested in a larger main clinical study. The latter was investigating Female Genital Schistosomiasis (FGS) and other sexually transmitted infections including HPV . HPV wasFemale participants aged 16 years and above were recruited from randomly selected high schools from the rural district in the northern coastal region of KwaZulu-Natal, South Africa. This district has minimal infrastructure; much of the population lives in informal settlements and has little sanitation and other infrastructure. Young women were targeted for this study because of the assumption that they are sexually active and would provide enough HPV positive samples to be able to compare the sampling devices used.The study population consisted of young women, recruited from high schools from a rural area in the northern coastal region of KwaZulu-Natal, South Africa. Inclusion criteria for the study were 1) attending the largest (more than 300 pupils) government high schools situated more than 10 kilometres from the coastline and below 300 m above sea level, (2) provided written informed consent, (3) sexually active, (4) not pregnant (5) above 16 years of age and (6) prepared to undergo self-sampling. Female participants were recruited from randomly selected high schools. Enrolment of participants into the study and group allocation was carried out following an outline in Fig.  attendinTrained research assistants interviewed the study participants in their local language, isiZulu. For sample collection each participant was given verbal, written and graphical instructions to follow when collecting a sample. The self-sampling devices, the Dacron swab-EZ  and the Viba-brush , were alternated as first and second sampling device respectively per group. One group collected using a Dacron swab first while the other group of participants used a Viba brush first. After a sample was collected, it was transferred to a solid carrier, the indicator FTA elute card , which changes color from purple to white when enough material has been transferred. Cards were kept at room temperature until they were processed.After the sample collection, participants self-administered a questionnaire on pain experienced and their preferred specimen-collection method. The reasons for their preferences were asked using open ended questions. The questionnaire was completed confidentially in the waiting room area of the research site clinic.Deoxyribonucleic acid obtained using swabs and Viba-brushes was extracted from FTA cards following the FTA elution method described previously . Briefly2, 25 mMol MgCl2, 2.5 µM GP5+ and 2.5 µM GP6+ primers, 10 mM nucleotides and 5 units taq polymerase [Human papillomavirus was detected by GP5+/6+ HPV PCR  followedlymerase . For PCRSix negative (DNA isolated from lung cancer cell line A549) and two positive controls  were included in each run. The cut-off value for hr-HPV positivity was calculated as the mean plus three times the standard deviation of six negative controls in each plate. Samples with a ratio of 2 or more were regarded as positive.All the specimens from which hr-HPV was detected, as described above, were further analyzed to identify the genotypes. For genotyping, individual probes were used instead of a probe cocktail mix used for the EIA test. Individual probes for the most common hr-HPV genotypes in cervical cancer  were useThe sample size was calculated based on the hypothesis that 50% young women will prefer a self-sampling procedure over the gynaecological examination and that 10% would prefer the gynaecological examination. In order to detect the difference with a significance level of 5% and power of 80% a sample size of 25 participants would be needed in each of the three groups for sample collection sequence and randomisation. In order to accommodate for uncertainties, and increase the power of the investigation, 125 participants were approached and of these, 98 were included in this study.The data were captured and analyzed by the IBM SPSS Statistics 22 package.The results on the preferred device were compared using 2 × 2 tables and Pearson Chi square test. The correlation of hr-HPV detection with Viba-brush and Dacron swab on the FTA card was determined using kappa statistics to determine the correlation beyond that expected by chance. A p value of < 0.05 was considered to imply a statistically significant difference between the variables. Kappa values were considered as poor (< 0.20), fair (0.21–0.4), moderate (0.41–0.60), good (0.61–0.80) and very good (0.81–1.00 and above) agreement, respectively . DiscordAll 98 participants were females attending rural high schools (grades 10–12), and residing in low resource areas of the KZN province. The women were aged between 16 and 22 years (median age 18) and had an average of two sexual partners in their lifetime (SD = 1.227). In all, 79  had heard about sexually transmitted infections (STI) whereas 17  denied any knowledge of STI. All study participants did the self-collection of vaginal samples using the Viba-brush and the Dacron swab.The questionnaire was completed by 91 participants , whereas seven women did not return the completed questionnaire (reasons unknown). Of the 91 participants, 58  preferred a Dacron swab  whereas 20  preferred a brush for self-sampling. Ten  were comfortable with both and three  did not indicate a preference. Only 5/91  women reported pain using a swab, whereas pain was reported by 25/91  using the brush. Of 91 participants 58 indicated a preference for self-sampling over collection with sampling by a doctor . They cited reasons against clinician-sampling related to time, cost, discomfort, embarrassment and some were worried about meeting male doctors. However, 21 out of 91 (23%) participants who preferred self-sampling also gave reasons in favor of clinician-sampling, which included fear of inadequate self-sampling, trust in the doctor’s expertise in sample collection and disease diagnosis. Of the 91 participants who answered the questionnaire 52  indicated that they were scared to hurt themselves when performing self-sampling.All 98 FTA cards had changed color, indicating that enough DNA had been transferred by the participants. Of the 98 participants, 37 were positive for hr-HPV, with 26 positive in both the brush and the swab and 11 were discordant. Of the 11 hr-HPV discordant results, three were negative in brush (positive in swab) and eight were negative in the swab (while positive in the brush) specimens. This resulted in a kappa value of 0.743, indicating good correlation of the HPV DNA detection between swab and brush specimens (Table At least one concordant genotype was found in 16 of the 26 participants that were hr-HPV positive in both brush and swab. In the brush we found a high number of hr-HPV genotype 39 (p = 0.011) and HPV 51 (p < 0.001) whereas in the swab type 66 was overrepresented (p = 0.046), for all other genotypes the prevalence was comparable in swab and brush specimens.The Dacron swab was the self-sampling device preferred by our study population, possibly because it was found to be less painful. The study showed that cards can be kept at room temperature in an area such as ours which was remote, hot and humid. Young sexually active women were targeted for this study because of the assumption that they would provide enough HPV positive samples to be able to compare the sampling devices used.The FTA cards have offered a useful collection and transportation tool for DNA test samples across a wide range of temperatures (22–45 °C) and humidity between 20 and 100% . FTA carThe fact that the swab specimens could be easily transferred to FTA cards may be beneficial for communities living in low resource areas provided the price is low. Self-sampling may be used to reach individuals who have difficulties reaching the health facilities, have religious reasons or are not comfortable with undergoing gynecological examination. Self-sampling for HPV screening has some advantages over collection by a health worker. These samples can be cost-effective, easy to collect and less demanding in terms of storage and transportation methods. For self-sampling to be effective it is necessary that the sampling tools or devices are user-friendly, that the targeted population is comfortable with using them, and that the laboratory test is able to detect the viral infection from such samples.Reasons for discordant hr-HPV results between the preferred device and the brush could not be ascertained. Therefore, further research to work out the sensitivity and specificity of the Dacron swab specimen on FTA card for HPV test is necessary since this type of specimen was preferred. FTA cartridges and self-collected brush specimens have been tested previously for HPV molecular analysis .Although self-sampling may seem easy and comfortable for women and is well-accepted by some , 28, it Successful implementation of self-sampling should be viewed in the light of the sampling tool used. In this study, two-thirds of the participants preferred using the swab thus suggesting that a swab can be used comfortably for self-collection of genital samples. Previous reports of self-sampling using swab specimens for HPV DNA analysis made use of transport media which can preserve the specimen  whereas Although we cannot preclude a type 2 error we found that neither device out-performed the other for hr-HPV detection. A good correlation was observed between the swab and the brush with regards to the number of positive and negative samples in this study, however, discordant results were found in hr-HPV genotypes in the specimens. Other reports have shown a higher prevalence of HPV detection from specimens collected in FTA cards than those in liquid medium from Costa Rica women involved in phase III vaccine trial . In addiThe cytological tests (Pap smears or liquid based cytology) are currently the most widely used and available conventional test for cervical cancer screening , 39. HowWhereas cervical cancer screening would normally aim at older women, we included young women for self-sampling to increase the chances of detecting HPV as infection is most common in this age group. It is not advisable to advocate HPV screening on young women since HPV infection will usually regress . The HPVLimitations in this study were the sample size, which due to financial constraints was kept at a  minimum and consistency in the sampling technique. For privacy reasons, we could not ascertain the consistency in the self-sampling technique between participants regarding the anatomical part of the genitalia each woman touched during sampling.The HIV status was not included in the study since it was not a prevalence study but it was merely to assess FTA card in combination with the Dacron swab against a Viba brush for HPV analysis. However, given the high HIV prevalence in South Africa, and hence, the potential impact, it would have been interesting to know if HIV infection had any significant effect on participant acceptability for self-sampling.Self-sampling was accepted by our rural study participants and the Dacron swab was comfortable for self-collection of female genital samples. Use of the Dacron swab coupled with the FTA indicator cards showed that sufficient DNA was collected and preserved. This study showed that the method can be used for identifying HPV and is acceptable to rural women. However, we would not encourage use of self-sampling for HPV screening in young women because a positive HPV result may cause anxiety and fear that they have cancer. Further investigation is necessary involving older age groups from rural KZN province on the acceptance and usefulness of self-sampling. However, this study shows that self-sampling is accepted and feasible, and therefore, self-sampling collection of specimens for HPV analysis may be of value for HPV vaccine rollout follow-up investigations in KZN. There will also be a need for proper training of women on the technique self-sampling technique.