We conducted a prospective, randomized two-arm, parallel group, and open label trial to investigate whether the use of LINE would increase HPV vaccine intention among not completely vaccinated university students. In June 2020, we recruited students aged between 18 and 35 years from four universities in Japan. Among the 357 enrollees , 178 and 179 participants were randomized into the LINE and Mail groups, respectively. At baseline, within three years, vaccine intention was observed in 40% vs. 42% of participants, respectively. At the first intervention, which provided similar PDF leaflets about HPV vaccine and cervical cancer prevention, there was no significant difference in vaccine intention between the two groups. However, at the second intervention of LINE-assisted knowledge intervention for 5 days per week for 7 weeks, the LINE group had a higher proportion of vaccine intention than the no intervention group : 1.59–4.35) in per-protocol analysis. The significance remained in the intention-to-treat analysis of multiply imputed datasets. Although LINE did not directly increase HPV vaccine intention compared to conventional posts, the LINE-assisted provision of information was effective in improving HPV vaccine intention among Japanese university and college students. Under the Japanese Immunization Program in 2013, the human papillomavirus (HPV) vaccine program was launched for girls aged between 12 and 16 years . The vacIt has been previously reported that knowledge, health literacy, health belief models, and awareness were important promotors of HPV vaccine intention ,10,11,12Today, social media is central to communication among young people, and hence it is highly expected that the use of social media may influence HPV vaccine uptake ,22. SociThis study was a prospective, randomized two-arm, parallel group, and open-label trial with equal allocation to each arm see .We recruited students from three universities  and one nursing college in Akita Prefecture, the northern part of Japan. We recruited participants during the first round (Round 1) between June and December 2020, and the second round (Round 2) between April and June 2021. The inclusion criteria were as follows: a male or female student aged between 18 and 35 years, a student who belonged to any of the four schools, and who had not been vaccinated. In the Akita prefecture, the majority of university and college students were aged 18–22 years and had missed active recommendations because they were ineligible for the publicly subsidized HPV immunization program in 2013 due to their age.Participants who were not of Japanese ethnicity and those who did not use LINE were excluded. In Japan, at the time of this study’s investigation, LINE was the most popular social networking service for young people to communicate with friends, family, school mates, and any other community members. The recruitment methods included campus electronic bulletin boards that addressed individual students, classroom websites, and on-campus notice boards. The students accessed the Google form by scanning the QR code attached to the flyer and read the outline of the study protocol. The purpose of the study and survey procedures were also included on the website of the Department of Public Health at Akita University. If a student agreed to participate, they submitted a consent form to the research administrative office; these forms were initially sent by e-mail from the office. Immediately after the submission of the informed consent form, they were asked to answer a questionnaire created in Google forms using a link attached to an e-mail from the office. The questionnaire was sent at baseline, and during the first and second interventions. We sent reminder e-mails and called at least twice on each occasion. The participants were given a JPY 500 (USD 5) equivalent gift card for each questionnaire. If the participants completed all the questionnaires, they received three gift cards of JPY 500 (USD 5).Participants were randomly assigned to the LINE and Mail groups in a 1:1 ratio  by an independent administrator . The randomization with a sex-stratified block size of 4 and 6 was generated beforehand by a statistician (not involved in the enrolment and intervention). Researchers, except for the statisticians (Kengo Nagashima and N.F.), were blinded to the randomization and allocation. We created a private open chat in which the LINE participants were regulated by the administrative office under an anonymous nickname and asked the participants to follow the ground rules for concealed allocation, which were: (1) not to talk outside the chat room and (2) not to talk to anyone about their participation in this study. Participants were asked to not talk to other LINE participants until the second intervention, interaction between enrollees was permitted at the second intervention, and the detailed study aims related to the allocation were not disclosed to the participants. Randomization and intervention were conducted twice according to Rounds 1 and 2.The study was approved by the Research Ethics Committee of Akita University (No. 2387), and it follows the ethical international standards established in the Declaration of Helsinki. Written informed consent was obtained from each participant prior to the study, and the present trial was registered at the University hospital Medical Information at Tokyo University with the registration number: UMIN000044750.We conducted the first intervention to observe whether there was a difference in HPV vaccine intention, HPV knowledge, health literacy, and health beliefs between the LINE and Mail groups. First, we sent an A1 sized two-page leaflet on HPV and cervical cancer prevention to both groups. The leaflet was created by the authors by referring to the relevant sources from the Ministry of Health, Labour, and Welfare ; Japan PSubsequently, we conducted the second intervention to observe whether there was a difference in HPV vaccine intention, HPV knowledge, health literacy, and health beliefs between the intervention and no intervention groups. During this period, we sent information on HPV and cervical cancer prevention 5 days a week for a period of 7 weeks only to the LINE group , and considered the Mail group as the no intervention group. Participants in the LINE-assisted intervention group were instructed in advance that they would be able to consult or tweet freely within the LINE community specifically created for this purpose. The administration office issued ground rules before each participant entered the chat room, which prohibited any critical, intimidating, or harsh comments. Membership would be forcefully terminated if there was no change in behavior after a warning had been issued for any offence. All the participants were kept anonymous and allowed to communicate with each other freely at any time during the second intervention. The administration office for this specific LINE closed group consisted of five medical students  and one faculty (K.N.), who were also members of this chat room. The five members dealt with any queries or comments and escalated more complex issues to the faculty. The medical students received basic training on potential interactions or concerns with participants and held a meeting at least once a week. The image of the intervention is shown in The participants completed the Google form-based surveys at three time-points: the baseline, first intervention, and second intervention.We asked the participants about HPV vaccine intention, HPV knowledge, health literacy, and health beliefs related to the HPV vaccine and cervical cancer prevention using a Google form-based questionnaire at the baseline and at the first and second intervention. The list of questions in the Google form-based questionnaire can be seen in This study’s primary outcome is the students’ intention to receive the HPV vaccine. Thus, the study participants were asked the question, “At what time do you intend to receive the HPV vaccine?” They answered based on a five-point scale: “immediately” = 1, “within six months” = 2, “within a year” = 3, “within three years” = 4, “no intention to receive a vaccine”, or “not sure” = 5. As this study was conducted during the COVID-19 pandemic, there was only a small percentage of participants who answered “immediately.” Subsequently, because of statistical reasons, the response patterns were grouped into binary with “immediately” to “within 3 years” as one group and the rest as “otherwise.” This study’s secondary outcome is HPV knowledge, health literacy, and health beliefs related to the HPV vaccine and cervical cancer prevention.We asked the participants to answer 20 knowledge questions on the HPV vaccine. The first 10 questions were basic, but the next 10 questions would require that students study individually by collecting further accurate information. The question items from 1–10 were as follows: (1) HPV is a human immunodeficiency virus (correct answer: incorrect); (2) There are four types of HPV (incorrect); (3) HPV causes cancer of the uterine body (incorrect); (4) HPV cannot be transmitted by a single sexual intercourse (incorrect); (5) HPV can only be transmitted by males, not by females (incorrect); (6) Types 6 and 11 cause more than two-thirds of all cervical cancer (correct); (7) Cervical cancer can be prevented by using vaccines (correct); (8) Cervical cancer screening is done by cytology (correct); (9) The eligible age for the HPV vaccine to be taken at public expense is up to 35 years old in Japan (incorrect); (10) Cervical cancer is caused by persistent infection with high-risk HPV (correct). The response patterns for the first 10 questions were either “correct,” “incorrect,” or “do not know.” The answer to question (8) has now changed in Japan, but other countries use HPV typing or cytology and HPV typing. The question items from 11–20 were as follows: (11) What do you think is the screening rate for cervical cancer in Japan?  (correct answer: 4); (12) In some foreign countries, HPV vaccination is also given to males  (correct answer: 1); (13) What percentage of females with a history of sexually transmitted infections do you think will experience HPV infection by the time they are 50 years old?  (correct answer: 2); (14) Which types of HPV are less likely to cause cancer?  (correct answer: 5); (15) What is the leading cause of cancer death among females in their 20 s and 30 s?  (correct answer: 5); (16) It is necessary to have a regular checkup for cervical cancer even if you have HPV vaccine  (correct answer: 1); (17) How effective do you think the cervical cancer vaccination is in preventing cervical cancer?  (correct answer: 2); (18) How often do you think serious adverse reactions will occur due to vaccination compared to flu vaccine?  (correct answer: 2 or 3); (19) What do you think is the probability for the occurrence of cervical cancer in the lifetime of a female?  (correct answer: 3); (20) Which of the following do you think is used to confirm the diagnosis of cervical cancer? , 5. Pap smear) (correct answer: 4). The total score was divided into binary at median.Considering a previous study , we inveWe exploited the health belief model  to identOther covariates measured by the self-administered questionnaire were sex, age, university, faculty , smoking  and drinking , sleep duration, exercise volume and intensity based on a unit of metabolic equivalents, frequency of breakfast per week, and the reasons for not getting the vaccine. The reasons for not getting the vaccine included concerns about side-effects, parents’ opposition, I do not know where to get vaccinated, expensive, sexually inactive, I don’t want to bother getting vaccinated , I would not get infected, friends not vaccinated, vaccination is unnecessary if I go for regular check-ups , schools do not recommend, afraid of get infected with COVID-19, and others, and the participants were allowed to choose as many as they had.Sample size calculation revealed a total sample size of 194, with 10% dropout, α = 0.05, 1-β = 0.8, and 5% difference in proportion. The descriptive analysis included the number and content of the tweet in the LINE open chat during the 3-month follow-up period.HPV vaccine intention, HPV knowledge, health literacy, and health belief model related to HPV were assessed using a logistic regression model that was stratified by sex at the first (LINE vs. Mail groups) and second (LINE-assisted intervention vs. no intervention groups) interventions. We also assessed the 20 questions on HPV knowledge and 10 questions on health beliefs using a logistic regression model that was stratified by sex at the first intervention. We applied both per-protocol (PP) and intention-to-treat (ITT) analyses using a multiple imputation technique  to creatAll statistical analyses were performed using SAS version 9.4 .n = 178) and Mail (n = 179) groups. The participants’ characteristics are presented in n = 86, 24%) and “parents’ opposition” , followed by “I do not know where to get vaccinated” , and “expensive” .Among the 395 undergraduate or graduate students who agreed to participate in this study, 38 participants did not meet the inclusion criteria. Furthermore, after excluding 4 students who did not report their age, 357 students were randomly assigned to the LINE  in the LINE group and 76 participants (42%) in the Mail group reported that they had HPV vaccine intention sometime between “soon” and “within 3 years”, and no significant difference was observed. No significant difference in knowledge, literacy, and health belief model was consistently observed, both in PP and ITT. At the first intervention, there was no statistical difference in HPV vaccine intention, knowledge, literacy, and health belief model between the LINE and Mail groups. Although the data were not shown due to small numbers, the number of participants who answered that they got vaccinated was six (4%) and three (2%) in the LINE and Mail groups, respectively.At the second intervention, HPV vaccine intention in the LINE-assisted intervention group had a higher proportion of HPV vaccine intention than in the no intervention group: the odds ratios ) were 2.62 (1.59–4.35) and 2.08 (1.32–3.28) in the PP and ITT analyses, respectively. Although the data were not shown due to small numbers, the number of participants who answered that they got vaccinated was four (3%) and five (4%) in the LINE-assisted intervention and no intervention groups, respectively. No statistical difference was observed in knowledge, literacy, and health belief model between the LINE-assisted intervention and no intervention groups.We compared the percentage of people who answered all 20 knowledge questions correctly. At the baseline, “Cervical cancer can be prevented by using vaccines” in the LINE group had a significantly lower rate than in the Mail group (10% vs. 18%). At the first intervention, we found no significant difference between the LINE and Mail groups . At the When we compared the agreement rate of each item of the health belief models at the baseline, those of “I am worried about the side effects of the vaccine” in the LINE group was significantly higher than the Mail (60% vs. 50%) group, and those of “I do not know where I can get the vaccine” in the LINE group was lower than in the Mail group (51% vs. 62%). At the first intervention, those of “I am worried about the side effect of the vaccine”  or no intervention (second intervention) groups. At the first intervention, where PDF leaflets were provided, there were no significant differences in vaccine intention, knowledge, health literacy, and health belief models between the LINE and Mail groups. However, three months after the second intervention, which consisted of a seven-week provision of information, vaccine intention was significantly higher in the LINE-assisted intervention group than in the no intervention group. We have discussed our results based on the context of the COVID-19 pandemic in Japan and on previous studies.We need to emphasize that our primary outcome was HPV vaccine intention and not the actual uptake. When this study was being conducted, COVID-19 vaccination was more aggressively recommended than HPV vaccination because the COVID-19 vaccine uptake was low, especially among the young generation, who were considered critical to ease the local pandemic spread. In Japan, we experienced the first national state of emergency, which was declared on 16 April 2020, and lasted until the end of May . SubsequAmong various social networking services, we used LINE as a communication platform to change vaccine intention and knowledge, literacy, and health belief model in this study. Previous studies ,36,37,38For the second intervention, the LINE assisted seven-week provision of information significantly improved the HPV intention compared to the no intervention group. There was also no significant difference in knowledge, literacy, and health belief models between the intervention and no intervention groups. However, the time series analysis demonstrated that the scores of knowledge, literacy, and susceptibility significantly increased in the LINE-assisted intervention group, while only literacy scores significantly increased in the no intervention group. Such difference within each allocated groups may explain HPV vaccine intention. Furthermore, students in the LINE-assisted intervention group were allowed to freely make any comments and interact with other participants in the closed LINE chat room. Content analysis of the dialogues revealed that the majority of the interaction was dominated by one-way queries addressed to the administrative office, which was run by the Department of Environmental Health Science and Public Health, Akita University Graduate School of Medicine, and all participants knew that the members were medical students. Furthermore, Ireland and Denmark, whose HPV uptake previously plummeted to 50%, but successfully increased their immunization rate to 80% ,46, attrThere are some limitations that should be addressed. First, as our participants were limited to students belonging to the four universities in Akita prefecture, sampling bias may exist. In addition, because this study was conducted during the COVID-19 pandemic, when there was a state of emergency, the numbers of participants were relatively small. Second, participants who were more interested in HPV might have participated in our study, which may have led to the overestimation of HPV vaccine intention. Nevertheless, here, HPV vaccine intention was much lower than those previously reported in other Asian countries . In factThe present study demonstrated that there is no advantage in using LINE over Mail to provide HPV-related information to increase HPV vaccine intention. However, the LINE-assisted seven-week provision of information about the HPV vaccine and cervical cancer prevention may increase HPV vaccine intention, although the role of health professionals might influence HPV vaccine intention.


Para-social Relationships, Social Presence, and gift-giving in the context of China. Based on the survey and interview of the viewer on Douyu, a Chinese live streaming platform, this study found that there is only a weak to a medium correlation between para-social relationships and viewers' gift-giving behavior. The correlation between social presence and gift-giving was even weaker. Although the conclusion of this study may be affected by the sample size limitation, it can still provide a reference for the current research on gift-giving on Chinese live streaming platforms.In China, live streaming has grown rapidly in recent years, with gift-giving, a unique business model in live streaming, driving the development of many industries. This article explores the association between gift-giving behavior in game live streaming and viewers' live streaming experience. Specifically, this study aims to explore the correlation between  Viewers can then comments on the streamers' behavior or interact directly with the streamers through the real-time comments function on the platform . The remaining four options had a combined response rate of 30 percent. They are: viewers think “streamer is someone they admire”; “Someone who understands them”; “A person who wants to meet in person”; And “someone like a friend”. The overall response rate of these five options was over 50 percent, proving that PSR exists between the viewers and game streamers on Douyu.In the questionnaire, there were eight prediction options that measured the existence of PSR, five of which were adapted from the prediction model established by previous scholars see . They haTwo of the three remaining prediction options deny PSR's presence on Douyu: Viewers perceive the game streamer as “Someone sophisticated” and “Someone distant”. However, the combined response rate of these two options was just over 10 percent, which is not representative. Furthermore, one of the remaining indicates viewers' recognition of game streamers' technical prowess in live streaming, with a one in three response rate. In conclusion, although the response rate of the remaining prediction options accounted for nearly 50 percentages, the total response rate of the two options, which predicted the non-existence of PSR, was insignificant, and the other option could not strongly predict the existence of PSR. Therefore, the finding that PSR exists between viewers and game streamers on Douyu still holds.During the interview, the author asked the interviewees about their choices in the questionnaire. For the PSR prediction option with the highest response rate, “Viewers perceive streamers as someone who is authentic”, respondents' replies focused on game streamers' performance during live streaming. The author named these recurring patterns and themes “Interactions that break the boundaries of fantasy.” by the author. For example, one of those interviewed wrote:The Streamers I watch often interact with the viewers… He can see what viewers have said in the real-time comment system, and he will select some of them to respond. And sometimes his responses to the audience go on for a while.” (participant 6)“This statement confirms Wulf's findings that the game streamer's interaction with the viewers during the live streaming is authentic, bringing the viewer into a “dialogue”. This “dialogue” has two dimensions. First, it refers to the fast, two-way connection (interaction) between viewers and streamers. Viewers are immersed in this connection and are encouraged to stay longer in this live streaming. In other words, this dimension of dialogue represents the increasing possibility of a two-way connection between the viewer and the game streamer, and these possibilities motivate the viewer to engage in more PSI “Unlike the patient and persistent interactions of game streamers reported by Participant 6, participant 9 did not have a strong sense of the game streamer's interaction. A key reason for such differences lies in the different reputations of the game streamers that the two participants often watch, which can also be understood as related to the size of the audiences on different live-streaming channels. Specifically, participant 6's most-watched game streamer has only moderate popularity on the Douyu, while Participant 9's most-watched game streamer has over 19 million subscribers on Douyu. The channels of game streamers that are extremely popular have numerous viewers participating at all times. As a result, even if the game streamers want to interact with viewers, the sheer volume of chats makes it impossible for the most popular streamers to engage effectively with any or all of their viewers. The researcher named this recurring pattern and theme as “Always control the power of interaction”—such situations confirm that PSI is typically one-sided and controlled by the streamer.Although the absence of two-way connections in large channels may affect PSI, PSR is noted to occur still. The reason is that the game streamers utilize different methods and tactics to maintain a sense of intimacy with their viewers during live streaming, ultimately leading to PSR. For example, as another interviewee noted:It is funny to watch his live streaming… I like his style of discourse, which has a sense of humor and at the same time stands on its own. There are many memes about his words in our community of followers… And, obviously, you can see a lot of awkward moments, and for the most part, he makes fun of his own awkward moments. That is what I like about him.” (Participant 1)“The response of Participant 1 outlines a way game streamers cultivate intimacy with their viewers—by communicating with their viewers in a friendly, accessible manner, for example, by making fun of their embarrassing moments , and more than a third of participants said they spent between 0 and 100RMB (N = 24) on gifts. The remaining participant's answers were distributed across the remaining four questionnaire options. Since those numbers were too small to be represent, the study did not analyze that data see .2 = 60.284 p = 0.033 < 0.05). For the first group of participants who only gave free gifts, the option that received the most responses was “Someone who is talented”. As noted hereinbefore, this option could not effectively predict PSR, so the high prevalence rate of this option does not prove a correlation between PSR and viewers' gift-gifting behavior.In this cross-analysis, there was a significant difference in the proportion of options selected . Combined with the high response rate of the option “Someone who is talented” mentioned hereinbefore, this study suggests a positive correlation between the viewers gifting behavior and the viewer's appreciation and admiration of game streamer's game technology.The interview results indicated a weak positive correlation between PSR and gift-giving behavior. Although most interviewees reported their positive perception of PSR in the interview, they did not express the relationship between this perception and their gift-giving behavior. Such as:I love this streamer… I feel like he is like a brother to me… As for I did not send a virtual gift, I personally feel there is no need, and my closeness to the streamer does not drive me to give gifts. I just watch his live streaming more, or pay more attention to his real life.“ (Participant 7)“Six interviewees (out of 10) presented the same idea. Based on the questionnaire answers of these six interviewees, they all positively felt the existence of PSR. This suggests that, for most viewers, the presence of the PSR only has a weak correlation with their gift-giving behavior. A possible reason may be related to the consumption power of the interviewees because nearly 90% of the participants in this study claimed their monthly income is less than 3000RMB, and they are between 18 and 25 years old. So they may not have a strong consumption power. However, interviewees who gave gifts also said:A big reason for my gift-giving is because I like his live streaming style. I only subscribed him on Douyu, and generally I will only watch his live streaming“ (Participant 2)”This statement is the same as Yu et al.'s  results,N = 439). Although the average response rate of the other five prediction options is not more than 15%, these options are actively predicting the viewers' SP experience, so this study assumes that the viewers can perceive a large number of SP while watching live streaming.This study found that viewers experience different SP when watching live streaming see . In the As for the correlation between SP and gift-giving behavior, the researcher found only a very weak association between SP and gift-giving behavior. In this research, six options in the questionnaire predicted the correlation between SP and gift-giving behavior. However, these six options did not have much response rate see . The higThe researcher questioned the participants in the following interview. Most of the participants who had gift-giving behavior indicated that SP was not what motivated them to give gifts to game streamers. Instead, their perception of SP motivated them to participate in other live streaming elements. Participants who had not given gifts expressed the same opinion. As one wrote:I see the streamer interacting with other viewers and it actually inspires me to get involved, and I want the streamer to talk to me, but I'm not going to give him a gift just to have him interact with me… And that will make me watch a little bit more… As for wanting to reflect my identity in the follower community, I don't think this is the motivation for me to give gifts “(Participant 10)”Therefore, this study believes that there is a significant positive correlation between the majority of viewers' live streaming participation and SP, such as sending real-time comments. However, gift-giving is only weakly correlated with SP. Thus, the second research question has been answered.In conclusion, this study explored the correlation between viewers' gift-giving behavior and PSI/SP on Douyu by using mixed methods of survey and interviews. Through the analysis of the results, it was found that, first of all, PSR does exist on Douyu, but there is only a weak to a moderate positive correlation between PSR and viewers' gift-giving behavior. Moreover, this study found a certain degree of positive correlation between viewers' appreciation of game streamers' game skills and viewers' gift-giving behavior. Finally, this study also found that although viewers perceived different experiences about SP in the live streaming, there was little to no correlation between SP and audience gift behavior.Different from previous research, although PSR and SP in this research showed positive impacts on other viewers' live streaming participation, they do not have a significant correlation with gift-giving behavior. One possible reason is that participants in this research were mainly in the age range of 18–25. A large percentage of them were students whose consumption power was limited. Future research could further refine age stratification based on the average graduation ages for different degrees. This research also has other limitations. First, the participants were primarily women, so that the data may lack representation. Second, this research only studied Douyu, a specific platform. Future studies can conduct a mixed study on different Chinese live streaming platforms to interpret the gift-giving behavior of the Chinese viewers in a more macroscopic way.However, this research innovatively adopted mixed methods to study the relationship between different social relations and gift-giving behavior in live streaming. Although researchers have conducted research in this domain, most of them have not studied China's live streaming platforms. This research still fills the gap in current research on the Chinese viewers' gift-giving behavior and provides a potential direction for future game live streaming studies.While the study was completed, the researcher also identified challenges in this research direction. First, Chinese streaming platforms have different styles and elements from those in Europe and the United States, such as Twitch. Future research can explore the gift-giving behavior in game live streaming in combination with the platform economy of Chinese live streaming platforms. Second, as mentioned hereinbefore, the graduation time of students with different degrees may lead to differences in their consumption power. Future studies could break down age groups, or divide participants between 18 and 30 into whether they are working or not.The original contributions presented in the study are included in the article/Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants or legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.The conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, software, supervision, validation, visualization, roles/writing—original draft, and writing—review and editing of this paper were all completed by the WZ.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Sudden cardiac death (SCD) caused by ventricular arrhythmias is the leading cause of mortality of cardiovascular disease. Mutation in TECRL, an endoplasmic reticulum protein, was first reported in catecholaminergic polymorphic ventricular tachycardia during which a patient succumbed to SCD. Using loss- and gain-of-function approaches, we investigated the role of TECRL in murine and human cardiomyocytes. Tecrl  mouse shows significantly aggravated cardiac dysfunction, evidenced by the decrease of ejection fraction and fractional shortening. Mechanistically, TECRL deficiency impairs mitochondrial respiration, which is characterized by reduced adenosine triphosphate production, increased fatty acid synthase (FAS) and reactive oxygen species production, along with decreased MFN2, p-AKT (Ser473), and NRF2 expressions. Overexpression of TECRL induces mitochondrial respiration, in PI3K/AKT dependent manner. TECRL regulates mitochondrial function mainly through PI3K/AKT signaling and the mitochondrial fusion protein MFN2. Apoptosis inducing factor (AIF) and cytochrome C (Cyc) is released from the mitochondria into the cytoplasm after siTECRL infection, as demonstrated by immunofluorescent staining and western blotting. Herein, we propose a previously unrecognized TECRL mechanism in regulating CPVT and may provide possible support for therapeutic target in CPVT. The endoplasmic reticulum protein TECRL promotes mitochondrial function in cardiomyocytes and its knockout in mice leads to cardiac dysfunction, decreased mitochondria function, and elevated levels of reactive oxygen species. Catecholaminergic polymorphic ventricular tachycardia (CPVT), a prevalent cause of SCD during childhood and adolescence, is characterized by ventricular tachycardia or syncope due to emotional overstimulation or physical exercise2. CPVT is a life-threatening genetic disorder with an estimated prevalence of 1:10,000 in the general population2. Current treatments for CPVT include exercise restriction, β-blocker, class 1c sodium channel blockers, cardiac sympathetic denervation, and implantable cardiac defibrillators3. The treatments mentioned above can delay and reduce the incidence of SCD, but it still needs more effective therapeutic options to increase the quality of life in treating SCD.Sudden cardiac death (SCD) due to ventricular arrhythmias is the leading cause of mortality in patients with heart disease4. Recently, a mutation in trans-2, 3-enoyl-CoA reductase-like (TECRL) gene, coded for endoplasmic reticulum (ER) protein, was first identified in CPVT patients5. Our group identified a compound heterozygosity mutation in the TECRL gene (c.587 C > T and c.918 + 3 T > G) in a 13-year-old CPVT patient6. Even though the TECRL gene mutation has been identified with CPVT7, the functions and pathogenic mechanisms of TECRL remain largely unknown and still need to be studied further.Since 1970, several CPVT mutations in ion channel genes or calcium-binding proteins have been identified: ryanodine receptor 2 (RYR2), calsequestrin 2 (CASQ2), and triadin (TRDN)5. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), TECRL deficient cardiomyocytes exhibited decreased calcium transient amplitude, elevated diastolic calcium, and increased Tau decay that was associated with abnormal SERCA and NCX activities5.TECRL encodes 363-amino acids and contains an n-terminal ubiquitin-like domain. The last two trans-membrane regions overlap with the 3-oxo-5-alpha steroid 4-dehydrogenase domain. TECRL mRNA is mostly expressed in the heart and skeletal muscles and is generally undetectable in other human tissues8. Priori et al. first reported that Ryr2 mutated mice (R4496C) exhibited a reduction in the percentage of cardiac cells with severe mitochondrial abnormalities9. Miragoli et al. reported that displaced mitochondria due to myocardial infarction resulted in aberrant mitochondrial Ca2+ propagation10. Mitochondria-ER structure plays a key role in myocardial mitochondrial homeostasis11. TECRL may have other regulatory roles in CPVT, besides the regulation of calcium transients5. We hypothesize that there may be a strong link between impaired TECRL signaling and the resultant mitochondrial dysfunction in cardiomyocytes, which may play a crucial role in CPVT disease.Mitochondria account for 20–35% of the volume of a single cardiomyocyte and is central to energy production and metabolismTo determine the role of Tecrl in mitochondrial homeostasis and cardiac function, we generated Tecrl-null (Tecrl KO) mice. Tecrl deficiency results in impaired cardiac function, altered cardiac metabolism, decreased mitochondria function, and elevated levels of reactive oxygen species (ROS). Mitochondrial fusion proteins such as MFN2 were decreased both in the Tecrl KO mice hearts and Tecrl- deficient cardiomyocytes (hiPSC-CMs). It is possible that TECRL overexpression can partially reverse MFN2 decline. Our results show that TECRL maintains mitochondrial homeostasis through regulation of MFN2, suggesting that TECRL may be a potential target for treating CPVT.To disrupt the open reading frame of Tecrl in mice, we designed a pair of sgRNAs targeting the murine Tecrl gene Fig. . The CRITo assess the cardiac function of the KO mice, we performed echocardiography on 8 weeks old WT and Tecrl KO mice using M-mode imaging Fig. . Comparep < 0.05). Similarly, KEGG pathway analysis indicated that mitogen-activated protein kinase (MAPK) and AMP-activated protein kinase (AMPK) signaling pathways were altered in the Tecrl KO hearts . Together, these data suggest Tecrl deficiency is associated with myocardial metabolism.To determine the impact of Tecrl deficiency on cardiac gene expression, we profiled the heart transcriptome of WT and Tecrl KO mice using RNA-seq. We identified 181 differentially expressed genes between the Tecrl KO and WT mice. Of these, 78 (43.1%) were upregulated and 103 (56.9%) were downregulated in the Tecrl KO mice compared to WT mice Table . Using vrts Fig.  . Tecrl KO murine hearts exhibited mitochondria ultrastructural changes, including irregular arrangement and loss of cristae Fig. . In Tecr12. To quantify ROS levels, we used a dihydroethidium (DHE) fluorescence assay. DHE intensity was elevated in the 8-week-old Tecrl KO hearts compared to WT hearts  in the Tecrl KO mitochondria compared to WT mitochondria. Using KEGG enrichment analysis, we observed that these altered proteins were involved in metabolism, oxidative phosphorylation, fatty acid degradation, and arrhythmogenic right ventricular cardiomyopathy (ARVC), which were significantly decreased in the Tecrl KO mice compared to WT .To determine the impact of Tecrl deficiency on mitochondrial protein composition, we performed proteomic analysis on purified mitochondria isolated from the Tecrl KO mice and WT hearts. We identified a total of 1891 proteins that differed between the Tecrl KO and WT hearts. Using a 1.3-fold cutoff and an FDR < 0.01, we found that 263 (85.9%) were upregulated and 43 (14.1%) were downregulated in the Tecrl KO mice compared to WT mice Table . As show14. To determine the relationship between the Tecrl and PI3K/Akt signaling during mitochondrial homeostasis, we measured the expression of mitochondrial fusion regulators: mitofusin1 (Mfn1), mitofusin2 (Mfn2), nuclear factor erythroid 2-related factor 2 (Nrf2), and PI3K/Akt. Compared to WT hearts, the Tecrl KO hearts exhibited a decrease in Mfn2, p-Akt (Ser473), and Nrf2; an increase in Fas; and a stable level of Mfn1 protein, which was measured by immunoblotting /Akt murine thymoma viral oncogene homolog (Akt), MAPK, and AMPK pathways, all play essential roles in cardiovascular homeostasising Fig. .Fig. 5Te15. As shown in Fig. Next, we used hiPSC-CMs to validate whether the TECRL-PI3K/Akt signaling axis is evolutionary conserved. Human-induced pluripotent stem cells (hiPSCs), which stained positive for pluripotent marker OCT4 Fig. , were diHiPSC-CMs and H9C2 cells were utilized to measure mitochondrial respiration. Maximum mitochondrial respiration was significantly enhanced after TECRL overexpression in hiPSC-CMs, but was blocked after PI3K/Akt inhibitor LY294002 treatment Fig. . SimilarThe Tecrl KO Mice exhibited significant heart contraction defects in 8 weeks, even though the mice hearts were normal at 4 to 5 weeks old. In the Tecrl KO mice, we demonstrated that Tecrl can regulate mitochondrial homeostasis through PI3K/Akt. We showed that in the absence of Tecrl, cardiomyocytes exhibited decreased Mfn2, p-Akt (Ser473), and Nrf2 protein expressions; there was also an increase in apoptosis-inducing factor (AIF) and cytochrome C (Cyc) released from the mitochondria into the cytoplasm. These resulted in a decrease in mitochondrial respiration. Overexpression of TECRL enhances maximum mitochondrial respiratory capacity and this increase is in a PI3K/Akt-dependent manner.6 and others5, reported that mutations in TECRL are linked to CPVT. And an international project to collate these findings has been launched16. CPVT patients are currently treated with conventional therapies, including β-blockers and channel blockers, which have limited efficacy in preventing arrhythmic events and SCD18. Currently, mutations in RYR2 and CASQ2 account for 50% and 5% of CPVT cases respectively19. Mouse models with Ryr2 mutations 23 and Casq2 mutations 24 can effectively replicate the CPVT phenotype. Yet clinically, the prescription of channel blockers does not prevent SCD, suggesting an alternative pathogenic mechanism may be at play. It has been demonstrated that mitochondrial dysfunction may lead to calcium leakage10, which can induce mitochondrial ROS production through an RYR2 mutation25, further dampening mitochondrial function. TECRL deficient cardiomyocytes have reduced expression of RYR2 and CASQ25. Here, we demonstrated that TECRL deficiency results in a late-onset mitochondrial dysfunction .Previous studies, conducted by our team2+ signaling, lipid transport, energy metabolism, and cell survival27. Wu et al. found that hyperglycemia or high glucose-driven inactivation of AMPK results in aberrant MAM formation, mitochondrial Ca2+ increase, mitochondrial dysfunction, and cardiac dysfunction28. Santulli et al. showed that 27 RYR2 CPVT patients displayed abnormal oral glucose tolerance and attributed this observation to sarcoplasmic reticulum stress-induced b-cell apoptosis23. TECRL expression is restricted to cardiac and skeletal muscles and is mostly localized to ER5. Bioinformatics analysis showed that the function of TECRL protein focused on very-long-chain fatty acid biosynthetic processes and oxidoreductase activity. We showed that loss of Tecrl results in altered Nrf2 as well as Fas expression . MAM structures play a pivotal role in Caon Figs. . Given tin Figs. , and 7b.33. The PI3K/Akt pathway has been implicated in survival signaling in response to ischemia and reperfusion, oxidative stress, hypoxia, and β-adrenergic stimulation33. It was reported that drug everolimus could reverse aberrant Aktser473 andp70S6 signaling to slow mutant the proliferation of mutant fibroblasts and reverse mitochondrial abnormalities34. The Mohanraj group also showed that cannabidiol attenuated the generation of high glucose-induced reactive oxygen species, nuclear factor-κB activation, and cell death in primary human cardiomyocytes35. We hypothesize that mitochondrial dysfunction may cause apoptosis-inducing factor (AIF) and cytochrome C (Cyc) to be released from mitochondria into the cytoplasm, which then leads to apoptosis , and NRF2 protein levels. Overexpression of TECRL enhanced mitochondrial respiration in a PI3K/AKT-dependent manner. Our work provides support for targeting TECRL in treating CPVT.In conclusion, we revealed that TECRL deficiency amplifies oxidative stress, results in loss of mitochondria ultrastructure and function, which is a hallmark of heart failure and aging6, we found that the TECRL protein level of the patient was much lower than his parents’ , indicating that a Tecrl KO mouse model can mimic clinical phenotypes of CPVT disease. However, we must still construct the point mutation mouse model and further test them for arrhythmias.From the CPVT family clinical blood samplesWT and Tecrl KO mice were used for all experiments at the age of 4 to 5 and 8 weeks old. Animals were raised under controlled conditions  and housed with a maximum of five animals per cage, with unrestricted access to food and water. All animal studies were approved by the Institutional Review Board of Shanghai Children’s Hospital, Shanghai Jiaotong University.http://www.sanger.ac.uk/htgt/wge/). The sequences of sgRNAs 9 were as follows: Tecrl-T1, TTAGGATATGCCCTAGATAA + AGG; Tecrl-T2, GATCGAAATACCTACGATCG + AGG. The in vitro-synthesized sgRNAs and Cas9 mRNA was co-injected into C57BL/6 mouse zygotes. After injection, surviving zygotes were then implanted to induce pregnancy. The genotype of Tecrl KO mice was confirmed by PCR amplification and DNA sequencing. Tail tip of each mouse was collected to isolate genomic DNA. The targeting region was amplified by PCR . Primer sequences of the WT and Tecrl KO mice for tail DNA identification are shown in Fig. S6a. And their positions are shown in Fig. S6b.The Tecrl KO mice were generated by Biocytogen  using CRISPR/Cas9. Two single guide RNAs (sgRNAs) were designed to target the upstream of exon 3 and downstream of exon 4 of Tecrl using the CRISPR design tool  and moved to a workbench. A linear array probe and 40.0 MHz of center frequency were utilized. All measurements were averaged over five consecutive cardiac cycles and M-mode images of the left ventricle were recorded. LVIDs, LVIDd, LVESV, LVEDV, LVEF, and LVFS were measured to evaluate heart function.Two-dimensional mouse echocardiography  was performed to test the left ventricular function, as previously described by Hu et al.Animals were anesthetized by intraperitoneal injection of pentobarbital (100 mg/kg) and placed on a heating pad. Respiratory rate and loss of toe-press reflex were used to monitor the level of anesthesia. Non-invasive small animal ECG was performed in mice using equipment purchased from INDUS Technology . After a stable baseline was reached, the ECG was recorded for 5 min. Epinephrine (2 mg/kg) and caffeine (120 mg/kg) were administered by intraperitoneal injection and ECG was continuously recorded in a time frame of 15 min. The entire post-epinephrine and caffeine ECG traces were used to analyze the presence or absence of ventricular arrhythmia. Information on the frequency and duration of arrhythmias was double-blind recorded and analyzed by skilled clinicians.http://www.genome.jp/kegg/) were used to identify the aberrantly expressed genes. Benjamini-corrected p < 0.05 was used as the cut-off for significantly enriched biological processes.RNAiso  was utilized to extract total RNA from mouse ventricle tissues. RNA integrity was evaluated by the Bio-analyzer 2100 system . Ribosomal RNA was isolated from 3 μg of RNA using a commercially available RNA Removal Kit . Thereafter, the sequencing library was constructed. PCR products were purified and library qualification was detected. The library was sequenced using the Illumina Hiseq 3500 platform to generate 150 bps long paired-end reads. Raw and clean data were obtained after filtering for quality control. Reading counts for every sample were analyzed using HTSeq v6.0. RPKM (reads per kilobase million mapped reads) and computed to estimate gene expression levels. The GO and KEGG database  and 50 mg/mL Collagenase Type IV  for 30 min. Afterward, the ventricle was cut into small pieces for digestion under gentle agitation in the enzyme solution. Rod-shaped adult cardiomyocytes were collected by centrifugation, followed by the gradual addition of CaCl2. Freshly isolated cardiomyocytes were plated on 25-mm cover-slips pre-coated with 40 μg/mL laminin for 1 h . All cardiomyocytes were cultured in M199 medium  supplemented with 10% fetal bovine serum  at 37 °C and 5% CO2 in a humidified incubator. Only excitable, rod-shaped, quiescent cells were used for the experiments. Mitochondria were isolated from heart tissues (both right and left ventricles) using a Mitochondria Isolation Kit  according to the manufacturer’s instructions. Heart tissue was ground with a grinding rod and washed with phosphate-buffered saline (PBS) twice at room temperature. Homogenate and cells were pelleted by centrifugation at 1000 × g for 15 min at 4 °C to break the cell membranes. Then the supernatant was recentrifuged three times to collect mitochondrial fractions.Adult mice cardiomyocytes were isolated from the WT and Tecrl KO mouse ventricle (both right and left ventricles) tissues, following a previously published protocolTEM for morphological analysis was performed at Shanghai Institute Precision Medicine, Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, according to standard operating procedures. For morphological TEM, heart tissues were cut into 1 × 1 × 1 mm sized patches and fixed with ice-cold 2.5% glutaraldehyde at 4 °C overnight. Ultrathin sections were stained with uranyl acetate and lead citrate. After sample preparation, 90–100 nm thick sections were mounted on a 200-mesh copper grid and imprinted using an FEI Tecnai G2 Spirit transmission electron microscope. All analyses were performed blind to the observer.2 incubator overnight. On the day of the assay, the XF Cell Mito Stress Test medium was prepared . The above cells were transferred and incubated with the appropriate assay medium for 1 h in a non-CO2 incubator at 37 °C. During the incubation time, the pouches containing the compounds  were set at room temperature for 15 min. Compounds were resuspended with the prepared assay medium, then diluted in the same medium to obtain the following final concentrations: 10 μM oligomycin, 10 μM FCCP, and 5 μM rotenone/antimycin for the Mito Stress Test. The assay was conducted in a Seahorse XFp System and analyzed using Wave software .Analyses of mitochondrial fuel usage were performed using Agilent Seahorse XF Cell Mito Stress Test Kits  according to the manufacturer’s instructions. The day prior to the assay, H9C2 cells (5000/well)/hiPSC-CMs (4000/well) were seeded in Seahorse XFp96 cell culture miniplates 48 h before measurements (isolated mice cardiomyocytes (3000/well) were seeded 1 h before measurements). A sensor cartridge was hydrated in Seahorse XF Calibrant at 37 °C in a non-CO41 [10]. Experiments were approved by the Experimental Research Ethics Committee of Shanghai Children’s Hospital affiliated to Shanghai Jiaotong University. Briefly, hearts from 1-day-old Sprague-Dawley rats were extracted and washed with cold PBS three times to remove blood. The hearts were sliced and then incubated multiple times in trypsin solution at 37 ∘C. After centrifugation, cell pellets were resuspended in Dulbecco’s modified Eagle medium  containing 10% heat-inactivated fetal bovine serum . To increase the purity of cardiomyocytes, dissociated cells were plated in 100 mm culture dishes and stored in a 5% CO2 incubator at 37 °C for 1 h. Nonmyocytes were readily attached to the bottom of the dish, while most cardiomyocytes remained suspended in the medium. The resulting suspension of cardiomyocytes was diluted and plated in confocal dishes for ROS detection. 5-Bromo2′-deoxyuridine (100 μM) was added during the first 48 h to prevent the proliferation of residual nonmyocytes.The primary culture of neonatal cardiomyocytes was established according to the method described aboveIntracellular ROS levels in the heart (both right and left ventricles) and neonatal cardiomyocytes were measured using DHE staining . DHE powder was dissolved in dimethyl sulfoxide and diluted with PBS at 55 °C. Mouse heart tissue sections (7 μm) were obtained by using a frozen tissue slicer . Neonatal cardiomyocytes were incubated with 1 μM DHE for 30 min and observed under a laser confocal microscope  at a wavelength of 488/610 nm. For Mitosox assay, neonatal cardiomyocytes were incubated with 5 μM Mitosox for 30 min and analyzed using a PE PerkinElmer Operetta CLS at a maximum excitation/emission of approximately 510/580 nm.g at 4 °C. We then discarded the supernatant and added 1.5 mL extraction buffer to every 2–5 × 107 cells before incubating them in ice for 10–15 min. The supernatant was transferred to a fresh tube after homogenization, and centrifugation was carried out at 11,000 × g for 10 min at 4 °C. The supernatant was carefully removed, for mitochondrial protein identification using a 100 µL cell lysis reagent and a cocktail of protease inhibitors (1:100 [V/V]) in the form of suspended particles.H9C2 cells were trypsinized and centrifuged for 5 min at 1000 rpm. H9C2 cells were washed and resuspended with ice-cold PBS, followed by counting and centrifugation for 5 min at 600 × 42. For GO-term pathway analysis, enrichment was reported as the percentage of total genes that annotate a given pathway (% enrichment), along with the Bonferroni Hochberg adjusted p value.Whole ventricular muscle mitochondria were lysed in ice for 30 min and centrifuged at 15000 rpm for 15 min at 4 °C. Then the supernatant was collected. The peptides were dissolved in a 5% acetonitrile aqueous solution containing 0.5% formic acid and analyzed by online nanospray LC-MS/MS on Q Exactive™  coupled to the Nano ACQUITY UPLC system . The mass spectrometer was run in the data-dependent acquisition mode, and it automatically switched between MS and MS/MS mode. The data of the proteomics analysis were subjected to functional network and pathway enrichment analyses. Protein-protein network analysis and visualizations were performed using Cytoscape (3.7.2). Literature-curated pathway enrichment was achieved using QIAGEN’s Ingenuity Pathway AnalysisTo acquire cell proteins for immunoblotting analysis, hiPSC-CMs and H9C2 cells were washed twice in PBS and incubated with cell lysate buffer at 4 °C for 5 min while rocking gently. Proteins were extracted from left ventricular myocardial tissue and lysed with cell lysate buffer. The cell lysate buffer contained RIPA, protease inhibitors, and phosphatase inhibitors. Proteins were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membrane . The membranes were incubated with primary antibodies specific to Tecrl , Mfn1, Mfn2, Nrf2, Akt, p-Akt (Ser473), Fas, and GAPDH  in blocking buffer at dilutions of 1:1000. Membranes were incubated with secondary antibodies in a blocking buffer at 1:2000 for horseradish peroxidase (HRP)-conjugated antibodies . After washing, membranes were visualized using chemiluminescent substrate (ECL). Densities of immunology bands were analyzed using a scanning densitometer  coupled with Bio-Rad personal computer analysis software.18. All the hiPSCs were maintained in Nutristem medium  and passaged in Accutase enzyme cell detachment medium  every three to five days. Culture dishes were pre-coated with Matrigel  and diluted at 1:400. After at least 20 to 30 passages, hiPSCs were seeded to differentiation to hiPSC-CMs based on the timeline. On days 1–2, we added Chir (4–6 μM) to the aliquot of media 1. On days 3–4, we added IWR (5 μM) to the aliquot of media 1. On days 5–6, we replaced the media in each well with media 1 without added chemicals. On days 7–10, we replaced the media in each well with media 3. On days 11–13, we replaced the media in each well with media 2. During days 10–15, we checked for contraction under a microscope, as this should be visible if differentiation has been successful. Between day 14 and day 15, the cells were replaced onto a fresh plate to remove fibroblasts and other cell types that were generated during cardiomyocyte differentiation. A video of cardiomyocytes (hiPSC-CMs) spontaneously beating was captured using an Olympus inverted microscope  and played with the Windows media player. MatLab and ImageJ, both open source softwares, were used to analyze the hiPSC-CMs beating video and evaluate the contractions. For a period of 2 s, the hiPSC-CMs beating video was recorded, and the contraction speed was determined using ImageJ Musclemotion-plugin, as described in a previously published method43.A healthy volunteer provided informed consent to participate in this study, under protocols approved by the Shanghai Children’s Hospital Institutional Review Board. Peripheral blood mononuclear cells were reprogrammed to pluripotency using the CytoTune Sendai reprogramming kit . These colonies were then stained for the pluripotency marker OCT4. HiPSCs were differentiated from hiPSC-CMs according to the manufacturer’s protocolAfter permeabilization with triton-X, the hiPSCs or hiPSC-CMs were stained with OCT4 antibody , or α-actinin antibody . Images were acquired with a laser confocal microscope .p < 0.05), and if their fold change was over 1.3. The results were expressed as mean ± SEM. Statistical analysis was performed using SPSS software, version 21.0 . Comparisons among groups were performed by one-way ANOVA. Paired data were evaluated using two-tailed Student’s t-tests. Statistical significance was set at p < 0.05.Tandem mass spectra were processed in PEAKS Studio version X . Peptides were filtered using 1% FDR and one unique peptide. Differently expressed proteins were filtered if they contained at least one unique peptide with significance over 13 (Further information on research design is available in the Peer Review FileSupplementary InformationReporting Summary


The management of patients with L-SIL/ASCUS cytology is controversial and not clearly standardized. Objective: To analyze the risk factors associated with H-SIL/CIN2+ in a cohort of patients with ASCUS or L-SIL in a Pap smear. Methods: Between 2012 and 2022, 1259 eligible women with ASCUS/L-SIL were referred for colposcopy. The risk factors associated with H-SIL/CIN2+ were analyzed. The colposcopic study, conventional or assisted with dynamic spectral imaging (DSI), was performed in all cases. Guided biopsies were performed in cases of abnormal examination or random biopsies when no lesions were found. A LEEP was performed in H-SIL/CIN2+ results or persistent LSIL/CIN. Results: A normal or metaplastic specimen was found in 750 women (63.2%), LSIL/CIN1 in 346 (29.1%), and H-SIL/CIN2+ in 92 (7.7%). The presence of HR-HPV , smoking habits , and the performance of DSI combined with colposcopy  were the factors associated with the detection of H-SIL/CIN2+. A summative effect of HR-HPV and smoking habit  was observed in the detection of H-SIL/CIN2+. In multivariate analysis, the presence of HPV 16/18 was the unique independent factor associated with H-SIL/CIN2+. Conclusion: In women carrying an ASCUS/LSIL in the Pap smear, the unique independent factor predictive of H-SIL/CIN2+ is the presence of the HPV 16/18 genotype. Smoking women carrying ASCUS/LSIL with HR-HPV should be targeted for stricter follow-up to avoid an unsuspected H-SIL/CIN2+. Cervical cancer is considered the fourth most common malignant tumor in women around the world in terms of incidence and mortality .Cervical cytology is an acceptable method for screening and is recommended in most European and American clinical guidelines. Nevertheless, its sensitivity for the detection of cervical intraepithelial neoplasia 2 or higher (H-SIL/CIN2+) is around 50%, not exceeding 80% in the best quality conditions [Women with abnormal cervical screening results or suspicious symptoms are usually assessed by conventional colposcopy. However, there is controversy regarding the proper evaluation and management of low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of undetermined significance (ASCUS) in cervical cytologic diagnoses .Colposcopy can evaluate the cervical lesions and detect the characteristics and type of precancerous or cancerous lesions. Indeed, it could help in orienting the biopsies of any area that seems abnormal. Furthermore, depending on the lesions’ extension, it could also help to determine which is the most appropriate treatment  ,4.It is of the utmost importance to implement specific criteria for women with ASCUS/L-SIL, specifically for more cost-effective approaches to triaging women for colposcopy . L-SIL iThe aim of this populational study was to describe the features of a specific cohort of patients with a result of ASCUS/L-SIL in cervical cancer screening, and to analyze the risk factors associated with the detection of H-SIL/CIN2+.A transversal observational clinical study in a population of women with cervical screening results of ASCUS/L-SIL referred to colposcopy at Hospital Clínico San Carlos in Madrid, Spain, between 2012 and 2022 was performed. ® HPV2 test, which detects and genotypes with high sensitivity and specificity 35 different types of HPV, including high-risk and low-risk types, in one assay. As a DNA control, it includes a set of primers to amplify a fragment of the human CFTR gene, while a spiked plasmid (1202 bp) is included as a PCR process control. The resulting visualized microarray is analyzed on a computer-guided reader with automated reading software. The genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 were considered HR-HPV. Clinical data and the personal backgrounds of each patient were recorded. All women were managed following the guidelines of the Spanish Society of Cervical Pathology and Colposcopy (AEPCC) available at the time of the colposcopic assessment ,8. CerviColposcopies with 3% acetic acid were performed on all women. The Schiller’s test was performed for cervical and vaginal evaluation ,8. A cohThe sample size was calculated using a poblational reference of 2000 patients. Considering a poblational proportion of H-SIL/CIN2+ of 7%, with a CI of 95% and a 99% statistical power, the required sample size is 1112 patients.p < 0.05. The computations were performed using IBM SPSS Statistics version 25  and Epidat 3.1 . Qualitative variables were summarized by their frequency distribution as well as quantitative variables by their mean and standard deviation (SD). The continuous, non-normally distributed variables were summarized by the median and interquartile range (IQR: P25–P75). In the case of qualitative variables, the chi-square test was used to assess the factors associated with H-SIL/CIN2+, or the Fisher’s exact test if more than 25% of the expected values were less than five. Quantitative variables were evaluated using the Student´s t-test. The magnitude of the association was evaluated using the odds ratio (OR) and its 95% confidence interval (CI). All statistical tests were two-sided. The statistical significance level was defined as p-value less than 0.10 in the univariate analysis, which were entered into the model using a forward/backward stepwise procedure.Different risk factors for cervical lesions were evaluated in the statistical analysis, considering the age, the presence of HPV, or other factors, as well as the number of sexual partners, smoking, or anal intercourse. A univariate analysis was performed to detect the factors associated with H-SIL/CIN2+. A multiple linear regression analysis was performed to identify the independent variables associated with H-SIL/CIN2+ among the variables with a The study was performed according to the STROBE guidelines and was approved by the Clinical Research Ethics Committee of Hospital Clínico San Carlos (C.I. 13/314-E). Signed informed consent was obtained from all participants.A population of 1219 patients was analyzed; 31 cases were excluded due to wrong or missing data. A final sample of 1188 women was evaluated for the study. In the univariate analysis of the factors associated to H-SIL/CIN2+ there were found significant differences in the presence of HR-HPV , smoking habit , and the performance of the DSI map combined with conventional colposcopy . In cases of HPV 16/18, the sensitivity to detect H-SIL/CIN2+ was 33.9% (IC 95% 24.3–43.6) with a specificity of 89.5% (IC 95% = 87.6–91.3). H-SIL/CIN2+ was diagnosed in 6.8% of the cases with no HPV 16/18, with a sensitivity to detect H-SIL/CIN2+ of 6.7% (IC 95% 3.6–9.8) and a specificity of 97.7% (IC 95% = 95.8–99.5). p < 0.1) to be analyzed using a logistic regression model: HR-HPV, HVP 16/18, HR-HPV No. 16/18, anal intercourse, tobacco, and the DSI + CC study  in order to avoid their progression to cancer. The detailed analysis of the potential risk factors associated with a H-SIL/CIN2+ could personalize the potential risk of being affected, improving patient experience, exposing fewer patients to diagnostic procedures, and lowering healthcare costs .In women with HR-HPV, different cofactors have been described in the literature that increase the risk of cervical cancer and its precancerous lesions. Tobacco and HR-HPV infection, particularly by genotype 16/18 ,14, are The immediate risk of H-SIL/CIN2+ in women with cytological L-SIL and positive HPV is 15% . In our Charlton et al.  found a As in our study, McIntyre-Seltman et al.  found a In our study, the presence of HR-HPV and current smoking habits were associated with an increase in H-SIL/CIN2+ detection. Furthermore, when analyzing together the presence of a smoking habit and the presence of HR-HPV, the detection of H-SIL/CIN2+ was higher than when considering both factors separately, which highlights the importance of insisting on stopping smoking in patients with HR-HPV. The fact that HPV 16/18 was the unique independent factor of the presence of occult H-SIL/CIN2+, HPV is the most recommended test for its detection. In patients with ASCUS/LSIL and the presence of HVP 16/18, the immediate risk of H-SIL/CIN3+ can reach 11%; on the contrary, genotypes different from 16 or 18 present an approximate risk of 3.7% [The performance of DSI added to the CC was found to be a protective factor  in the detection of H-SIL/CIN2+. We did not find significant differences in other factors evaluated, such as HPV vaccination, postmenopausal status, or anal intercourse, although some of them have already been described as risk factors for developing cervical cancer (age or immunocompromise) .The strength of this study is the large sample included, which allows for high precision in the results obtained. The patients have been evaluated by a unique and senior colposcopist, who followed the same protocols and criteria for every patient, which increased the feasibility of the study and reduced bias. A senior colposcopist’s performance of the colposcopy implies an increase in sensitivity compared to a junior , though The factor associated with an unsuspected H-SIL/CIN2+ in women with ASCUS/LSIL in cervical cancer screening cytology was the presence of HPV 16/18 genotypes. Women carrying ASUS/LSIL with HR-HPV and who smoke should be targeted by meticulous medical evaluation, diagnostic testing, and follow-up in order to avoid an unsuspected H-SIL/CIN2+.


Background: Human Papilloma Virus (HPV) represents the most prevalent genital infection in young women of reproductive age. Objective: This systematic review aims to estimate the effect of HPV infection during pregnancy and assess the correlation between HPV and adverse pregnancy outcomes. Materials and methods: The search strategy has been developed based on the PICOS framework: Population (pregnant women infected with HPV), Intervention (HPV infection confirmed by molecular tests), Comparator (pregnant women without HPV infection), Outcomes (adverse pregnancy outcomes) and Study design . We searched PubMed, Web of Science, and Scopus databases on 8 January 2022 by using the following keywords: “HPV”, “prematurity”, “preterm birth”, “miscarriage”, “premature rupture of membranes”, “adverse pregnancy outcome”, “low birth weight”, “fetal growth restriction”, “pregnancy-induced hypertensive disorders”, “preeclampsia”. Selection criteria were HPV infection confirmed within maximum 2 years before pregnancy with a molecular test and adverse pregnancy outcomes. (Results: Although numerous studies are conducted on this topic, data are still controversial regarding identifying maternal HPV infection as a risk factor for adverse pregnancy outcomes. More prospective large cohort studies are needed to prove a causative relationship. HPV is a double-stranded DNA virus from the Papillomaviridae family, specific to humankind. It is composed of a family of over 200 subtypes with specific tissue tropism  . A totalPregnant women, due to their immunological and endocrinological status, have a higher risk of HPV infection, with an infection rate reported to be 82% compared with a rate of 10.4% in non-pregnant women . HPV cerViral infections are recognized as a risk factor for adverse pregnancy outcomes , and as This systematic review is conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines  and aimsOn 8 January 2022, we searched several databases: PubMed, Web of Science and Scopus, targeting literature published between 1 January 2012 and 1 January 2022. We used the following keywords: “HPV”, “prematurity”, “preterm birth”, “miscarriage”, “premature rupture of membranes”, “adverse pregnancy outcome”, “low birth weight”, “fetal growth restriction”, “pregnancy-induced hypertensive disorders”, “preeclampsia”.The search strategy has been developed based on the PICOS framework: Population (pregnant women infected with HPV), Intervention (HPV infection confirmed by molecular tests), Comparator (pregnant women without HPV infection), Outcomes (adverse pregnancy outcomes), and Study design . The inclusion criteria applied for the elected population were: HPV infection confirmed within maximum two years before pregnancy or during pregnancy with a molecular test and adverse pregnancy outcomes.The exposure of interest is the association of HPV infection in a pregnant women with adverse pregnancy outcome. The presence of HPV infection in all genital sites  must be confirmed by a molecular test within maximum two years before pregnancy or during the pregnancy.The comparison group will include pregnant women that are not HPV infected or are not documented with infection.We have excluded: editorials, opinions, case reports, studies without a control group, and studies in which HPV infection was not molecularly documented. We have also excluded studies that explored the post-surgical treatment of cervical HPV during pregnancy associated with adverse pregnancy outcomes, post-vaccine studies, HPV sperm-related studies, and co-infection studies.The primary outcome of this review is the link between HPV infection and premature birth. The secondary outcomes were miscarriage, premature rupture of membranes (PROM), pregnancy-induced hypertensive disorders (PIHD), and fetal growth restriction (FGR).Two reviewers (SDP and AGB) independently screened titles/abstracts independently and in duplicate and extracted data from selected full-text reports using a predesigned form. Disagreements were resolved by discussion with the coauthors.We included in this systematic review 21 studies. From which 3 were systematic reviews and meta-analyses ,29,30, 4Preterm birth is assumed as any birth before 37 weeks and after 24 weeks of gestation. About 15 million newborns are born preterm worldwide, with a higher incidence in low- and middle-income countries (LMIC). It is the primary cause of an estimated one million neonatal deaths annually, and is a significant contributor to childhood morbidity. Starting from the fact that intrauterine infections act as a determinant factor for adverse pregnancy outcomes, especially for preterm birth, numerous studies have tried to investigate the relationship between maternal HPV infection and adverse pregnancy outcomes. However, not all studies published on this theme have proven this correlation keeping controversies in this field.A number of 11 studies have investigated as primary outcome the link between preterm birth and HPV infection ,40,44,45You et al.  have demSlatter et al.  in theirNiyibizi J et al.  in a latp = 0.019). 60% of HPV positive women were HR-HPV (16.18) infected and 40% were LR-HPV (6.11) infected, so they finally concluded that HPV infection represents a risk factor for preterm birth among the Egyptian population, especially HR-HPV infection [Mosbah et al.  conductep = 0.002), and premature rupture of membranes  but the reliability of the results may be affected due to differences in retrieval mechanisms, differences in risk factors in distinct references and potential publication bias. The authors recommend a multi-center, large sample original research in order to prove the theoretical data [In another systematic review and meta-analyses that included seven studies and 45,603 patients conducted by Wu et al.  publishecal data .Into another systematic review and meta-analyses  the authOther studies have identified no link between HPV infection and adverse pregnancy outcomes i.e., preterm birth and miscarriage. Ambühl et al.  in a DanCaballero et al.  conducteThe link between HPV infection and spontaneous miscarriage is investigated in 9 studies ,41,43,45Miscarriage or spontaneous abortion is defined as a pregnancy loss before 20 weeks gestation. The World Health Organization (WHO) estimates that about 26% of all pregnancies miscarriages. Miscarriage etiology is multifactorial being involved and demonstrated genetic factors, teratogen factors, immunological factors, anatomical factors, environmental factors and infectious factors. Many studies in the field have tried to assume HPV infection as a risk factor for miscarriage, but the data up to date are controversial. Due to the immunological status of a pregnant patient and the proof that HPV may infect the cervix, the placenta, the membranes, and the amniotic fluid, the concern of researchers regarding HPV involvement in miscarriage is legitimate.Ambühl, in the systematic review conducted, have identified a higher HPV incidence among women with miscarriage with an incidence of 24.9% HPV positive, but without assigning a clear, determining role of HPV infection . SimilarNo significant association between HPV infection and miscarriage has been reported by Niyibizi in his systematic review and meta-analyses .Basonidis have resumed in his work several studies regarding HPV correlation with spontaneous abortion and presented conflicting findings and limited conclusions due to the small sample size and/or methodological limitations of the studies they included. The link between HPV and miscarriage is still unclear, and more studies are needed .Conde-Ferraez et al.  have conRegarding recurrent miscarriage and HPV infection, Ticconi et al.  found loThe data published to date are contradictory and limited by the small sample size. More studies are needed to prove a correlation between maternal HPV infection and the risk for spontaneous abortion.Premature rupture of membranes (PROM) is defined as the rupture of amniotic membranes before labor onset; it mostly occurs near term. If the membrane rupture occurs before 37 weeks of gestation, it is called preterm PROM (PPROM). Preterm PROM complicates approximately 3 percent of all pregnancies and is the generating cause of one-third of preterm births . Both Prp = 0.029) [p < 0.00001) [The linkage of maternal HPV infection with premature rupture of membranes is evaluated in 6 studies ,35,38,42= 0.029) ,25,35. I0.00001)  Niyibizi0.00001)  ThereforPregnancy-induced hypertensive disorders are classified by the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy into 4 categories: (1) chronic hypertension, (2) preeclampsia-eclampsia, (3) preeclampsia superimposed on chronic hypertension, and (4) gestational hypertension  .Hypertension during pregnancy complicates 2–3% of all pregnancies. Gestational hypertension is defined as the new onset of hypertension after 20 weeks of gestation, without having hypertension before pregnancy, proteinuria, or other manifestation of preeclampsia/eclampsia. Preeclampsia develops hypertension after 20 weeks of gestation associated with proteinuria and is a multiorgan disease process . The patMcDonnold and his team have conducted a retrospective cohort study in which have compared a group of pregnant women with HR-HPV infection (314) with a control group (628) and discovered a twofold higher risk of developing preeclampsia in the positive group (10.19% vs. 4.94%). However, the study has limitation and only reveal an association, not a causality, in order to prove causality, more extensive prospective studies are needed and adjustment secondary risk factors must be done: for ethnicity, smoking, BMI, chronic renal diseases or chronic high blood pressure . Reily-BFetal Growth Restriction (FGR) is defined as the inability of the fetus to reach its biological growth potential, and it is diagnosed when the estimated ultrasonographic fetal weight is less than the 10th percentile for gestational age . The patIt is notorious for specific infectious agents (TORCH) the causality with FGR. Going along with this parallel and having studies demonstrating the infection of trophoblastic cells with HPV ,45 materThere is a high interest in the correlation between HPV infection and pregnancy outcomes, and there are some studies that investigate this subject published at the date; most of them are investigating preterm birth, premature rupture of the membranes and the miscarriage rates.Our systematic review is a clear reflection of the literature published in the last ten years on the involvement of HPV infection in adverse pregnancy outcomes and brings with it a brief presentation of all the conclusions from the included studies and a review of their limitations and potential bias Some infectious pathogens are implicated in pregnancy adverse outcomes by interfering with the intrauterine environment. The updated literature identifies multiple pathways that HPV may infect the conception product and its annexes—as by ascending infection from the cervix with contiguous infection of the uterine contents or as hematogenous disemmination ,49. InfeThe pathophisiological pathway of preeclampsia and fetal growth restriction is based on abnormal placentation. As in vitro and in vivo studies have shown that HPV can infect the trophoblastic cells and therefore alter the early placental development and determine malfunction of the placenta, maternal HPV infection may be suspected as a potential risk factor for both preeclampsia and fetal growth restriction ,30,36,40HPV may affect the pregnancy outcomes by affecting the cervical competence, altering the vaginal microbiota, ascending infection of the chorionic villi and causing placental dysfunction, infecting the amnitoic fluid causing amniotitis, or secondary to surgical treatments of the cervical HPV dysplasia; co-existence with other genital infection may also intefere with a normal pregnancy outcome; also semen infection may seem to have a potential role in generating adverse pregnancy outcomes. If there is a causative relationship between genital HPV infection and adverse pregnancy outcomes, we should see an improvement with the vaccination programs.The studies included in our systematic review suggest that HR-HPV infection in pregnant women may be a risk factor for preterm rupture of membranes and preterm birth. Regarding miscarriage, fetal growth restriction, preeclampsia the small number of studies and the small sample sizes investigated prevents us from drawing any conclusions.Further studies are needed to complete the hypothesis that maternal HPV infection acts as a negative prognostic factor in pregnancy courses and to fully elucidate the pathophysiological mechanism of HPV infection in pregnancyIn addition to the preclinical studies needed to elucidate these mechanisms, retrospective observational studies are needed to identify potential cofactors of adverse pregnancy outcomes  .HPV is the most prevalent infection in both women and men in their reproductive years, and it represents a global health problem when discussing genital cancers. This systematic review highlights the idea that maternal HPV infection during pregnancy may be a risk factor for adverse pregnancy outcomes like spontaneous miscarriage, prematurity, pregnancy-induce hypertensive disrders, fetal growth restriction, premature rupture of membranes, and even fetal death. Although there is a growing interest in this field and more and more studies are published there have been no clear conclusions yet and the data are still controversial. HPV infection is an infection that can impact both the mother and the fetus negativelyso further epidemiological studies are required to prove this causality. Also, standardized HPV testing protocols should be developed to have common conclusions when discussing the HPV causality of adverse pregnancy outcomes.


Numerous studies support that Human papillomavirus (HPV) can cause cervical cancer. However, few studies have surveyed the heterogeneity of HPV infected or uninfected (HPV+ and HPV-) cervical cancer (CESC) patients. Integration of scRNA-seq and TCGA data to analyze the heterogeneity of HPV+ and HPV- cervical cancer patients on a single-cell level could improve understanding of the cellular mechanisms during HPV-induced cervical cancer.CESC scRNA-seq data obtained from the Gene Expression Omnibus (GEO) database and the Seurat, Monocle3 package were used for scRNA-seq data analysis. The ESTIMATE package was used for single-sample gene immune score, CIBERSORT package was used to identify immune scores of cells, and the “WGCNA” package for the weighted correlation network analysis. Univariate Cox and LASSO regression were performed to establish survival and relapse signatures. KEGG and GO analyses were performed for the signature gene. Gene Expression Profiling Interactive Analysis was used for Pan-cancer analysis.In the HPV+ CESC group, CD8+ T cells and B cells were down-regulated, whereas T reg cells, CD4+ T cells, and epithelial cells were up-regulated according to scRNA-seq data. Survival analysis of TCGA-CESC revealed that increased expression of naive B cells or CD8+ T cells favors the survival probability of CESC patients. WGCNA, univariate Cox, and LASSO Cox regression established a 9-genes survival signature and a 7-gene relapse model. Pan-cancer analysis identified IKZF3, FOXP3, and JAK3 had a similar distribution and effects in HPV-associated HNSC.Analysis of scRNA-seq and bulk RNA-seq of HPV+ and HPV- CESC samples revealed heterogeneity from transcriptional state to immune infiltration. Survival and relapse models were adjusted according to the heterogeneity of HPV+ and HPV- CESC immune cells to assess the prognostic risk accurately. Hub genes represent similar protection in HPV- associated HNSC while showing irrelevant to other potential HPV-related cancers. In 2020, the World Health Organization (WHO) reported 604,127 new cases of cervical cancer (CESC) and 341,831 CESC-related casualties (Single-cell RNA sequencing (scRNA-seq) is a technique used to define the global gene expression profile of a single cell utilizing optimized next-generation sequencing. This allows the exploration of previously hidden heterogeneity in a cell population . In prevWe hypothesized that HPV has an effect at multiple levels. Hence, we aim to reveal the heterogeneity from transcriptional to immune infiltration between HPV+ and HPV- samples by scRNA-seq as well as bulk RNA sequencing. Then, we try to link the survival and relapse with genes based on the difference between HPV+ and HPV- samples. Further, we analyzed the role of hub genes in other potential HPV- associated cancers to know whether HPV infections have a consistent effect on different cancers.http://www.ncbi.nlm.nih.gov/geo/). Single-cell RNA sequencing data of HNSCC with HPV+ and HPV- (GSE139324) were downloaded from Gene Expression Omnibus (GEO) database Transcriptome profiling data and the corresponding clinical data were collected from GSE GEO database  and TCGA-CESC via the Sangerbox tools, a free online platform for data analysis (http://www.sangerbox.com/tool).Single-cell RNA sequencing data of cervical cancer with HPV+ and HPV-  were downloaded from Gene Expression Omnibus (GEO) database  . Immune Differentially expressed RNA (DeRNA) among HPV+ and HPV- groups were screened out through Linear Models for Microarray Analysis (limma) package in R software . The cutThe survival package’s coxph  function was used to fit the Cox risk regression, and a p-value of 0.05 was considered survival/relapse related . The leahttp://www.sangerbox.com/tool).Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to identify biological processes and pathways that were most related to hub genes with p < 0.05 as cutoff value , a web-based tool, analyzed the hub genes expressions and overall survival in HPV-associated cancers . ScRNA-sPatients’ information was summarized from the corresponding clinical data in 46 samples with known HPV states Table 1.+ T cell and B cell clusters, while an increase in Treg -, CD4+ T -, and epithelial cell clusters was observed within HPV+ CESC patients. Pseudotime trajectory analysis highlighted the heterogeneity of immune cell development between HPV+ and HPV- CESC patients   and a standard p-value (<0.05) were set  proportional risk models were taken to analyze the hub genes, which related to CD8+ T cells, Naive B cells, and differently expressed between HPV+ and HPV- CESC. Two hundred genes were significantly associated with overall survival  curves revealed significant differences between the low- and high-risk groups , HNSC, Colon adenocarcinoma (COAD), Prostate Adenocarcinoma (PRAD)), an HPV-associated pan cancer analysis of IKZF3, FOXP3, and JAK3 was performed. Since these genes were represented in both risk models , their potential impact on cancerogenesis was estimated higher than for genes appearing in only one model. The expressions of genes are shown in The survival analysis of IKZF3 showed that IKZF3 is favorable in BLCA  with the highest correlation to CD8+ cells and B cells and used for subsequent construction of survival and relapse models. Finally, we identified JAK3, FOXP3, and IKZF3, three genes with reduced expression in the HPV+ group, as protective factors for survival and relapse. The lower expression of these three, the higher the RiskScore in the model, which means a higher risk of death and recurrence.via RNA-Seq data. For this result, we concluded that hub genes are affected by HPV+ and that high expression of hub genes in non-HPV-infected tumors is not protective.The higher expression of three hub genes shows a higher percentage of survival in HNSC, which is consistent with our analyzed difference between HPV+ and HPV- HNSC samples. However, this result could not be observed in other potential HPV-associated cancers, only IKZF3 was found to be favorable in BLCA. As Jørgensen KR and Jensen JB found out, most studies fail to prove clear-cut relevance of HPV in BLCA irrespectively of histological subtype . No hub The functions of FOXP3, JAK3, and IKZF3 are widely researched in the field of tumor immunology. As the most specific and reliable biomarker of T reg cells, the studies of FOXP3 are conflicted. The infiltration of tumors with FOXP3+ Tregs was considered unfavorable for patient survival in many types of cancer. However, more recent work has described Helios as a new marker of Tregs. Tregs only have an immunosuppressive function when both FOXP3 and Helios are expressed . In the In conclusion, analysis of scRNA-seq and bulk RNA-seq of HPV+ and HPV- CESC samples revealed heterogeneity from transcriptional state to immune infiltration. Based on the difference, we found prognosis-related genes where expression was affected by HPV+. The LASSO Cox regression analysis of these genes constructed survival and recurrence models. IKZF3, FOXP3, and JAK3 represented in both risk models had the similar distribution and protective effects in HPV-associated HNSC, which means the expression of the hub genes are affected by HPV+.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171894, PRJNA721342, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE139324, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE142583, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE6791, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE181805, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE190224. In addtion, GSE139324, GSE 6791, GSE190224.Publicly available datasets were analyzed in this study. This data can be found here: BC-E and MR conceived and designed the study. EW, AR, and JL analyzed the data and drafted the manuscript. EW and LF prepared the figures. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.