The aims of this study on human papilloma virus (HPV) 6/11/16/18 infection among females in Chengdu were to provide more targeted strategies for the prevention and treatment of cervical cancer and genital warts. In this study, the infection status of 20 genotypes was analysed by gene chip technology. The prevalence rates of HPV-6, -11, -16, and -18 infection among 180,276 cases were 0.94%, 0.57%, 3.22%, and 1.28%, respectively. The prevalence of HPV 6/11/16/18 showed a bimodal U-shaped curve with age; the first and second peak occurred among females < 20 and ≥ 60 years old, respectively. As the multiplicity of infections involving HPV6/11/16/18 increases, the infection rate decreases. The ratios of HPV16 single infection showed a yearly increase. The top five genotypes with HPV-16, -18, -6, and -11 in coinfection were HPV52/58/53/51/33, HPV 52/16/53/58/51, HPV52/16/58/51/53 and HPV16/52/58/59/18, respectively, HPV16/18/6/11 were mainly coinfected with HR-HPV. In sum, among the five vaccines available, nonavalent vaccine is more suitable for Chengdu females. For young females prioritizing vaccination is essential in the current context, while HPV screening remains an effective approach for older females. Additionally, in patients with genital warts, it is necessary to assess the presence of high-risk HPV infection and manage it appropriately in patients with genital warts. HPVs belong to the Papovaviridae family, and humans are the sole host. HPVs are classified into five diverse genera based on tissue tropism and subsequent host pathogenesis, which are Alphapapillomaviruses , Betapapillomaviruses (beta), Gammapapillomaviruses (Gamma), Mupapillomaviruses (mu) and Nupapillomaviruses (nu)2. HPV genomes are further classified into intratypic variants according to the diversity of their genome sequence4. The HPVs that infect the mucosal epithelium are classified into the genus Alphapapillomaviruses 5. To date, 231 types of HPV have been identified, on the basis of the genomic sequence of L16, with more than 40 types infecting the genital area1. Based on their carcinogenic potential, mucosal alpha-PVs are designated as high-risk and low-risk (HR-HPV and LR-HPV). Currently, at least 14 HPV genotypes have been described as high-risk 7, while LR-HPV includes HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and CP61088. Based on HPV activities, HPV genome is classified into three regions: the long control region (LCR) or the non-coding upstream regulatory region (URR), the late (L) region, and the early (E) region5among them, three oncogenes, E5, E6, and E7, modulate the transformation process, two regulatory proteins, E1 and E2, modulate transcription and replication, and two structural proteins, L1 and L2, compose the viral icosahedral capsid. Viral capsids have evolved to fulfil numerous roles that are critical to the establishment of viral infection9. HPVs infect host basal layer of the epithelium via micro-wounds10, and then in order to initiate viral infection, the L1 capsid protein binds to herparin sulfate proteoglycans (HSPGs) or interact with laminin-332 on the extracellular matrix Viral and capsids have evolved to fulfil numerous roles that are critical to the establishment of viral infection11. 60% of HPV infection in one year will be cleared spontaneously, and the great majority of infected women (more than 90%) resolve the infection spontaneously in two years13, while only a very small number of cases progress into cervical intraepithelial neoplasia (CIN), precancerous lesions and cancer. One of the reasons HPV causes cancer is that they have established molecular mechanisms to prevent cell cycle arrest and apoptosis14. Viral E6 and E7 oncoproteins inactivate the tumor suppressor proteins p53 and pRB, respectively, and are involved in uncontrolled cell proliferation and deliver their genetic material into the nucleus of the target cell16, Another cause of cancer is through the integration of HPV DNA into the host genome. When the E6 and E7 genes of the viral DNA remain intact and integrated into the host chromosome18, which causes additional chromosomal damages and genome destabilization in the infected cells, whereas the E6 and E7 oncogenes are extensively expressed. Approximately 98.7% of cervical cancers are attributable to HPV19. HR-HPV genotypes 16 and 18 are responsible for approximately 71% of cervical cancer cases20.Human papillomaviruses (HPVs) are non-enveloped, double-stranded circular DNA viruses of 8 kb in size that causes epithelial hyperplasia of the skin and is primarily transmitted through sexual contact or close contact and causes cervical cancer and other diseases 21. Nearly 70% of the global burden of cervical cancer occurs in less developed countries. As the most populous developing country in the world, China bears a serious burden of cervical cancer. According to the latest statistics from the ICO/IARC HPV Information Centre in 2021, China experiences approximately 109,700 new cases and 59,100 deaths related to cervical cancer annually. The newly diagnosed cervical cancer cases in China constitute approximately 18.2% of the global incidence, while the mortality figures account for approximately 17.2% of global deaths attributed to cervical cancer22.Cervical cancer is the second most common cancer in women worldwide and the third most common cause of cancer-related death worldwide. In 2020, an estimated 604127 new cases of cervical cancer and 341831 deaths worldwide were attributed to HPV infection1, of which anogenital warts are more common. Ninety percent of genital warts are caused by noncarcinogenic LR-HPV genotypes 6 or/and 11, with HPV6 being the most common type of the disease, but HPV11 accounting for approximately a quarter of cases. In addition to anogenital warts, HPV types 6 and 11 are also associated with conjunctival, nasal, oral, and laryngeal warts1. Globally, genital warts are estimated to affect approximately 1% of the population23. The global incidence of genital warts in females (including both new and recurrent cases) is between 76 and 191 per 100,000 population 24. Although genital warts are benign, they cause discomfort and affect psychological well-being. Even when addressed with physical therapy, they persist due to HPV infection and may recur upon external stimulation, which poses considerable challenges for patients and treatment.LR-HPV primarily causes genital warts and recurrent respiratory papillomatosis14. As of now, the China Food and Drug Administration (CFDA) has approved a total of five vaccines, including the three mentioned above , which were respectively marketed in mainland China in 2016, 2017, and 2018. Subsequently, in 2019 and 2022, the CFDA granted approval for two locally produced bivalent vaccines to be marketed in mainland China, namely Cecolin produced by Xiamen Wantai Canghai Biotechnology Co., Ltd. and "Wozehui" by Yunnan Walvax Biotechnology Co., Ltd. HPV vaccines have shown 98% to 100.0% protective efficacy in clinical trials against HPV type-related diseases, offering effective protection for both vaccinated and unvaccinated females27. Due to various factors, the HPV vaccine was introduced to Chinese females a decade after their global counterparts, and owing to economic constraints, the vaccine is only promoted as a national secondary vaccine. Nationally, financial constraints have resulted in less than 0.05 percent coverage of the HPV vaccine among eligible women aged 9–45 years, with women aged 9–14 years accounting for less than 5 percent of the vaccinated population28. Alongside vaccination, HPV DNA and cytologicals creening is crucial for alleviating the burden of cervical cancer and other HPV-related diseases. The World Health Organization's "Global Strategy to Accelerate the Elimination of Cervical Cancer" aims to have 70% of women undergo regular HPV screening by 2030, with HPV DNA testing as the preferred method for cervical cancer screening30. China conducts limited large-scale screening for female HPV DNA, with most of it taking place in hospitals. Given the high incidence of cervical cancer and related diseases in Chinese women, coupled with low HPV vaccination rates and limited HPV-DNA testing, obtaining prevalence data on HPV infection in Chinese women is vital and timely. Although infection with genotypes other than HPV6/11/16/18 can also cause genital warts and cervical cancer in women, HPV6/11/16/18 infection is the primary cause, so in-depth analysis of these four genotypes is absolutely necessary. Currently, there is a lack of large-scale research data on HPV6/11/16/18 in Chengdu or even the entire nation. The data for this study were derived from a national tertiary-grade hospital in southwest China, comprising a central institution and three branch hospitals, covering virtually the entire Chengdu urban and suburban area. This study has provided a comprehensive analysis of the situation of HPV6/11/16/18 in different age groups, infection modes, and years.The most effective approach for controlling cervical cancer and genital warts is prioritizing HPV vaccination. Currently, the US Food and Drug Administration (FDA) has approved three prophylactic HPV vaccines, and they are respectively Gardasil®4, a quadrivalent vaccine  available in 2006, Cervarix™, a bivalent vaccine  available in 2007, and Gardasil®9, a nonavalent vaccine  available in 2014Patients came from Chengdu Women's and Children's Central Hospital (CWCCH) for HPV screening motivated by diverse reasons, including patient requests, diagnostic needs prescribed by doctors, and opportunistic screening by doctors. Specific reasons included health examinations, abnormal vaginal bleeding, lower genital tract inflammation, genital warts, infertility, unknown lower abdominal pain, gynaecological tumours, and urethritis. Inclusion criteria consisted of a history of sexual activity, absence from menstruation and pregnancy, while exclusion criteria included women with no sex, menstruating and pregnant women, and women who had undergone uterine surgery. For cases that were reviewed, we only considered the results of the first screening. A total of 6254 cases were excluded from the study. Finally, a total of 180,276 participants were included in this retrospective study conducted at CWCCH between January 2015 and December 2021. The mean age was 38.8 ± 9.1 years (ranging from 12 to 91 years). categorized into age groups of < 20, 20–29, 30–39, 40–49, 50–59, and ≥ 60. This study was approved by the Medical Ethics Committee of Chengdu Women’s and Children’s Central Hospital, and all methods were performed in accordance with the relevant guidelines and regulations.This research received approval and a waiver for participant informed consent from the Medical Ethics Committee of Chengdu Women's and Children's Central Hospital due to the retrospective nature of the study, where patient identities were deliberately anonymized, rendering individual informed consent unnecessary.A gynaecologist wipes cervical secretions with a cotton swab before sampling, places the cervical brush head on the cervix and rotates the brush head clockwise 5 times to obtain a sufficient amount of cervical epithelial cells. Then, the cervical brush head is placed into the sample tube marked with the patient's name, the cap is tightened, and the sample is quickly sent for examination.DNA extraction and HPV genotyping were performed using an HPV genotyping test kit . The kit detected 20 types of HPV: HPV6, 11, 42, 43, 16, 18, 31,33, 35, 39, 45, 51, 52, 53.56, 58, 59, 66, 68, and 73. The process involved three steps: HPV-DNA extraction, PCR amplification and HPV-DNA hybridization. The Haema9600  was used for gene amplification. The amplification parameters were set as follows: ①50 °C for 2 min; ②95 °C for 10 min; ③40 cycles were performed at 95 °C for 30 s, 52 °C for 45 s and 65 °C for 30 s; and ④ 65 °C for 5 min. GL-HB-9600  was used for DNA hybridization. After color rendering, positive detection results were indicated by clear blue dots. To ensure the reliability of each HPV test result, six probe arrays  were used as the internal control (IC), and HPV16 was added as a positive control. The testing doctor strictly follows the HPV interpretation rules to issue the patient's HPV test results. This study completely ruled out the impact of reproductive pathogens  other than HPV on the HPV detection results. Specific HPV extraction solution was added during the DNA extraction stage to ensure specificity. During the DNA amplification stage, HPV primers and probes were added to the reaction mixture to ensure the specific amplification of HPV fragments. The probes on the gene chip were specifically designed for HPV genotypes, ensuring no cross-reactivity between different genotypes during the DNA hybridization process.Microsoft's Excel 2021 was used to process and analyse the data, the chi-square test was used to compare the sample rate between the groups, SPSS 26  was used to calculate chi-square data and P values, and two-sided P values of less than 0.05 were considered statistically significant.The total infection rate of in 180,276 cases was 21.97% . the 16 high-risk types were HPV-52, -16, -58, -53, -51, -68, -56, -18, -59, -33, -39, -66, -31, -35, -45 and -73 in order. The prevalence of the 20 genotypes is shown in Fig. The 30–39 years group made up the largest proportion of screened participants, accounting for 38.54% of the total population, yet the < 20 and ≥ 60 age groups made up fewer proportions, accounting for 0.44% and 14.4%, respectively , and the L1 spontaneously formed VLPs are highly immunogenic and produce high titers of neutralizing antibodies to prevent HPV infection32. VLPs mimic the organization and conformation of authentic native viruses but lack the viral genome, inducing an immune response without causing disease33. In contrast, attenuated or inactivated viruses may pose risks of incomplete attenuation or reversion to virulence, raising safety concerns. As a result, current HPV vaccines are safer than attenuated or inactivated viruses. A decade of HPV vaccination practice has demonstrated that HPV vaccine can reduce HPV infection rates, genital warts, low-grade and high-grade cervical intraepithelial neoplasia (CIN), and CIN2+27, moreover it has a strong group effect or indirect protection against unvaccinated females26. In terms of HPV screening, HPV DNA has a high sensitivity and negative predictive value (fewer high-grade lesions in the subsequent third year of screening) and is an effective screening method34.To date, vaccination and HPV screening for women constitute two major strategies in the prevention of cervical cancer and genital warts. The focus of HPV vaccine research lies within the L region35  and the more developed regions of Shanghai36  but lower than those of the economically comparable Tianjin37 . The prevalence rates of HPV-6 and -11 infections in Chengdu  were lower than those in Shanghai36  and Guangxi35  but higher than those in Tianjin37 . Disparities in HPV infection rates across different regions may be influenced by factors such as local economic conditions, awareness of prevention, lifestyle habits, and HPV detection methods. However, regardless, given the crucial roles of HPV 16/18 and HPV 6/11 in cervical cancer and genital warts, the aforementioned findings underscore the imperative need to enhance HPV vaccine coverage in the Chengdu region.In this study, the prevalence rates of HPV-16 and -18 infections in Chengdu were 3.22% and 1.28%, respectively. These rates were significantly higher than those of the less developed regions of Guangxi39. In this study, the top five genotypes in 180,276 cases were HPV-52,-16,-58,-53,-51, and the top five genotypes with HPV16 and HPV18 coinfection were HPV52, -58, -53, -51, -33 and HPV52, -16, -53, -58, -51, respectively, while the top five genotypes with HPV6 and HPV11 coinfection were HPV-52, -16, -58, -51, -53 and HPV-16, -52, -58, -59, -18, respectively. Thus it can be seen that HPV-52 and -58 are the top two genotypes among females in Chengdu. However, currently, among the five available HPV vaccines in the China mainland, only the nonavalent vaccine covers these two genotypes, so the nonavalent vaccine is more suitable for Chengdu females. These findings of this large-scale epidemiological investigation based on HPV genotyping screening hold significance for the prevention and control of diseases such as cervical cancer and genital warts in Chengdu. Additionally, they provide valuable insights for the future design of HPV vaccines.In terms of infection patterns, the ratios of HPV-6, -11, -16 and -18 single infection were 48.56%, 57.41%, 60.71% and 56.23%, respectively, which indicates that single infection is the primary mode of HPV infection, followed by double infection. As the multiplicity of coinfection increased, the prevalence of multiple infections gradually decreased. The occurrence of multiple infections is associated with having multiple sexual partners. The potential impact of multiple infections on cervical cancer risk remains debatable40 have indicated that the proportion of high-grade cervical lesions among HPV16 single infections is the highest. In response to the increasing trend of HPV16 single infections, a stronger push for vaccines covering HPV 16 is warranted. While the infection rate of HPV18 is lower than that of HPV16, its coinfections with HPV52, -58, -53, -51, and -33 genotypes in multiple infections probably enhance its carcinogenic potential.HPV16, as a highly carcinogenic genotype, was found to have the highest infection rates in HPV6/11/16/18. The single infection positivity rate of HPV16 showed a continuous increase yearly, and studies41 have pointed out that the most common HPV genotypes in genital warts in China are HPV-6, HPV-11 and HPV-16. In this study, HPV16 ranked second in HPV6 multiple infections (172 coinfections), and HPV16 ranked first in HPV11 multiple infections (97 coinfections), which also explains why HPV16 is the most frequent HR-HPV in genital warts. Moreover, multiple infections of HPV6/11 with HR-HPV may be the cause of genital warts appearing in LSIL, HSIL and tumour progression42; therefore, in patients with genital warts, it is necessary to test whether there is an HR-HPV infection and to address it appropriately.In this study, the coinfection of HPV6 and HPV11 was very low (42 cases), the single infection of HPV6 and HPV11 accounted for the majority , and most of them had multiple coinfections with HR-HPV. In cases of multiple infections involving HPV 6, the most frequently cooccurring genotype is HPV-52, -16, -58, -51, and -53, whereas for HPV 11, the most commonly cooccurring genotype is HPV-16, -52, 58, -59, and -18. It can be seen that low-risk HPV6 and HPV11 are the most common co-infected LR-HPV with HR-HPV. Studies44. The first peak of HPV infection rates in young women can be up to 80% in some populations45, with most women suffering from transient infections that clear rapidly46. The highest infection rate among young women (< 20 years old) may be related to less mature immune protection and/or multiple sexual partners in this age bracket47 but also to the fact that HPV screening is performed only if the patient has a sexual history and associated symptoms  or if the patient requests it, which results in a relatively high detection rate. In the face of the highest HPV6/11/16/18 infection rate among young girls, it is urgent to strengthen HPV vaccination among women who have not had sex before in Chengdu. After this first peak, the prevalence of infection gradually declined to a plateau in middle-aged women (20–49 years old), which is probably due to increased autoimmune function and stable sexual partners. For the second minor peak of infection, the higher rate of infection in old women (≥ 50 years old) may be due to immunosenescence, changes in sexual behaviour and sexual partner , and reappearance of past (latent) infections51. Some scholars52 point out that the geographic difference in this second peak may be partially explained by indirect indicators of menopausal hormonal patterns, such as body mass index (BMI) and ethnicity. Since young women (age ≤ 20 years) are HPV-susceptible because of high HPV infection rates, vaccination of women before sexual debut is the most cost-effective strategy to prevent cervical cancer. Data54 showed that the earlier young women were inoculated with the HPV vaccine, the higher the antibody titre and the better the protective effect of the vaccine. Studies55 showed that almost everywhere, most men and women started having sex in their late teens (ages 15–19), and most began having sex at 15. The crude median sexual debut age for the youngest age group was 17 years in China56. Considering Chinese cultural traditions and social factors such as nine-year compulsory education, Chinese scholars recommend that junior middle school girls (aged 13 to 15) to be the first group to be vaccinated. In mainland China, there is an ongoing initiative to gradually introduce free HPV vaccination services or provide subsidized HPV vaccine inoculation. Currently, 19 provinces, municipalities, or autonomous regions across China offer free HPV vaccine administration. As of now, locations within Sichuan Province where free HPV vaccination can be arranged for those junior middle school girls (aged 13 to 15) include the entire city of Chengdu, as well as Wenchuan County in Aba and the city of Barkam, the latter two being economically disadvantaged areas. Similarly, HPV vaccination is also necessary in women from less than the age of 20 to 45, and even in women already infected with HPV, data54 show that the immune response to the HPV vaccine appears to prevent reinfection or reactivation of disease with vaccine HPV type. Moreover, HPV 16 and 18 vaccines were well tolerated and highly efficacious against HPV 16 and 18-associated high-grade genital lesions and persistent infection57.In this study, the prevalence of HPV6/11/16/18 was bimodal with age, with young women (< 20 years old) having the highest prevalence, old women (≥ 60 years old) having higher prevalence, and middle-aged women (20–49 years old) having a modest decline in prevalence. In fact, HPV infection is bimodal with age and is very common in many regions, and almost all young women have the first peak, with the second peak occurring at different ages58. Given that HPV 16 or 18 infections have the highest risk for CIN 3 and occult cervical cancer, 2019 ASCCP59 specifically emphasized that additional evaluation  is necessary even when cytology results are negative if HPV 16 or 18 testing is positive and that additional laboratory testing of the same sample is not feasible, the patient should proceed directly to colposcopy.For the second minor peak of HPV infection, HPV and cytological screening is especially important in middle-aged and older women, as screening not only reduces the burden of precancerous lesions and related persistent HPV infections, but removal of lesions may have a direct antigen-presenting effect that could protect against subsequent HPV infections60. For middle-aged and older women, regular HPV screening is a better strategy to prevent cervical cancer and precancerous lesions.In sum, vaccination is of extreme importance in the current situation. The WHO global strategy to accelerate the elimination of cervical cancer as a public health problem highlights 90% vaccination coverage for girls younger than 15 years old by 2030 as one of the three targetsThe study possesses several limitations. First, it lacked cell or pathological data from positive cases, rendering comparative statistics unfeasible. Second, female vaccination rates were not taken into account, making it impossible to calculate the specific impact of the vaccine on HPV infection rates. Third, the study's subjects comprised solely patients from hospitals within a 6-year timeframe, which does not represent the typical population of Chengdu. Therefore, these findings are exploratory in nature and merit further investigation. Fourth, only four genotypes were involved in this study, obviously, some other HR-HPVs and LR-HPVs are involved in malignant disorders, especially cervical cancer and other genital cancers, However, due to the need for detailed and in-depth study of the four genes, few other genotypes are taken in account, which need to be further presented in future studies.In conclusion, the infection rate of HPV6/11/16/18 was high in Chengdu, with HPV16 exhibiting the highest infection rate and HPV16 single infections showing an annual increase. The prevalence of HPV6/11/16/18 infection in young women (< 20 years) was highest and in old women (≥ 60 years) was higher; therefore, prioritizing vaccination for young females is essential in the current context, while HPV screening remains an effective approach for older females. Among the available HPV vaccines in Chengdu, the nonavalent vaccine appears to be relatively most suitable for Chengdu females. Additionally, in patients with genital warts, it is necessary to assess the presence of HR-HPV infection and manage it appropriately.


GZMB, IFNG, IL12B, and IL8 and downregulation of NFATC4 and IL7 in CA samples. Immunohistochemical analysis showed increased FOXP3, IFN-γ, CD1a, and CD4 expression in CA than in the control tissue samples. In contrast, CD3 and CD8 expression was decreased in CA lesion samples. Increased levels of pro-inflammatory cytokines in HPV-positive patients compared with HPV-negative patients seem to reflect the elevated immunogenicity of HPV-positive CA lesions. Host defense against HPV begins during the early stages of the innate immune response and is followed by activation of T lymphocytes, which are mainly represented by CD4+ and regulatory T cells. The low CD8+ T cell count in CA may contribute to this recurrent behavior. Additional studies are needed to elucidate the mechanism of host defense against HPV infection in CA.Condyloma acuminata (CA) is a benign proliferative disease mainly affecting in non-keratinized epithelia. Most cases of CA are caused by low-risk human papillomavirus (HPV), mainly HPV 6 and 11. The aim of the current study was to highlight the candidate genes and pathways associated with immune alterations in individuals who did not spontaneously eliminate the virus and, thus, develop genital warts. Paraffin-embedded condyloma samples (n = 56) were analyzed by immunohistochemistry using antibodies against CD1a, FOXP3, CD3, CD4, CD8, and IFN-γ. The immunomarkers were chosen based on the evaluation of the innate and adaptive immune pathways using qPCR analysis of 92 immune-related genes, applying a TaqMan Array Immune Response assay in HPV 6 or HPV 11 positive samples (n = 27). Gene expression analysis revealed 31 differentially expressed genes in CA lesions. Gene expression validation revealed upregulation of  Human papillomavirus (HPV) infection is related to several benign and malignant diseases –4. The pCondylomata acuminata, or anogenital warts, is a sexually transmitted infection (STI) caused by HPV, most often by low-risk HPV types 6 and 11; however, coinfections with high-risk HPV types can also be observed [Anogenital warts are benign proliferative diseases that manifest as visible lesions, such as single or multiple papules on the vulva, vagina, cervix, penis, scrotum, perineum, and perianal area .2 laser vaporization, and interferon injections are also frequently used [Although condyloma acuminata is a benign disease, condylomas cause significant psychosocial distress and anxiety . The tretly used . Howevertly used , 8.In a generic viral skin infection, keratinocytes, considered as sentinels in the innate immune response, express pattern recognition receptors (PRR) that recognize viral DNA or RNA . These rThe immune response to HPV is based on recognition by epidermal dendritic cells, represented by Langerhans cells (LCs) . LangerhT cells, helper T (Th) lymphocytes, and cytotoxic T lymphocytes are responsible for regulating the adaptive immune response in cooperation with antigen-presenting cells. The release of different cytokines during infection regulates the synthesis and action of other cytokines. The Th1 immune response is responsible for the release of immunostimulatory cytokines, such as interferon gamma (IFNG), TNF alpha, interleukin 2, and interleukin 12, which mainly induce cell-mediated immunity. Th2-type cytokines  predominantly induce humoral immune responses and inhibit cell-mediated responses) –17.Previous studies have demonstrated that the occurrence, remission, relapse, and cancerization of condyloma acuminata are associated with immune disorders in patients , 19. EveIn the present study, we performed expression profiling of 92 immune-related genes in condyloma acuminata biopsies. We also evaluated the population of immune cells present in the condyloma microenvironment, aiming to highlight candidate genes and pathways associated with immune changes in individuals that did not spontaneously eliminate the virus and thus developed genital warts.All procedures involving human participants in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from all participants included in the study. The study was approved by the Research Ethics Committee of the Institute of Biosciences, Humanities and Exact Sciences of São Paulo State University, city of São José do Rio Preto, Brazil (license number 37830514.0.0000.5466). All the patients agreed to participate in the study.Gene expression analyses were carried out using 27 fresh condyloma acuminata (CA) biopsies with the respective adjacent normal tissues, obtained by surgical excision from female patients who visited the Clinical Hospital of the University of São Paulo Medical School, Ribeirão Preto, Brazil. For these analyses, only HIV-negative patients were considered, and the biopsies were subjected to histopathological analysis by an experienced pathologist who diagnosed CA.Seventy paraffin-embedded samples were used for immune cell quantification using immunohistochemistry. These included 56 biopsies of CA and 14 normal vulvovaginal tissue samples obtained from HPV-negative patients undergoing myomectomy, which were used as control samples. These samples were obtained from the Medical School (FMB) of São Paulo State University , and CA was histopathologically diagnosed by an experienced pathologist.The methodology for Total DNA and RNA isolation is described in the supplementary material.5-CGT CCM ARR GGA WAC TGATC- 3) and PGMY11 (5-GCM CAG GGW CAT AAY AAT GG-3) were used as degenerate primers to detect viral DNA. The amplification products were used for nested PCR with the primers GP5+ (5′-TTTGTTACTGTGGTAGATACTAC-3′) and GP6+ (5′-CTTATACTAAATGTCAAATAAAAA-3′), which amplify a sequence of 150 bp internal to the fragment produced by the PGMY09/PGMy11 pair of oligonucleotides [2, 0.2 mM dNTPs, 0.4 μM of each primer, 500 ng of DNA, and nuclease-free water. Thermocycling conditions included an initial denaturation step at 95°C for 9 min, followed by 40 cycles at 95°C for 1 min, 55°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 5 min. For nested PCR, the 25.0 μL amplification reaction comprised 2.5 U of Taq DNA Polymerase , 2.5 μL of 10X PCR Buffer, 7.6 μM magnesium chloride (MgCl2), 0.064 mM deoxynucleotides (dNTPs), 0.48 μM of each primer, 5.0 μL of the product from the PGMy09/PGMy11 reaction, and nuclease free water. An initial denaturation step was conducted at 95°C for 9 min, followed by 40 cycles at 94°C for 30 s, 45°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 8 min. Positive (Caski cell line) and negative controls (Nuclease-free water) were used in the PCR.Polymerase chain reaction (PCR) was used to amplify HPV DNA in 27 condyloma samples. The oligonucleotides PGMY09  [The obtained sequences were assessed for quality using PHRED/PHRAP/CONSED, available online at  System) . All seq System) .Gene expression in CA samples and adjacent tissue samples without lesions was compared. Lesion margins were used to mitigate the bias in gene expression analysis between CA and normal tissues.To analyze immune-related gene expression and the biological component of variation, 27 fresh CA samples were synthetized and combined in three cDNA pools; the first, second, and third pools were prepared with six samples of HPV 6 condylomas, six samples of HPV 11 condylomas, and six HPV-negative sample margins, respectively. Therefore, each pool was tested as a single sample  in TaqMan array RT-qPCR.Twenty-seven cDNA samples, including those used in the PCR array, were used to validate the results of the PCR array using RT-qPCR.12-18  (0.25 μM), RNaseOut (Recombinant Ribonuclease Inhibitor)  (5 mM), and MultiScribe Reverse Transcriptase . The tubes were maintained at 25°C for 10 min followed by incubation at 37°C for 120 min.cDNA samples were prepared from 1 μg of total RNA using the High-Capacity cDNA Reverse Transcription kit . The reactions were prepared in a final volume of 20 μL, containing 1x Buffer, 1x dNTP Mix, 1x Random Primers, Oligo(dT)™ Array, Human Immune, Fast 96-well plate , representing 92 immune-related genes and four reference genes  and TaqMan Gene Expression Assays  , accordiRT-qPCR was performed on a QuantStudio 12 K Flex Real-Time PCR System . All samples were tested in triplicate. The relative expression of each specific gene was calculated using the ΔΔCq method ; a cutofSeventy paraffin-embedded samples were arranged in two tissue microarrays (TMAs): one corresponding to case-patients with lesions, and the other from control-patients. The case-patients TMA contained 56 biopsies of CA. The control-patients TMA contained 14 vulvovaginal tissue samples obtained from healthy patients undergoing myomectomy.2O2 ready for use solution  for 10 min. Protein blocking solution was applied for 10 min, and after washing in TBS-T, the slides were incubated overnight at 4°C with primary antibodies . Slides for detecting FOXP3, CD1a, CD4, and CD8  were incubated with HRP polymer  for 30 min at 25°C, and those for CD3 and IFN-γ  for the same time  coupled with an Olympus VS120 Virtual Microscope Slide Scanning System (VS120-S5). For quantifying FOXP3 and IFN-γ proteins, which presented diffuse cytoplasmic staining in the epithelium, three random photomicrograph fields of each TMA sample were captured using OLYMPUS OlyVIA 2.9 software  at 40x magnification. Immunostaining was quantified by imaging analysis using Image-Pro Plus software , with the M130 multipoint system proposed by Weibel,  as previFor the cellular and punctual staining markers CD1a, CD3, CD4, and CD8, the number of positive cells per area was noted along with the values expressed in cells per square millimeter of tissue (cell/mm²). Quantification was also performed on the digitized slides. For each sample, positive brown-stained cells were counted individually using OLYMPUS OlyVIA 2.9 software . The total areas of the TMA samples were measured.p-value ≤ 0.05 was considered statistically significant.Data were subjected to the Kolmogorov-Smirnov normality test and found to be normally distributed; all groups (case-patients and control-patients) were compared using Analysis of Variance (ANOVA) followed by Tukey’s multiple comparison test. All values are expressed as the mean ± standard error using GraphPad Prism 8 . Any Among the 27 CA HPV-positive samples evaluated, 29.6% showed the HPV 11 genotype and 70.4% showed the HPV 6 genotype, which was the most prevalent .2)  were downregulated in the pools of both HPV 6 and HPV 11 condyloma samples. Seventeen genes  presented increased expression in the pool of HPV 6 condyloma samples and ten genes  showed increased expression in the pool of HPV 11 condyloma samples. Eight genes were upregulated in the pools of both HPV condyloma samples   . Eight o samples . Five ge7A, IL8) .GZMB, IFNG, NFATC4, IL12B, IL7, and IL8, were selected for qPCR validation using 27 condyloma samples (GZMB was upregulated in 63% of the samples (17/27), similar to the TaqMan Array result (IFNG was upregulated in 81.5% (22/27) of the samples (IL8 was upregulated in 78% (21/27) of the samples (NFATC4 was downregulated in 63% of the samples (17/27) (IL12B was upregulated in 55.5% of the biopsies (15/27) (IL7 was downregulated in only 30% (8/27) of the samples  . IL12B w (15/27) , whereas samples .Six different immunomarkers  were used to quantify immune cells in CA samples. From the genes validated by RT-qPCR, immunomarker selection was performed following the innate and adaptive immune pathways. For FOXP3 and INF-γ, a cytoplasmic pattern was observed among cells of the stratified squamous epithelium . For CD1FOXP3 expression presented a three-fold increase in the case-patients TMA compared to that from the control-patients TMA  p < 0.0. There w2 were obtained in TMAs from case- and control-patients, respectively. CD1a expression was approximately twice as high in case-patient samples compared to that in the control sample group  than in control samples (mean of 3.47 cells/mm2) (p = 0.0049) (CD4 levels increased by more than two-fold in case samples (mean of 7.53 cells/mm 0.0049) .CD8 expression showed a four-fold decrease in case patients (p < 0.0001)  comparedHPV types that cause genital warts do not generally cause cancerous changes. Most HPV infections are transient, but in 10% of patients, the infection persists and may produce late recurrence, especially in states of immune deficiency .In this study, 31 immune system genes were found to be differentially expressed in condyloma acuminata (CA) lesions. Some gene expression heterogeneity was also observed among the evaluated samples. This was probably attributed to the fact that human immune systems vary among individuals , 36. AnaAs the primary target of HPV, keratinocytes play an important role in the emergence of viral infection and subsequently promoting an effective immune response. As part of the innate response, the immune defense of the epithelium affects the participation of dendritic cells, represented by LCs and keratinocytes . KeratinEpidermal Langerhans cells (LCs) are immature skin dendritic cells. Changes in the number of local dendritic cells can result in immune system damage and contribute to viral persistence .To analyze the presence of LCs in condylomas, we used the CD1a antibody. CD1a serves as a surface marker for LCs, as it is expressed by LCs participating in antigen presentation. We found a higher expression of LCs in test samples than in control tissue samples.In contrast to our results, Pan and co-authors observed fewer LCs in CA lesions compared to those in normal tissues. The same result was observed by Feng and co-authors when investigating the frequency of LCs in CA lesions and normal controls using CD1a staining by immunohistochemistry , 43.GZMB, which is known to activate target cell apoptosis through caspase-dependent perforin [One reason for our contrasting results could be the control exerted by miRNAs, as evidenced by McBee and co-authors in 2011; they reported a possible control of the CD1A gene by miR-344 in cervical dysplasia samples. The same mechanism could be present in condyloma acuminatum, as miR-433 involvement in cellular proteins targeted by high-risk or low-risk E6 and E7 HPV proteins has not been examined. Thus, additional studies are required to confirm this hypothesis. Furthermore, differential expression of LCs in condyloma indicates the innate immune activity as an initial control for HPV. Similarly, our gene expression results also showed upregulation of perforin .IFNG overexpression in keratinocytes, which play an important role during the triggering of HPV infection and are considered immune sentinels [IFNG expression in HPV-positive tissues [IFN-γ is a soluble cytokine belonging to the IFN type II class and is secreted by cells of both the innate and adaptive immune systems . Based oentinels , 14. Hu  tissues .IFNG, IL12B, and IL8 in genital warts. Strong CD4+ T helper (Th) and cytotoxic T cell (CTL) responses are essential for effective HPV clearance. Th1 mechanisms  act against intracellular pathogens and promote pro-inflammatory responses [The involvement of T helper 1 (Th1) cells in host defense against HPV was evidenced in this study by the overexpression of CD4, along with overexpression of esponses . Furtheresponses .NFATC4, which encodes a member of the nuclear factor of activated T cell (NFAT) protein family [NFATC4 expression. The opposite result could be a consequence of IL-7 downregulation, which was also observed in the present study. IL-7 is reported to mediate the NFAT activation pathway independent of Ca2+/calcineurin signaling [Gene expression analysis showed downregulation of n family , 51. Then family . As we oignaling , an NFATignaling .Contrary to the higher number of CD4+ cells in CA samples, immunohistochemical analysis revealed fewer CD8+ cells in CA compared to those in the control tissues. Class I MHC complexes restricted to CD8+ T cells are a principal component of adaptive immune responses to intracellular pathogens and tumors . During + T-cell response is crucial for host defense against HPV persistence and suppression of HPV-associated disease progression [A powerful CD8gression –58.+ T-cell infiltration positively correlate with the regression of HPV-positive lesions [+ T-cell cytotoxicity in HPV-related lesions may be caused by suppression via E7-expressing keratinocytes [The levels of CD8 lesions . In 2017inocytes . Papilloinocytes  with E2 inocytes . TherefoCD3 staining represents the T cells in general , and itsGZMB, IFNG, NFATC4, IL7, IL8 and IL12B. Of these, GZMB, IFNG, IL8, and IL12B were upregulated, whereas NFATC4 and IL7 were downregulated in the analyzed CA samples. Thus, we observed an increase in the expression levels of pro-inflammatory cytokines and genes involved in the innate immune response and activation of adaptive immunity. The increased expression levels were reflected in the protein levels of the CA samples, as evidenced by the immunomarkers FOXP3, IFN-γ, CD1a, and CD4. These results suggested that the initial host defense against HPV was accompanied at some level by the activation of T lymphocytes (high expression of FOXP3 and CD4 immunomarkers). Downregulation of CD3 and CD8 expression was also observed.In summary, the results presented here show differentially expressed genes (DEGs) selected after the initial screening of 92 immune-related genes in CA samples. To confirm these results, six genes were validated: In conclusion, increased levels of pro-inflammatory cytokines in HPV-positive patients compared with those in HPV-negative patients seem to reflect the elevated immunogenicity of HPV-positive CA lesions. Host defense against HPV begins during the early stages of the innate immune response and is followed by the activation of T lymphocytes, which are mainly represented by CD4+ and regulatory T cells. The low CD8+ T cell count in CA may contribute to this recurrent behavior. Additional studies may contribute to further elucidate the mechanism of host defense against HPV infection in condyloma acuminata.S1 FigA numerical version is presented in (TIF)Click here for additional data file.S2 Fig(A) BLAST alignment analysis evidence HPV genotype 6 based on the database stored in the GenBank and (B) BLAST alignment analysis evidence HPV genotype 11 based on the database stored in the GenBank. The GP5+ oligonucleotide binding site is highlighted.(TIF)Click here for additional data file.S3 Fig(A) diagram illustrating the experimental plan of the study. (B) diagram summarizing the findings of the study.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table2). GAPDH, 18S, GUSB and HPRT1 are reference genes.Relative mRNA expression level (Log(DOCX)Click here for additional data file.S6 Table2).Relative mRNA expression level (Log(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


The study included 14 patients (group A = cases) who received 3 doses of an intramuscular HPV vaccine (Gardasil 4 or Gardasil 9) in addition to the standard treatments for AGWs and OWs, and 15 age- and sex-matched patients (group B = controls) with an analogous number of lesions to group A who received only standard therapies. After 12 months, 85% of patients of group A versus 33% of group B had positive clinical outcomes (0.004). Our findings suggest a possible therapeutic role of HPV vaccines in addition to standard treatments for AGWs/OWs. Preventive vaccines, blocking the viral entry through the induction of L1-specific antibodies, can prevent autologous reinfections (through auto-inoculation) and favor the elimination of the virus.Human papillomavirus (HPV) vaccines are preventive measures to decrease HPV infection rates. Knowledge of their efficacy as treatment options for anogenital warts (AGWs) and oral warts (OWs) is limited. To evaluate the efficacy of HPV vaccinations in recalcitrant AGWs and OWs , we compared a group of patients treated with standard therapies plus an HPV vaccine with a group of patients treated with standard therapies only. The response to treatment (in terms of the number of lesions) in the two groups was compared. Data were analyzed with the χ2 test and  Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) worldwide, affecting sexually active individuals at least once in their lifetime ,2,3. CerGlobally, over 200 HPV genotypes exist with a relevant genetic variability . Their cImportantly, an HPV infection may impact the patient’s psychological, physical, and sexual health as well as the patient’s global quality of life (QOL), even leading to depressive symptoms and changes in sexual functioning .®), containing protein antigens for HPV types 6, 11, 16, and 18, was commercialized in most industrialized countries. The bivalent vaccine (Cervarix®), licensed in 2007, targets HPV types 16 and 18. Lastly, the nonavalent HPV vaccine (Gardasil 9), licensed in 2014, targets HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 [The burden of HPV infections and HPV-related diseases has led to increasing efforts of the scientific community to develop efficacious strategies for infection prevention. Among these, HPV vaccines are being administered worldwide and have already proven effective in reducing HPV infection rates and HPV-related diseases ,19. In 2, and 58 ,22. The , and 58 .To date, knowledge of the possible therapeutic effect of the HPV vaccine on already-established HPV infections and HPV-related disease is limited ,18. ConsIn the present study, we aimed to evaluate the possible therapeutic efficacy of HPV vaccinations in the treatment of recalcitrant AGWs and OWs described as HPV-induced benign epithelial proliferations persisting for more than 6 months and not responding to at least one standard treatment.p values < 0.05 were considered to be statistically significant.We retrospectively studied patients who had visited the STI Outpatient Clinic  between January 2015 and July 2019 for recalcitrant AGWs/OWs. Patients who received 3 doses of an intramuscular HPV vaccine in addition to and simultaneously with standard treatments for AGWs and OWs were included in the study as the cases (group A). Controls (group B) were selected from patients affected by recalcitrant AGWs/OWs who received only standard therapies, who were matched for age and sex, and who had clinical characteristics comparable with group A . Demographic and clinical features were collected. The response to treatment was clinically evaluated at 12 months, based on the number of AGWs/OWs. The response was considered to be complete if there was a complete clearance of lesions, partial if there was a decrease > 50% in the number of lesions, and absent if there was a decrease < 50% in the number of lesions. The follow-up time was 12 months for all patients. Data were analyzed using the χ2 test and The study included 14 patients  in group A (cases) and 15 patients  in group B (controls).The clinical features of each patient are described in Clinically, the body area most frequently affected by warts was the genital area, followed by the perianal skin. The number of warts at the first visit ranged from 1 to 15  and their duration prior to the visit ranged from 6 months to 36 years . The most commonly used standard treatment was cryotherapy, followed by imiquimod 5% cream .p > 0.05).Comorbidities affecting the immune system were present in 3 patients of group A (2 human immunodeficiency virus (HIV)-infected patients, both on antiretroviral therapy for 2 and 4 years; 1 autologous bone marrow transplant patient for Hodgkin’s lymphoma) and in 2 patients of group B (both having received autologous bone marrow transplantations for Hodgkin’s lymphoma less than one year before the first visit to our outpatient clinic). In group A, all the patients received the 9vHPV vaccine at the time of the study; 3 of them had also received the 4vHPV quadrivalent vaccine several years earlier. The difference in the clinical outcome between patients who received only the 9vHPV vaccine and patients who had previously received the 4vHPV vaccine was not statistically significant (p > 0.05).Regarding the factors contributing to vaccine efficiency in group A, we found that an age < 45 years, a number of lesions less than 10, a duration of the lesions ≤ 2 years, and an absence of comorbidities affecting the immune system  were correlated with a better clinical outcome , although the difference was not statistically significant (p = 0.01).The response to treatment was clinically evaluated at 12 months. In group A, 9 patients showed a complete response (64%), 3 showed a partial response (21%), and 2 showed no response (15%). In group B, 4 patients showed complete responses (27%) and 9 responses were absent (60%). Taking the complete and partial responses to treatments together, 12 patients of group A (85%) versus 6 of group B (40%) had a positive clinical outcome . The difNowadays, HPV vaccines are successfully administered as a primary antiviral prophylaxis in presumed HPV-naïve patients (adolescents at age 11 or 12 years) and in adults aged >26 years not previously vaccinated. HPV vaccines can be administered regardless of a history of AGWs, an abnormal Pap test or HPV test, or anogenital precancerous conditions. A 2-dose vaccine schedule  is recommended for people who initiate vaccinations before their 15th birthday. A 3-dose vaccine schedule  is recommended for immunocompromised people, regardless of the age of initiation .Recent studies have assessed the effectiveness of HPV vaccines in reducing the prevalence and mortality of HPV-related diseases among men who have sex with men (MSM)  and womeLittle is known about the possible role of HPV vaccines as therapeutic agents in already-infected individuals with ongoing HPV-related cutaneous and/or mucosal conditions ,26.Numerous conventional and alternative treatments exist for AGWs, including topical agents and physically destructive procedures ,23. TherIn the context of the many available therapies for AGWs and OWs, a possible dual role of HPV vaccines, both preventive and therapeutic, would be of revolutionary clinical relevance and deserves in-depth investigations and clinical research.However, to date, only a few studies and sporadic case reports have focused on the possible therapeutic role of HPV vaccines (both the 2vHPVvaccine and 4vHPV vaccine); these obtained partly contradictory results ,33,34,35The therapeutic efficacy of the last commercialized 9vHPV vaccine on AGWs was assessed only recently in three small case series  and in t® [Bossart et al. reported 100% remission  of recalcitrant AGWs in five male patients, 21 to 58 years old, after three doses of Gardasil 9 . Similar® .Interestingly, in the present report, we observed a good response to HPV vaccines in both immunocompetent and immunocompromised patients (people living with HIV and patients who had recently received a bone marrow transplant).Moreover, our study is the first to show the encouraging therapeutic results of the 9vHPV vaccine in patients with OWs. Cyrus et al. reported the complete remission of HPV-32-related oral papillomas in a 60-year-old immunocompetent man after three doses of the 4vHPV vaccine .The exact immunologic phenomena underlying the therapeutic effect of HPV vaccines have not yet been elucidated. Whilst the preventive efficacy of commercially available HPV vaccines is based on the induction of a humoral immune response, it has been postulated that the regression of already-existing anal, vulvar, and cervical lesions is mainly associated with the activation of cellular, rather than humoral, immunity . OriginaMoreover, HPV vaccines have been associated with increased levels of cytokines  and with the induction of IFN-γ-producing CD4+ and CD8+ T-lymphocytes , cytotoxSeveral trials have indicated that the Bacillus Calmette–Guérin (BCG) vaccination has a protective and therapeutic efficacy against cutaneous and genital warts. Its efficacy is possibly due to a trained immunity’s cross-protection against other bacterial or viral infections through the efficient activation of the immune system, especially innate immune cells. Trained innate immunity is indeed characterized by providing cross-protection against infectious diseases following stimulation derived from bacteria, fungi, viruses, and parasites. This protective effect defends against a certain range of unrelated pathogens .Similarly, it could be speculated that through the mechanism of trained immunity, HPV vaccines may efficiently activate innate immune cells against an already-present HPV infection.HPV vaccines might be especially effective for AGWs as the DNA of LR HPVs is usually not integrated into the host genome and viral particles are produced and released from the granular layer of the epithelium at every viral replication . HPV vacConversely, in high-grade epithelial lesions, the HPV genome is often integrated into the host genome and infected cells neither express L1 nor produce viral particles. Therefore, as infected cells cannot be recognized by vaccine-induced antibodies, the HPV vaccination is ineffective in disease control .In conclusion, our findings suggest that the addition of HPV vaccines to the standard treatments for AGWs and OWs leads to positive clinical outcomes. HPV vaccine administration together with conventional treatments might reduce the use of alternative and off-label treatments such as podophyllin resin, intralesional interferon, photodynamic therapy, and topical cidofovir that might be aggressive and associated with side-effects . AlthougA limitation of our study was the small sample size. Larger randomized and controlled trials are needed to confirm the results of our study and to assess the mechanisms underlying a possible therapeutic effect of HPV vaccinations.


P < .001). Mean age of the patients from Mozambique and Spain was 45 ± 12 and 72 ± 14, respectively (P < .001). No differences were found in terms of HPV genotypes or multiple HPV infection rates. 1/35 tumors (3%) from Mozambique and 29/41 (70%) from Spain showed abnormal p53 immunostaining (P < .001). In contrast with the predominance of HPV‐independent VSCC affecting old women in Europe, most VSCC in sub‐Saharan Africa are HPV‐associated and arise in young women. This data may have important consequences for primary prevention of VSCC worldwide.Two pathways have been described for vulvar squamous cell carcinomas (VSCC), one associated with human papillomavirus (HPV), and the other HPV‐independent. We compared the etiopathogenic features of a series of VSCC from Mozambique, a sub‐Saharan country with high prevalence of HPV and HIV, with those of Spain, a European country with low prevalence of HPV and HIV. All VSCC diagnosed at the two institutions from January 2018 to December 2020 were included . HPV DNA detection and genotyping, and immunohistochemistry for p16 and p53 were performed. Tumors showing p16 positive staining and/or HPV DNA positivity were considered HPV‐associated. 34/35 tumors (97%) from Mozambique and 8/41 (19%) from Spain were HPV‐associated ( Vulvar squamous cell carcinoma (VSCC) can arise from HPV or non‐HPV causes, and that affects prognosis. Here, the authors compared VSCC samples from a population in Mozambique, where HPV prevalence is high, with those from Spain, where it is low. In Mozambique, 97% of VSCC were HPV‐associated, based on p16 staining and HPV DNA positivity, compared with only 19% of VSCC in Spain. Mean age of patients in Mozambique was 45, vs 72 in Spain. Vaccination programs, the authors suggest, could therefore help prevent the majority of VSCC in sub‐Saharan Africa. All cases diagnosed as VSCC during this period were retrieved, and the available material was reviewed. All cases fulfilling the following inclusion criteria were included in the study: (1) presence of invasive VSCC in the vulva and (2) available material for histological revision and HPV detection by polymerase chain reaction (PCR) and p16 IHC in the invasive tumor.2.2Hematoxylin and eosin sections of all tumors were reviewed. In the histological revision, the presence of invasive carcinoma was confirmed, the histological variant of the tumor was assessed  and the most representative and well‐preserved paraffin‐embedded block was selected for HPV detection and IHC staining.All the histological slides were evaluated by two gynecological pathologists with expertise in vulvar pathology . The observers were aware of the geographical origin of the cases but were blind to the HPV testing results.The clinical charts form all patients were reviewed.2.3DNA extraction was performed on whole sections of formalin‐fixed paraffin‐embedded tissue from surgical specimens or vulvar biopsies as previously described. In all cases, the analyzed tissue included the invasive tumor, but no microdissection was performed. Sectioning and sample preparation were carried out with the highest safety measures to avoid cross‐contamination.HPV DNA detection and typing were performed using SPF10 PCR and the LiPA25 system . A volume of 10 μl of isolated DNA was used for PCR amplification through the SPF10‐LiPA system . HPV genotyping was conducted using INNO‐LiPA HPV Genotyping Extra II kit . This system allows the amplification and genotyping of high‐risk HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68; of the probable high‐risk HPV 26, 53, 66, 70, 73 and 82; and of low‐risk HPVs 6, 11, 40, 42, 43, 44, 54, 61, 62, 67, 81, 83 and 89. Quality in each run was confirmed with both positive and negative controls for DNA isolation, amplification, hybridization and genotyping.2.4All cases were stained with a p16 monoclonal antibody using the CINtec Histology Kit . Tumors showing strong and diffuse block‐like staining were considered as positive (p16 upregulation), whereas patchy or completely negative p16 staining was considered as p16 negative.All cases were stained for p53 with the monoclonal antibody . The IHC staining was evaluated in the invasive tumor following the recent p53 pattern‐based interpretation framework,Normal patterns, suggestive of wild‐type p53 protein included: (1) scattered  and (2) mid‐epithelial staining with basal sparing .Abnormal p53 staining patterns, suggestive of mutant protein included: (1) basal overexpression , (2) diffuse  overexpression , (3) cytoplasmic (cytoplasmic staining with or without nuclear expression) and (4) null patterns .All IHC stains were performed using the automated BenchMark ULTRA platform . All IHC and molecular analysis were performed at the pathology department of the Hospital Clinic in Barcelona.2.5All tumors showing positive p16 IHC staining, independently of the results of the HPV detection and typing, or positive result of the HPV detection, independently of the results of p16 IHC, were considered as HPV‐associated, whereas cases negative for both techniques were considered as HPV‐independent. Histological features and p53 IHC staining were not considered for classifying a tumor as HPV‐associated or independent.2.6t or analysis of variance tests were used for comparisons.Data analyses were performed with the SPSS 23.0 statistical package . Categorical variables were expressed as absolute numbers and percentages. The chi‐square exact test was used to compare qualitative variables. Quantitative variables were expressed as mean ± SD, and the Student's 33.1P < .001). Tables P = .008).From January 2018 to December 2020, 41 VSCC were diagnosed at the Maputo Central Hospital (Mozambique) and 41 at the Hospital Clínic of Barcelona (Spain). From the 41 cases initially diagnosed in Mozambique, four were excluded because of the absence of invasive carcinoma, and two because of the absence of sufficient material for further analysis. None of the cases from Spain were excluded. Thus, finally 35 patients from Mozambique and 41 from Spain fulfilled the inclusion criteria. Mean age of the patients from Mozambique and Spain was 45 ± 12 and 72 ± 14, respectively (3.2P < .001).Thirty‐four out of 35 tumors (97.1%) from Mozambique and 8/41 (19.5%) from Spain were HPV‐associated (In the series from Mozambique HPV‐DNA was positive in 22 (62.9%) of the tumors. p16 IHC was positive in 33/35 tumors (94.3%), 12 (36.4%) of whom had negative HPV‐DNA. Only one tumor positive for HPV‐DNA showed negative p16 IHC. In this tumor HPV6 was identified. HPV16 (43%), HPV33 (29%), HPV58 (24%) and HPV18 (14%) were the HPV types more frequently identified in Mozambique. Seven tumors showed infection by multiple HPV types. Table In the series from Spain HPV‐DNA was positive in 7 17.1%) of the tumors. p16 IHC was positive in 8/41 tumors (19.5%), and only one of them 12.5%) had negative HPV‐DNA. No tumors were negative for p16 and positive for HPV‐DNA. HPV16 (50%), HPV33 (25%), HPV53 (7%) and HPV56 (7%) were the HPV types more frequently identified in Spain. Two tumors showed infection by multiple HPV types. Table .5% had n7.1% of tNo significant differences were found in terms of percentage of HPV genotypes between the two countries.3.3P < .001).One out of 35 tumors (3%) from Mozambique and 29/41 (70%) from Spain showed abnormal pattern of p53 immunostaining  “A way to make Europe.”The authors declare no conflicts of interest.The study was approved by the Ethics Committees of the Faculty of Medicine of Maputo Central Hospital (CIBS FM&HCM/071/2017) and Hospital Clinic of Barcelona (ref HCB/2020/1198).AIDSacquired immunodeficiency syndromeHPVhuman papillomavirusIHCimmunohistochemicalPCRpolymerase chain reactionSCCsquamous cell carcinomasVSCCvulvar squamous cell carcinomaWHOWorld Health Organization


We compared 2 human papillomavirus (HPV) assays to detect the 14 high-risk HPV (hrHPV) genotypes in self-collected anal samples. We found a good agreement and similar performance to detect HPV-16, HPV-18, and the 12 other hrHPV genotypes. The global performance to detect the 14 hrHPV genotypes was not significantly different between the 2 assays. Around 30 000 new cases of anal squamous cell carcinoma (ASCC) are diagnosed each year worldwide . Key popIn this study, we aim to compare the agreement and performance of the Cobas 4800 HPV test (Roche) and the Anyplex II HPV28 Detection assay (Seegene Inc) to detect hrHPV in self-sampling anal swabs from asymptomatic MSM and transgender women (TGW) in Phnom Penh, Cambodia, especially for HPV-16 detection, which was the most prevalent HPV genotype found in this population [A cross-sectional retrospective study was conducted between 1 October 2021 and 30 November 2021 in different clinics in Phnom Penh, Cambodia, and under the scope of activities of Men's Health Cambodia, a nongovernmental and nonprofit organization working with men, MSM, and TGW in Cambodia. During consultation, a questionnaire was used to collect patient data, and anal collection  was self-performed using anal swabs discharged in viral transport media (VTM). They were then stored at −80°C at the Institut Pasteur du Cambodge (IPC) until further analysis. A comprehensive explanation of eligibility criteria and procedures has been published previously [First analysis was performed at IPC on 400 μL of sample with the Cobas 4800 HPV test (Roche), which allows the detection of 14 hrHPV types by polymerase chain reaction (PCR) and nucleic acid hybridization. This test specifically identifies HPV-16, HPV-18, and 12 other hrHPV types in the same channel including HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Then, 1 mL of frozen VTM was sent to the Virology Department of the Pitié-Salpêtrière Hospital, Paris, France. DNA extraction (from 200 μL of sample to 100 μL) was performed with the E-mag device (bioMérieux), and both samples and extracted DNA were stored at −80°C. Then, HPV testing was performed with the Anyplex II HPV28 Detection assay (Seegene Inc), which detects and differentiates 28 distinct HPV types including the 14 hrHPV types detected by the Cobas 4800 HPV test and 14 other HPV types . Both HPV tests also detect a human gene as internal control to assess the quality of the sampling and the efficiency of the PCR amplification. If internal control was not amplified, samples were referred as invalid.rstatix from R software. McNemar and kappa indices were calculated to compare the proportion of positive samples and the degree of agreement between the 2 methods to detect the 14 hrHPV types, respectively. A 2-tailed P value of <.05 was considered significant.Statistical analysis was performed with the package Overall, 162 participants were included with a median (IQR) age of 28.1 (24.4–31.8) years, of whom 56% were MSM and 8% were HIV infected. None of them received any HPV vaccine.P = .52), 16% versus 17% (P = .86), and 50% versus 59% (P = .18), respectively  and 41 (25%) samples, respectively. The prevalence of HPV-16, HPV-18, and other hrHPV types was not significantly different between the 2 methods: 20% versus 25% (ectively .i) 3 for HPV-16 positivity, of which 2 HPV-16 were missed by Roche and 1 missed by Seegene; and (ii) 12 for other hrHPV positivity, of which 8 hrHPV types were missed by Roche and 4 by Seegene  (P = .44).The agreement (Cohen kappa) for detection of HPV-16, HPV-18, and other hrHPV types was 0.91  (.81–1.00), 1.00 (1.00–1.00), and 0.75 (.62–.88), respectively. The performance to detect HPV-16, HPV-18, and other hrHPV positivity were not significantly different between the 2 tests  . In totaTo our knowledge, this is the first study conducted in self-collected anal samples that compared the Cobas 4800 HPV test (Roche) and the Anyplex II HPV28 Detection (Seegene) tests for hrHPV detection.+/GP6+ PCR testing followed by genotyping with Luminex [First, comparing the 2 methods on 97 samples revealed a good agreement between them and comparable worldwide ability to identify any of the 14 hrHPV genotypes currently recommended for cervical cancer screening. This result was also consistent for HPV-16 alone, HPV-18 alone, and the other hrHPV genotypes detected with the 2 methods. However, compared to the Seegene approach, the Cobas test missed more anal hrHPV infections. In term of hrHPV prevalence in normal cervical tissue, a difference was also identified between the Cobas method and GP5The prevalence of HPV-16 was the highest in our high-risk population, as expected, and the 2 methods showed high agreement and performance to detect the most oncogenic genotype in the anal site. This is of importance as HPV-16 testing could be one of the biomarkers used as anal cancer screening in key populations in the near future. For instance in France, new guidelines propose to use HPV-16 testing in anal cytology specimen collected by physicians as an anal cancer screening marker in MSM living with HIV . If posiSimilarly to self-collected vaginal swabs for cervical cancer screening , self-coIn conclusion, both the Anyplex II HPV28 Detection assay and the Cobas 4800 HPV test are suitable to detect the 14 hrHPV genotypes, in particular HPV-16, in self-collected anal samples.ofad540_Supplementary_DataClick here for additional data file.


The PCRs were highly reproducible, with inter- and intra-assay coefficient of variations ranging 2.0–6.3% for HPV16 and 4.9–11.6% for HPV18. Testing self-collected anal samples positive for HPV16 (n = 29) or HPV18 (n = 33) and those negative for both HPV types (n = 22) with the ddPCR assay and Seegene Anyplex II HPV28  showed high agreements between the two assays. In conclusion, HPV broad-spectrum primers containing mismatches at their target binding sites and a touchdown thermal cycling approach resulted in sensitive and specific amplification of HPV genome using ddPCR. The assay has potential clinical diagnostic applications for detection, quantitation, and monitoring of anal HPV infections.Broad-spectrum droplet digital polymerase chain reaction (ddPCR) assays can be used to quantitate multiple human papillomavirus (HPV) types. However, the presence of nucleotide mismatches at the primer binding sites may result in different optimal annealing temperatures and, consequently, under detection of some HPV types. Touchdown PCR uses decreasing annealing temperature over the first PCR cycles allowing for specific and efficient amplification of the target sequence. Here, as a proof-of-concept, we assessed if touchdown ddPCR (TD-ddPCR) using broad-spectrum oligonucleotides and genotype-specific probes targeting HPV L1 can be used to quantitate HPV. We tested our hypothesis using HPV positive cell lines and anal samples which potentially contain PCR inhibitors. Using serial dilutions of HPV16 and HPV18 DNA, the TD-ddPCR assays were performed with linearity across the dilution series (Pearson’s correlation The quantity of the human papillomavirus (HPV) is associated with disease outcome. We designed an accurate and precise digital PCR assay for quantitating HPV in anal samples, a sample type that is typically problematic due to the presence of PCR inhibitors. Human papillomavirus (HPV) infection may result in a variety of benign and aggressive neoplastic lesions on the skin or mucosa of anogenital tract and oropharynx –4. HPVHPV16 and HPV18 are associated with 84.3% of anal cancers . AccordiConsistent with other HPV-associated cancers , 16, sevAlthough there are many commercially available HPV detection kits, the majority are not quantitative assays . The cur7 HPV genomic equivalents per milliliter.SiHa ATCC HTB35)  and HeLa  and HeLEighty-four self-collected anal swab samples were from the National HPV Monitoring Program (IMPACT), an ongoing surveillance program for monitoring the impact of nationwide vaccination on the prevalence of HPV types in Australia’s population , 39. Sam7 cells each) were pelleted separately by centrifugation at 1,500 × g for 5 minutes at room temperature. Cell pellets were resuspended in 200 µL of phosphate-buffered saline (pH 7.4). Using the Roche MagNA Pure 96 DNA and Viral NA Small (or Large) Volume Kit , we extracted total NA from cell lines (200 µL) and anal samples (500 µL) (HeLa and SiHa cells (10(500 µL)  then elu(500 µL) . The purAnal swab samples were tested using Seegene Anyplex II HPV28  as described previously , 42–44Previously reported modified versions of GP5+/6+ (MGP) primers with a 10-base stretch at their 5' ends were used  (Table SEach ddPCR reaction contained 10 µL of 2× ddPCR Supermix for Probes (No dUTP) (BioRad), 500 nM each of the three primers , 250 nM probe, and 2–5 μL of DNA extracts in a final volume of 20 µL. Droplets were generated with a BioRad QX200 Droplet Generator following the manufacturer’s instructions. The standard two-step ddPCR and touchdown (decreasing 2°C per two cycles over five cycles) thermal cycles were used for assay optimization (Table S2). Reactions were analyzed using the QX100 Droplet Reader (BioRad). The HPV genome copy number was quantified using the QuantaSoft Software which included a Poisson distribution correction step . PositivTo assess the reproducibility of the TD-ddPCR assays, we estimated intra-assay coefficient of variation using triplicates of SiHa or HeLa cell extracts and both FAM- and HEX-labeled probes. Standard deviations and averages were calculated and used to estimate intra-assay coefficient of variations for triplicate samples of SiHa and HeLa cells. Likewise, to calculate the inter-assay coefficient of variation, we evaluated the average of the above three measures and the results of two additional TD-ddPCR reactions (performed on two different occasions) for both HPV16 and HPV18 (FAM or HEX).The sensitivity of each assay, minimum detectable HPV copies by the assays, was evaluated using quantitated WHO DNA standards of HPV16 and HPV18. Four 10-fold serial dilutions  of the DNA standards were prepared in Tris-EDTA buffered saline  and tested in duplicate using TD-ddPCR and both the FAM- and HEX-labeled probes. To measure the HPV genomic copy number per SiHa and HeLa cells, we used a combination of specific primers and FAM-labeled probes for human β-globin  and HPV t-test (Microsoft Excel) was used and P < 0.05 was deemed significant. GraphPad Prism version 9.1.1 for Windows  was used to analyze the ddPCR HPV quantitation results from Anyplex28 HPV positive clinical samples. Microsoft Excel was used for the calculation of Pearson’s correlation.For assay comparison, the overall agreements, negative and positive percent agreements , and Cohen’s kappa statistic were calculated , 48. IntAs mentioned above, the broad-spectrum primers used in this study contained several nucleotide differences with their target HPV sequences that could potentially affect their optimal annealing temperatures. Using SiHa and HeLa cell lines harboring HPV type 16 (1–2 copies per cell) or 18 (10–50 copies per cell) genomes, our initial attempts using the standard two-step thermal cycles (Table S2) failed to generate any positive droplets (data not shown). We then tested a three-stepwise (decreasing/increasing temperatures) cycling program toward a defined annealing temperature (Table S2). The annealing temperatures were based on that previously described  whereby P = 0.46) or HPV18 (P = 0.06).To show the reproducibility of the results, using SiHa and HeLa cells, we calculated intra- and inter-assay coefficients of variation for HPV16 and HPV18 using FAM- and HEX-labeled probes for each target . There wPrecision was assessed using 10-fold serial dilutions of WHO HPV16 and HPV18 international NA standards. TD-ddPCR detected down to one genome copy of HPV16 and HPV18 , althougTo assess the cross-reactivity of each assay, initially we used DNA extracts from SiHa and HeLa cell lines containing HPV16 or HPV18 genomes. As shown in n = 22), HPV16 positive (n = 29), and HPV18 positive (n = 33) with valid IC signals (++ and +++) were analyzed by ddPCR    . Of the ian, 83) . For HPVian, 83) , with anian, 23) . Two of ian, 23) .In this study, we developed a TD-ddPCR protocol using broad-spectrum HPV primers and genotype-specific probes for HPV types 16 and 18, which detected as few as one copy of HPV genome and did not cross-react with any of the 26 non-HPV16/18 types, indicating high precision and accuracy. Anyplex28 and TD-ddPCR produced 100% concordant results when 51 anal swab samples were tested for HPV16. A slightly lower agreement was observed for HPV18 (95% PPA and 91% NPA).Development of broad-spectrum PCR assays combined with genotype-specific probes can facilitate the development of multiplex HPV detection assays. However, genetic diversity across HPV types with less than 90% similarities  is a majThe TD-ddPCR assays described in this study detected three and eight HPV genomes per each SiHa and Hela cell, respectively, which slightly differed from the known figures , and this discrepancy could be due to inter-laboratory variation in sample preparation and the cells used in each laboratory. When tested against the quantitated WHO standards, the TD-ddPCR assays detected fewer HPV genome copies than expected; such discrepancies have been reported previously , 57–59The HPV18 TD-ddPCR did not detect HPV in three samples that were HPV18 positive by Seegene Anyplex28. Of these, two had 1+ Anyplex28 signal (Ct < 40) and one had 3+ signal. PCR amplification and Sanger sequencing of the entire L1 and L2 genes of the HPV18 sample with an Anyplex28 3+ signal showed a large deletion containing the MGP binding site (independent manuscript in preparation), but the other two samples were inadequate for PCR amplification and sequencing. The failure to detect the remaining two samples by ddPCR could be due to the presence of nucleotide mismatches at primer binding sites or lower viral titer; lower sensitivity of ddPCR relative to other detection assays has been reported previously for clinical samples .Study limitations include using only one type of sample, although pilot experiments showed that the TD-ddPCR can be used for the detection of HPV in cervical and formalin-fixed paraffin-embedded specimens (data not shown). The assays were developed for the detection of just two HPV types (16 and 18), one at the time, from limited numbers of clinical samples, although these are by far the most important HPV types. The described methodology can be applied for the detection of other HPV types, but further studies are required to select combinations of appropriate fluorophores for the development of multiplex assays for simultaneous detection and quantitation of HPV types.In conclusion, the TD-ddPCR assay is a highly sensitive method with no cross-reactivity with other genotypes for the detection and absolute quantification of HPV in anal swab samples. These assays provide a method for assessing and monitoring HPV viral load over the course of infection and treatment. Additionally, due to the use of broad-spectrum primers, this test forms a platform for a multiplex TD-ddPCR for the detection of several HPV types from one small sample in one reaction. Additionally, the touchdown approach described here can be applied to any ddPCR aimed at the detection of a heterogenetic population of genomes with slight sequence variations at primer binding sites.


Blue shark populations are currently declining, caused mainly by the impact of overfishing from industrial fishing vessels in the open oceans. However, there is a paucity of comprehensive biological and habitat data concerning the reproductive characteristics of blue shark species in the Indian Ocean. In response to the call made by the Indian Ocean Tuna Commission (IOTC) to provide valuable parameters for the conservation of blue shark resources, this study collected observer data from the Indian Ocean longline fishery targeting tuna between 2010 and 2020. Through rigorous statistical analysis, it was determined that the estimated length at sexual maturity for male blue sharks is 161.4 cm and 179.3 cm for females. Moreover, the average litter size was found to be 33.7 pups. The study suggests the existence of a possible seasonal migratory pattern for pregnant blue sharks, with the first and fourth quarters of the year being potential mating grounds in proximity to the equatorial region of the Indian Ocean. Additionally, the temperate marine areas of the Indian Ocean were identified as crucial habitats for parturition and juvenile development of blue shark species. Consequently, it is strongly recommended to implement more scientifically informed and ecologically sustainable operational strategies in these designated areas.p < 0.05). This study suggests that the area near the equator in the Indian Ocean from October to March of the following year may be the mating ground for blue sharks, while the temperate waters in the Indian Ocean are the nursery ground and parturition ground for pregnant and juvenile throughout the whole year. Therefore, it is recommended to adopt a more scientific and reasonable operational method in these areas.Due to the limited biological research on the blue shark in the Indian Ocean, such as the lack of a clear understanding of its reproductive biology and distribution, our study analyzed and evaluated the fork length distribution, sexual maturity length, reproductive capacity, and spatiotemporal distribution of blue sharks based on biological data and capture location information collected in the western Indian Ocean from 2010 to 2020. The objective of this study is to provide reliable biological information important in performing future stock assessments vital for species conservation in this region. A total of 791 male (33–249.5 cm FL) and 803 female (12–349.6 cm FL) blue sharks were collected in the West Indian Ocean. We used the morphology of the sexual organs to ascertain their sexual maturity. Results show that the observed size at 50% sexual maturity of male blue sharks in the West Indian Ocean was 161.4cm FL (192.4 cm TL) for males and 179.3 cm FL (213.9 cm TL) for females based on logistic curve analysis. The average litter size of pregnant blue sharks was 33.7 pups. There were significant differences in the distribution of blue shark individuals with different sexual maturity levels in different quarters ( Prionace glauca) belongs to the family Carcharhinidae of the order Carcharhiniformes. As viviparous fish, blue sharks have lifespans and reproductive patterns similar to those of mammals [The blue shark  of blue sharks has been observed across all major oceans . The wesThe reproductive biology of the blue shark in the Atlantic and Pacific oceans has been extensively studied ,12. HoweThe present study aimed to enhance our understanding of blue sharks’ reproductive biology in the Indian Ocean and to provide a basis for effectively managing and conserving this vital resource. Specifically, with the research on key reproductive indicators including length distribution, sex ratios, pregnancy rates, litter size, sexual maturity, and spatiotemporal distribution of blue sharks, this study provided basic biological data for the assessment of blue shark stock and the conservation and management of blue shark resources in the Indian Ocean. Due to limitations in manpower and resources, we were only able to collect biological samples of blue sharks in the western Indian Ocean for analysis, providing only partial details for the study of blue shark reproductive biology in the Indian Ocean. The data used in this study were collected by observers from the Chinese distant-water fishing scientific observation project aboard longline vessels targeting Indian Ocean tuna. The observers received training per the requirements of IOTC for data collection. The recorded data include operation location, number of hooks, hook time, bait usage, weather conditions, sea surface temperature and individual biological information of the caught specimens . The data used in this study were obtained from observers onboard Chinese distant-water tuna longline vessels from July 2010 to June 2020 in the western Indian Ocean, covering the geographical range, from 10° N to 39° S and from 23° E to 90° E . The obsTo some extent, the CPUE reflects the abundance of the blue shark population in a research area. The higher the CPUE value, the more blue sharks there are in that area. The CPUE of the blue shark catch location data was divided into 2.5° × 2.5° grids. The CPUE of blue sharks was calculated and plotted using the following formula based on data from catches, distribution, hook numbers, and situations:t-test (two-tailed independent samples t-test) was used to examine whether the mean fork length of male and female samples followed a normal distribution and to see whether there were significant differences in the fork length distribution between male and female blue shark samples.With a grouping interval of 20 cm, a frequency distribution graph of fork length for male and female blue sharks was plotted. The two-sample Kolmogorov-Smirnov (K-S) test for goodness of fit was used to examine whether there were significant differences in the fork length distribution between male and female blue shark samples. The The sex and sexual maturity of the blue sharks were determined by dissecting their reproductive systems. For male blue sharks, sexual maturity was divided into three stages ,21: 1. JFor female blue sharks, sexual maturity was divided into five stages ,22: 1. JThe maturity stage of each sample was converted to a binomial distribution value  for statistical analysis. The ratio of mature individuals in each clasper length group was calculated for both male and female blue sharks using 20 cm intervals. A logistic model was used to fit the relationship between the mean values of each clasper length group and the ratio of sexually mature individuals, and the length at which 50% of blue sharks reached sexual maturity was calculated .P = 1/, and no significant difference in the distribution between the two samples (D = 0.048 < 0.05) was observed. In the 180–200 cm FL group, the number of females is significantly higher than that of males, while in the 120–140 cm FL, 160–180 cm FL, and 200–240 cm FL groups, the number of males and females is almost the same. The group with the highest number of male samples is 200–220 cm FL, accounting for 26%; the group with the highest number of female samples is 180–200 cm FL, accounting for 20%.This study collected a total of 1594 blue sharks, including 813 male samples, accounting for 51%, with an average fork length of 192.8 cm, and 781 female samples, accounting for 49%, with an average fork length of 194.3 cm. The length frequency distribution analysis of male and female samples is indicated in The pattern of changes in the sexual maturity rate of male and female blue sharks with body length can be well-fitted using a logistic model . The logα = 0.044 and β = −7.1069.The parameters are The logistic equation for the sex maturity rate of female blue sharks in relation to the median fork length of each length group:α = 0.033 and β = −5.990. When P is equal to 0.5, we can take this into the equation and conclude that the sizes at 50% maturity of males and females were 161.4 cm fork length (FL) and 179.3 cm FL, respectively. Within the body length range of 200–240 cm FL, there was still a certain proportion of females that had not reached sexual maturity, while most males reached sexual maturity in this length range. The length at which females reached sexual maturity was greater than that of males.The parameters are Analysis of the relationship between fork length and clasper length in male blue sharks  shows tht-test analysis showed that the average liver weight of female blue sharks was larger than that of males. The exponential model was used to fit the relationship between body length and liver weight for male and female blue sharks, and the results showed .The range of length at sexual maturity of female samples of the blue shark was 100–287 cm. The width of the oviducal gland of female specimens changed with the reproductive cycle. It increased during ovulation and gestation periods, and 95% of sexual maturity females have an oviducal gland width greater than 2.5 cm. The number of embryos carried by pregnant blue sharks ranged from 20 to 54 , with anThe overall sex ratio of blue shark samples was close to 1:1. The monthly distribution of the sexual maturity ratio of male and female blue shark samples was similar , with a This study divided the overall samples of the blue sharks into four groups for comparative analysis : juvenilWhen analyzing the location and hooking situation of juvenile blue sharks below 125 cm FL captured in each season, it was found that between April and September, the frequency of occurrence was higher in the temperate waters between 25° S and 40° S in the western Indian Ocean. However, in the other quarters, juvenile blue sharks below 125 cm FL were not captured in large numbers in the waters north above 25° S .By analyzing the distribution of immature individuals of both sexes greater than 125 cm FL in each season, it was found that the distribution pattern was the same throughout the year. In the first quarter, they were mainly concentrated in the waters near the equator, then left the area between April and June, and appeared more frequently in the temperate waters between July and September. Between October and December, they were mainly concentrated along the coast of Tanzania and the eastern coast of Madagascar .When analyzing the location and total number of hooks of captured matured females and males in each season, it was found that the activity ranges of matured males and females highly overlapped from October to March of the following year, but not in the second and third quarters. From July to September, matured females migrated southward to temperate waters, while their numbers near the equator were relatively low, during which time the number of males near the equator predominated. From October to December, sexually mature female individuals were concentrated in waters south of the equator. It is worth noting that between July and September, a certain number of matured females appeared in the southwest coastal waters of South Africa, while matured males did not appear in this area but were distributed in offshore waters .When analyzing the captured pregnant female blue sharks in each season and the total number of hooks, it was found that between January and March, pregnant females were mainly distributed in the waters near the equator, while no pregnant females were captured in this area between April and June. From October to December, the frequency of pregnant blue sharks near the equator was low, while their frequency was high in the waters near 30° S in the temperate zone. From October to December, pregnant blue sharks were mainly distributed in the coastal waters of Tanzania and the east coast of Madagascar, and no pregnant females were found in the waters near the equator .At present, the method of determining the sexual maturity of male and female blue sharks through the development of their reproductive organs has been widely applied. Previous studies mainly relied on measuring the length and calcification level of the claspers, the development of testes, and the presence of sperm in the testes to determine the sexual maturity of male individuals; while the development of the uterus and ovaries were used to determine the sexual maturity of female individuals ,20,23,26This study converted the preliminary 50% sexual maturity fork length for males (161.4 cm FL) and females 179.3 cm FL) of the blue shark into 50% sexual maturity total length  using the relationship equation between fork length and total length (FL = 1.73872 + 0.82995 TL) . The res9.3 cm FLThis study obtained an average litter size of 33.7 pups. As of now, data on the reproductive capacity of the blue shark is only available for the Atlantic and Pacific oceans, with an average litter size of 30 pups . This study found that the relationship between male length and liver weight fits an exponential model, while the relationship between female length and liver weight does not fit the exponential model well. Typically, the energy required for embryonic development during early pregnancy is relatively low, while a significant amount of energy is needed for embryonic development during the juvenile stage, primarily derived from the liver ,24. AddiThis study suggests that juvenile blue sharks below 125 cm FL are usually located in temperate waters between 35° S and 40° S. Pregnant matured females, and immature juveniles over 125 cm FL have the same distribution pattern in each season, concentrating near the equator between January and March, migrating south to temperate waters between April and September, and migrating north to coastal waters of Tanzania and Madagascar from October to December. Earlier research has indicated that while blue sharks residing in the Indian Ocean exhibit variation in their body sizes and sex ratios, they still fall under the same stock and do not experience seasonal reproductive isolation ,34. LargThe migration of blue sharks is mainly accomplished through ocean current systems ,41. TaggCarrera-Fernández  found thThis study observed that the distribution of matured male and female blue sharks overlapped highly in the first and fourth quarters, and was concentrated in certain areas. There may be two mating areas for blue sharks in the western Indian Ocean. The first being near the equator in the first quarter, and the second in the coastal waters of Tanzania and eastern Madagascar in the second quarter. Pregnant blue sharks captured in the western Indian Ocean are most abundant from September to March of the following year, with the highest proportion from January to March. Although the total number of recorded catches was lower from January to March, more pregnant blue sharks were captured during this period. Pregnant blue sharks mainly concentrated in the area between 40° E and 60° E and between 5° N and 10° S in the first quarter. The frequency of pregnant individuals in the sample was lowest from December to January . PregnanThis study found that the proportion of immature individuals was higher in the temperate waters of the southern Indian Ocean, indicating that the activity range of male and juvenile blue sharks is usually closer to the southern latitudes, which is consistent with the results of studies by Coelho and Vandeperre ,35. ThisMany studies have suggested that blue sharks have a seasonal reproduction scheme, with an embryo development period of 9–12 months, and pregnant females usually give birth in spring or summer ,19,20,23Currently, little is known about the bycatch of sharks, and the data sources for assessment models are limited, resulting in a limited understanding of the population status of sharks. IOTC uses a stock synthesis (SS) model to assess the status of blue shark resources in the Indian Ocean . Age, grThis study observed that the average litter size of pregnant blue sharks was 33.7 pups, and its fecundity was significantly higher than that of other pelagic sharks, which may be the reason why its population still did not decline significantly under the influence of bycatch. This study suggests that the area near the equator of the Indian Ocean from October to March of next year may be the mating area of blue sharks, while the temperate waters of the Indian Ocean are the area where blue sharks spawn and grow. Therefore, it is suggested that more scientific and reasonable management methods should be adopted when fishing activities are carried out in these waters to reduce the bycatch of blue sharks and ensure the survival of the released sharks.