Falcaria vulgaris (FV) is a medicinal plant rich in antioxidants. This research aimed to assess the FV hydroalcoholic extract effects on kidney toxicity induced by Merc.Mercuric chloride , and 150 mg/kg FV mitigated these outcomes . Urea, creatinine, nitric oxide, and the level of apoptosis revealed a significant increment in the kidney of the rats that received Merc , and 150 mg/kg FV decreased these results. Furthermore, FV ameliorated histological changes induced by Merc . The FV hydroalcoholic extract protects the kidneys against Merc-induced nephrotoxicity. Antioxidant and anti-apoptotic FV hydroalcoholic extract properties were involved in this healing effect. It causes damage to the brain, liver, respiratory system, skin, eyes, nervous system, and kidneys . It is believed that Merc toxicity in various tissues is an outcome of reactive oxygen species (ROS) making, resulting in the promotion of inflammatory cytokines and production and increase in nitric oxide (NO) . The kidneys are one of the tissues which can damage by Merc poisoning via ROS production and induction of oxidative stress . Although there is no efficient method to eschew the toxins' entry into the body, today, natural compounds have enticed the consideration of therapists and researchers to help suffering persons. Previous investigations illustrated the positive role of plant antioxidants on the renal injury caused by methotrexate, nicotine, and Merc-induced damage .Mercury  due to industrial pollution and its deposition in ecosystems is ubiquitous in the environment and has serious risks to human health. Mercuric chloride  is a medicinal plant is a member of the Apiaceae (Umbelliferae) family, growing in the west and southwest of Iran as an annual wild plant , purity≥99.5% was purchased from Sigma-Aldrich . Xylazine was obtained from Alfasan co (Netherlands). Kits from Pars Azmoon, Iran were used to evaluating the serum creatinine and blood urea nitrogen levels. NO level was measured by Griess Reagent System, Promega (Griess reagent). Superoxide Dismutase (SOD) activity in the renal tissue was assayed via Zell Bio kit (GmbH) and spectrophotometric devices. A kit (Cat No: TAC-96A) ZellBioGmbH-Germany was used to estimate the total antioxidant capacity (TAC). Cell death detection was evaluated via ELISA-kit .Merc . After removing the impurities, the leaves and stems of the plant were placed in the shade for five days to dry and then powdered. One hundred grams of this powder was combined with 70% ethanol (1:4 ratios) and kept in a warm water bath at 35°C for 48 hr. Afterward, it was stored in dark conditions. Next, we used Buchner funnels filter paper, a vacuum pump, and a rotary device to obtain the concentrated extract. Finally, the extract was dissolved in distilled water.Animals and experimental protocolForty-eight male Wistar rats (220-250 g) were bought from the Razi Institute of Iran. One week before the start of the experiment, standard cages were used for animal care at 22±2°C temperature and 12-hour light-dark periods. Rats had free access to water and food. Tests were done according to the Helsinki guidelines as well as the Ethics Committee of Kermanshah University of Medical Sciences (IR.KUMS.REC.1398.088).We randomly divided the 48 rats into eight groups : Group 1: Control, each animal in this group received 0.9% saline intraperitoneally (I.P), once a day for 14 days; Group 2: Merc, the rats were exposed to a dose of Merc (0.5 mL/day of 0.5 ppm aqueous) dissolved in drinking water once a day for 14 days ; Groups 3-5: FV1, 2, and 3; rats were injected I.P with FV at doses of 50, 100, and 150 mg/kg, respectively once a day for 14 days; and Groups 6-8: Merc+FV1, 2, and 3; the animals received Merc (0.5 ml/day of 0.5 ppm aqueous)  and FV at doses of 50, 100, and 150 mg/kg once a day for 14 days (I.P). The doses of Merc and FV were selected based on preliminary studies . After the last treatment, animals were anesthetized by one I.P injection of ketamine and xylazine at the doses of 100 mg/kg and 10 mg/kg, respectively. Then the kidneys were removed and weighed by digital scales. In the next step, kidney tissue was prepared for macroscopic studies and nitric oxide (NO), SOD, TAC, and apoptosis evaluations. The blood was taken from the hearts and gradually added to the test tubes until the clot formation was maintained at lab temperature. Then serum was separated by centrifugation (1000 g) for 15 min. Blood serums were collected by centrifuging the samples separately and stored at −20°C until blood serum urea nitrogen and creatinine analysis.Kidney index evaluationThe Renal index (mg/g) was estimated using this formula : Kidney index = Kidney weight (mg)/Body weight (g).Histopathological assessmentsThe renal tissue was stabilized in buffered formalin (10%) for 24 hr. Afterward, a longitudinal incision was used to split each kidney into two sections from the middle. Paraffinized tissue blocks were provided for partitioning at 5-7 μm thickness. 5 microscopic sections of each sample were randomly selected and followed by hematoxylin-eosin (H&E) staining. The slices were studied by a histologist and pathologist using light microscopy . For estimation of the diameter of renal corpuscles five fields  of 400× and 100× magnifications were captured by a camera connected to the microscope. The field's selection was done via a zigzag form monitoring the sections to achieve a randomized inclusion. Then, at least 150 captured pictures (400×) of the round or nearly round renal corpuscles by a blind observer were selected, and a specialized software package  was used to estimate this parameter. Evaluation of serum urea nitrogen and creatinine .Serum urea concentration was determined using a commercial kit , according to the manufacturer's instructions. Serum creatinine was measured by Jaffe's method, using a commercial Kit NO level of the kidney tissue measurementRenal tissue NO was assessed by Griess reaction using zinc sulfate powder for deleting proteins from tissue samples. For this aim, 6 mg of zinc sulfate powder was blended with 400 ml of micro tubes of samples. Then, they were homogenized by a vortex for one minute. Afterward, the samples were centrifuged for 10 min at 12,000 revolutions per minute (rpm) at 4 °C. In the next step, an upper solution (supernatant) was utilized to evaluate NO (NOx). In summary, a 50 ml sample was added with 100 ml of Promega (Griess reagent) and the reaction mixture was incubated for about 30 min at room temperature, being preserved from light. The optical density was estimated at 450 nm in a microplate reader in accordance with the reagent manufacturer's instructions.Evaluation of the SOD activity The reaction between 3--2,5 diphenyltetrazolium bromide (MTT) and anion superoxide  is blocked via the SOD enzyme. In the following, the other steps were done base on the manufacturers' instructions. ELISA at 570 nm was utilized to read the light absorption of this compound. The percentage of inhibition caused by the SOD enzyme was obtained using the kit manufacturer's instructions.Estimation of the kidney tissue TAC levelFor measuring the renal tissue TAC, a ZellBioGmbH-Germany kit (Cat No: TAC-96A) was used. The kit contents include a reagent ready to use, dye powder, buffer X 100, reaction suspension solution, standard, and a microplate of 96 wells. The TAC level was assayed by comparing the amount of antioxidants present in one gram of homogenized renal tissue and ascorbic acid was used as the standard (The kit's sensitivity = 0.1 mM). In the end, absorbance was determined using a microplate reader  at 490 nm.The apoptosis levelApoptosis level in the renal tissues was assayed by a cell death detection ELISA kit . Briefly, 1 mg of the kidney tissue was mixed with 100 µl of lysis buffer and vortexed. The specimens were centrifuged at 1000 g for 10 min and washed with phosphate buffered saline (PBS) in triplicate. The cells suspended in 20 µl of PBS were moved to a 96-well plate filled with 80 µl of immunoreagent. The wells were lidded by a foil for 2 hr at 20°C under vibration (300 rpm). After washing the samples, the absorbance of the samples at a wavelength of 490 nm was read.Statistical analysis Statistical analysis was done by SPSS (version 16), and the comparisons were performed using one-way ANOVA followed by Tukey's post hoc analysis. The outcomes are demonstrated as mean±SEM (standard error of means). A p<0.05 was assumed statistically significant.Effects of FV and Merc on the kidney index of ratsEvaluation of kidney index displayed a considerable decrement in the Merc (p<0.01) and Merc + FV1 (p<0.05) groups in comparison with the control group. This parameter showed no significant difference for intra-group comparison of the FV1, FV2, and FV3 groups . Relative to the Merc group, the kidney index revealed a significant increment in the FV1, FV2, FV3, Merc + FV2, and Merc + FV3 groups . Based on Merc + FV intra-groups comparison, the kidney index in the Merc + FV1 group was significantly lower than in the Merc + FV3 group (p<0.05) .Effects of FV and Merc on the kidney histology As offered in Effects of FV and Merc on urea and creatinineserum levels There was a significant increase in urea serum level  and creaEffects of FV and Merc on NO level, SOD activity, and TAC level in renal tissue of ratsThere was a significant increase in renal tissue NO levels in the Merc (p<0.01), Merc + FV1 (p<0.05), and 2 (p<0.05) groups compared to the control group. The alterations of this parameter in the intra-group comparison of FV alone recipients were not statistically significant . FV administration in the FV1, FV2, FV3, Merc + FV2, and Merc + FV3 groups significantly decreased renal tissue NO levels than the Merc group . In the Merc + FV groups intra-group comparison, this parameter in the Merc + FV1 group was significantly higher relative to the Merc + FV3 group (p<0.05) . The renCompared to the Merc group, there were remarkable increases in the renal SOD and TAC levels in the FV 1,2,3 and Merc + FV3 groups. Also, renal SOD levels showed a considerable enhancement in the Merc + FV2 group in comparison with the Merc group (p<0.05). In the Merc + FV intra-group comparison, the renal SOD and TAC in the Merc + FV1 group were remarkably lower than in the Merc + FV3 group  .Effects of FV and Merc on the number of apoptotic cellsAccording to Merc poisoning is one of the factors that increase ROS production in cells. It induces organ injury by incrementing the free radicals' production and oxidative stress . Since oxidative stress is involved in Merc poisoning, it has been suggested that antioxidants could help in its treatment. Therefore, researchers have focused on finding natural antioxidants of plant origin. FV is a medical plant with antioxidant activity . Thus, in this attempt, we studied the effect of FV hydroalcoholic extract on Merc-induced toxicity in renal tissue of male rats. Generally, our results demonstrated that FV administration protected the studied rats against Merc-induced nephrotoxicity by enhancing antioxidant defense status.In this study, the kidney index decrement in the Merc alone receiving group was seen to occur due to their weight loss which is described via kidney parenchyma atrophy and decline of the renal corpuscle size as a result of apoptosis induction. Similar to our results, Abdel-Moneim and colleagues have reported a reduced kidney index of rats following lead acetate administration . On the other hand, the FV extract effectively restored kidney and body weight loss in rats receiving Merc. After administrating FV in Merc-induced groups, the diameter of renal corpuscles elevated. In line with our results, a study revealed that the renal corpuscles diameter decreased in Merc-induced mice . The histological estimation affirmed the ameliorative efficacy of FV on Merc-induced nephrotoxicity. It seemed that Merc via oxidative stress induction caused renal tissue injury. Other investigations have reported Merc-induced histological damages . Merc via enhancement of ROS production could activate nuclear factor kappa B and lead to the over secretion of proinflammatory cytokines. It seemed that inflammation was an important mechanism involved in Merc-induced nephrotoxicity. Previous researchers reported an increment in the concentration of inflammation factors in animal renal tissue after Merc treatment .Outcomes of the current study show that Merc-induced kidney injury is evidenced by elevation in the serum urea and creatinine, as revealed in another research . The elevated value of urea and creatinine is due to oxidative stress-induced cellular injury and reduced glomerular filtration rate which shows the loss of kidney function . The free radicals produced by oxidative stress change the enzymatic activity via assaulting biological molecules like DNA, proteins, lipids, and proteins, which finally cause cell injury or death . In contrast, FV administration significantly restored the changed levels of urea and creatinine that might be due to the repair of tissue injury mediated by its antioxidants' effects. It seems that polyphenol hydroxyl groups existing in FV arrested the free radicals . In conformance to these results, previous research showed that FV hydroalcoholic extract treatment reduced urea and creatinine levels and improved kidney tissue and kidney function mediated by reducing renal biochemical markers levels . Based on our results, the FV hydroalcoholic extract effectively reduced renal tissue NO level and increased tissue levels of SOD and TAC in rats receiving Merc. Mitochondrial dysfunction in most body tissues increases the production of free radicals such as NO and ultimately induces tissue damage . NO has a role in acute kidney failure because its free-radical nature might participate in tubular harm . As reported previously, Merc intoxication enhances the creation of pro-inflammatory factors and NO in mice . Also, the findings of Jalili and colleagues confirmed that Merc treatment increased NO levels in renal tissue of mice while reducing the production of TAC and SOD activity in this tissue, which is consistent with the results of the present study . According to our results, FV can reduce the levels of free radicals causing reduced levels of NO and enhancing the levels of antioxidant activity. FV appears to exert protective effects against Merc-induced nephrotoxicity via enhancing the kidney antioxidant capacity and inhibiting oxidative stress in this tissue. In line with these results, other studies have shown the antioxidant properties of FV .Our findings revealed the increment in the renal tissue level of apoptosis following Merc administration which is consistent with previous reports . Based on the current study, the administration of FV in Merc receiving groups significantly decreased the level of apoptotic cells. It indicated the role of FV in apoptosis inhibition. In line with our results, Hosseini and colleagues have reported that FV has significant cytotoxic effects on SW-872 cells via apoptosis induction . Based on the results of the present study, there was no significant difference between the FV alone receiving groups for any investigated parameters. Moreover, there were remarkable differences between the groups that received Merc + FV at 50 mg/kg and Merc + FV at 150 mg/kg for all the investigated parameters. The data showed there are no differences between groups of Merc + FV at 50 mg/kg and 100 mg/kg. Therefore, we suggested that a higher dose of FV (150 mg/kg) was more impressive effects on the Merc nephrotoxicity.According to the outcomes of this study, Merc triggers oxidative stress and the production of free radicals in kidney tissue. In contrast, the FV plant's hydroalcoholic extract reduced Merc poisoning's effects on kidney tissue. This reduction in toxicity is justifiable due to the saponin, flavonoid, and antioxidant compounds in FV extract that decline the NO level, increase the SOD activity, and down-regulation of apoptosis. Thus, our results help to understand how environmental doses of Merc interact in their biological effects on kidney tissue. Research on the effective mechanism for applying this protective effect can help develop appropriate treatments with the minimum side effects for humans and animals exposed to heavy metals.The authors have declared that there is no conflict of interest.


Salvelinus namaycush). Because gills are responsible for metabolic gas exchange, are crucial sites of acid–base homeostasis and respond to stressors quickly, we hypothesized that the relative mRNA abundance of genes related to these three physiological processes would be altered after angling. We took gill samples of live lake trout at 0, 2 or 48 h after fish were angled by rod and reel, and then used quantitative PCR (qPCR) to measure the relative abundance of nine candidate mRNA transcripts. Heat shock protein 70 (hsp70) mRNA levels significantly increased over 5-fold 2 h after angling, indicating a potential activation of a cytoprotective response. However, contrary to our hypothesis, we observed no change in the relative mRNA abundance of genes related to metabolism or acid–base regulation in response to C&R angling within a 48-h period. As C&R angling can negatively impact fish populations, further use of transcript-level studies will allow us to understand the impact C&R has on specific tissues and improve our knowledge of how C&R influences overall fish health.Catch-and-release (C&R) angling is a conservation-oriented practice intended to reduce the impact recreational angling has on fish populations. Even though most recreationally angled fish are released, little is known about how C&R angling impacts fish at the cellular or tissue level. As the first to explore the impacts of C&R angling on mRNA abundances, our study aimed to identify how the stress of angling influenced metabolism, acid–base regulation and cellular stress in the gills of lake trout ( In some instances, recreational anglers can negatively influence wild fish populations by harvesting their catch . As overSome fish die as a result of C&R, but there are also examples of recreational fisheries where mortality is relatively low, especially when anglers use good handling practices . HoweverChanges in gene expression indicate how cellular processes of a specific tissue are regulated in response to a stressor. In the event of a stressor, select genes are differentially expressed allowing for a coordinated cellular response and return to homeostasis e.g. . As an iGills are the major site of metabolic gas exchange in teleost fishes  and are Salvelinus namaycush). We collected gill samples of wild-caught lake trout 0, 2 or 48 h after they were angled by rod and reel. We then used quantitative PCR (qPCR) to measure the relative mRNA abundance of nine candidate genes associated with metabolism, acid–base regulation and the cellular stress response. We hypothesized that transcript levels of genes associated with these three key cellular processes of gill tissue would be altered after C&R angling. Understanding the impacts of C&R angling on gill tissue will greatly improve our knowledge of how C&R angling impacts fish gills at the molecular level as well as overall fish health.As the first to use a transcript-level approach to study the effects of C&R angling, we aimed to identify how the stress of angling impacts three key cellular processes in the gills of lake trout  and a Manitoban Provincial Scientific Collection Permit (#22758865). Fieldwork was completed on Treaty 5 Territory, within the homeland of the Swampy Cree of the Opaskwayak Cree Nation.Salvelinus namaycush; derived from the Cree word nemekos, meaning ‘dweller of the deep’) from boat by rod and reel on Clearwater Lake, Manitoba  between June 23 and July 18, 2022. We used 2.1 m medium-heavy spinning rods spooled with 9.1 kg line. We vertically jigged barbless lures/baits at or near the bottom of the lake (depth: 22.9 ± 7.6 m (mean ± SD)) where the mean water temperature and dissolved oxygen was 7.3 ± 1.2°C and 8.9 ± 1.8 mg/L, respectively . Specifically, we used a jigging spoon as an artificial lure or two baits. Both baits consisted of cut-up longnose sucker (Catostomus catostomus) presented either on a 10.2-cm tube jig or on a 2/0 treble hook. When we hooked-up to a fish, we recorded fight time (the time elapsed from hook-up to when we netted the fish). After landing the fish, we recorded the fork length (± 1 mm), total length (± 1 mm), weight , hooking location , and level of bleeding at the hook site . We also recorded air exposure time as the time elapsed while we were sampling the fish. Before fish were released, we attached a coloured T-bar anchor tag to the left side of the dorsal fin to ensure we only sampled each fish once.We angled lake trout . We kept samples at 4°C for 24 h, and then froze them at −20°C until we could store them long term at −80°C. To determine if gill mRNA levels of various genes change after angling, we sampled each fish at one of three sampling periods, 0, 2 or 48 h after capture. Fish at 0 h (n = 8)  were sampled immediately after capture, before the transcripts we were interested in have been shown to change in abundance  following manufacturer's instructions. We determined the quantity, quality and integrity of RNA using a NanoDrop One  and by visualization on a 1% w/v agarose gel. We converted 500 ng of total RNA into cDNA using High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) as per manufacturer's protocols. We determined the relative mRNA abundances of nine candidate genes using qPCR. We chose these targets based on their rapid induction in response to stressors similar to C&R angling e.g.  for eachT method . SamplinWe determined differences in the relative mRNA abundances of target genes among sampling periods using a one-way analysis of variance (ANOVA), followed by a Tukey's honestly significant difference (HSD) test. The assumptions of equal variance and normality were tested for each target using Levene's and Shapiro–Wilk's tests, respectively. We performed all statistical tests using R version 4.0.2  with sigheat shock protein 70 mRNA relative transcript abundance . We observed that hsp70 levels significantly increased by approximately 5-fold 2 h after angling and then returned to near 0 h levels 48 h after angling  or heat shock protein 11b  after angling , citrate synthase  and cytochrome c oxidase 5b  after angling , carbonic anhydrase  and sodium-hydrogen exchanger 1  did not change in response to angling . We analysed mRNA levels in gill samples taken from 24 fish. The mean fight time was 32 ± 12 s (range: 13 to 60 s) and the mean air exposure time was 125 ± 57 s (range: 45 to 258 s). Angling significantly altered  angling . We obse angling . There w angling . Similar angling .hsp90aa1 and hsp11b, hsp70 mRNA levels significantly increased by over 5-fold 2 h after C&R angling, indicating a potential activation of cytoprotective responses in lake trout gills.We examined the consequences of C&R angling on molecular markers in the gills of lake trout. Overall, contrary to our hypothesis, there was limited evidence for a meaningful impact of C&R angling on the molecular factors we quantified. We observed no change in the relative transcript abundances of metabolic or acid–base regulatory mRNAs, suggesting that C&R angling has little impact on these cellular processes in lake trout gills, when fight times are less than 60 s and air exposure times less than 258 s. While we observed no change in hsp mRNAs showed some activation of a cellular stress response. Heat shock proteins are ubiquitously expressed molecular chaperones, playing roles in protein transport, folding, assembly and degradation  (Albula spp.) (The recovery of la spp.) , also suhsp70 mRNA levels responded to C&R angling. As a chaperone, HSP70 targets include nascent polypeptide chains as well as degraded or altered proteins  and brook trout   (Larimichthys crocea) . Whereas it is well known that C&R induces metabolic acidosis in salmonids and other fish because of increased anaerobic production of lactate e.g. , less knxcretion . Additiohsp70 in response to C&R angling reflected activation of the cellular stress response.Analysis of fish blood, the most common physiological tool used to assess impacts of C&R angling . A metabFuture C&R angling transcript-level studies should examine mRNA levels in red blood cells, use a larger sample size and sample at more timepoints. Red blood cells, like gill tissue, can be obtained non-lethally (hsp70 mRNA levels. Thus, using hsp70 transcript levels, other studies could now compare the impacts C&R angling has on cellular stress between species of fish or different angling techniques. Conversely, we found no impact on metabolic or acid–base regulatory mRNA levels. Future research might be able to identify which mRNA markers change in response to these processes or find that they do change when studying a different fish species or when using different angling techniques. Further incorporation of transcript-level approaches that provide insight into how specific fish tissues are affected by angling will yield mechanistic insight needed to develop best practices for C&R.Currently, the biggest challenge associated with using a transcript-level approach in C&R science is a lack of directly comparable literature. Although many studies use stressors similar in nature to C&R, they subject fish to stressors of much higher duration or strength than angling does. Thus, currently, we can only conclude that C&R has less of an impact on metabolic or acid–base regulatory mRNA levels than does a 10-min hypoxia (


Papillomaviridae family account for 27.9–30% of all infectious agents associated with human cancer. The aim of our study was to investigate the presence of high-risk HPV (human papilloma virus) genotypes in patients with periodontitis and a pronounced clinical picture. To achieve this goal, after proving the bacterial etiology of periodontitis, the samples positive for bacteria were examined for the presence of HPV. The genotype of HPV is also determined in samples with the presence of the virus proven by PCR (polymerase chain reaction). All positive tests for bacteria associated with the development of periodontitis indicated the presence of HPV. There was a statistically significant difference in HPV positive results between the periodontitis positive target group and the control group. The higher presence of high-risk HPV genotypes in the target group, which was also positive for the presence of periodontitis-causing bacteria, has been proven. A statistically significant relationship was established between the presence of periodontitis-causing bacteria and high-risk strains of HPV. The most common HPV genotype that tests positive for bacteria associated with the development of periodontitis is HPV58.Members of the  Papillomaviridae family make up 27.9–30% of all infectious agents associated with human cancer [Representatives of the n cancer . Althougn cancer ,5. In adn cancer . There in cancer ,9.It is estimated that about 16% of the world’s population is infected with HPV. According to a study by K.J. Syrjanen, nearly 80% of women are at risk for HPV infection. This is not the case with regard to affecting the oral epithelium; for example, in the United States, it has been found that 7% of people aged 14–69 years are infected with HPV, and the infection in men is almost three times more common . The majOncogenic serotypes of human papillomaviruses are used as a tumor marker associated with cervical cancer due to their safe participation in the etiology of this cancer, and in many cases, they are responsible for the development of neoplastic lesions of the penis, tongue, and more rarely of mucosal lesions with other localization. An Italian team of authors conducted a study that reports that women (20.4%) with a diagnosis of cervical-HPV and their male partners (30.7%) are at high risk for subclinical oral HPV infection .The presence of HPV in the oral cavity has been associated with various lesions, but the presence of viruses has also been found without clinical manifestation . The linTwo hundred and thirty samples were used from patients with clinical manifestations of gingivitis and periodontitis without a pronounced clinical picture of oropharyngeal lesions in the oral cavity (target group). The age of the target group patients ranges from 25 to 59 years. All of them required periodontal procedures and placement of implants. There is no evidence in the target group of the presence of immunosuppressed individuals. Samples for the presence of bacteria causing periodontitis were taken only from the gingival margin due to the specificity of the disease. Samples were taken using 0.5 mm dental paper pins using 5 pins per patient. For a control group, 460 buccal mucosa and coronal margin samples from 230 people without clinical signs of periodontitis were used. The representatives of the control group were of a similar age: from 30 to 60 years old. They were screened for high-risk HPV. We used sterile brushes  for the buccal mucosa samples and thin, metallic, sterile swabs  for those from the gingival margin. Various primer systems were used in the present work to detect genomic fragments of HPV. Consensus primers MY09/11 cover a region of the DNA of the virus with a size of 450 nucleotides (nt) encoding the L2 and L1 proteins and the triple primers GPE—the genes encoding proteins E6 and E7  of diffeAn ISOLATE II DNA Mini kit with a silica membrane was used to isolate the DNA . The isolation protocol was according to the manufacturer’s instructions.®plus11 kit . Reactions were performed according to the manufacturer’s instructions using a FluoroCycler® 12 PCR apparatus , and a TwinCubator  was used to perform the hybridization. The pathogenic bacteria associated with periodontitis were detected by micro-IDentAn Amplisens kit  was used to detect HPV types 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, and 66. The protocols were according to the manufacturer’s instructions. GenBank—National Center for Biotechnology Information, USA) were used to perform the reactions. Minor changes were made to the reaction protocols according to the melting temperature of the primers. To determine the temperature range of the primers, a gradient PCR was performed at the following temperatures: 51.0, 52.9, 54.4, 56.2, 58.4, and 60.3 °C. MayTaq HS mix  was used for PCR.The commercial in vitro diagnostic kits and primers described in Primers PC04 and GH20 for the beta globin molecule gene  forming a fragment with a size of 268 base pairs (bp) were used as internal control of the system. Consensus primers MY09-MY11 (10 pmol/µL) adopted as the gold standard were used to detect HPV, yielding a 450 bp fragment .Consensus primers covering E6/E7 genes were also used to detect HPV: GP-E6-3F, GP-E6-5B, and GP-E6-6B primers yielding a 650–700 bp fragment depending on of the genotype.The parameters of the reactions for proving HPV were as follows: initial denaturation—3 min, 40 cycles: denaturation 95 °C—45 s, connection 55 °C—45 s, extension 72 °C—45 s, final extension 72 °C—7 min, and storage 4 °C. Gel electrophoresis was used for qualitative analysis of the extracted DNA and PCR products. Gel electrophoresis was performed with 2% agarose , 10 ng/mL of ethidium bromide , 1 × TAE buffer, 100 bp DNA Ladder , and 1 kb DNA ladder  at 120–150 V and 70–120 mA for 30 min. For statistical processing of the results, chi-square test and McNemar’s test were used to determine the level of significance.DNA obtained from all tested samples had a purity greater than 1000 and an appropriate concentration greater than 20 ng/µL. Evidence of pathogenic bacteria associated with the development of periodontitis.Pm (Peptostreptococcus micros), Fn (Fusobacterium nucleatum/periodo) En (Eubacterium nodatum), and Cr (Campylobacter rectus) was recorded. A total of 230 samples from the coronary margin, isolated from the control group, were examined for the presence of the relevant bacterial pathogens. Samples for the presence of bacteria were taken only from the gingival margin due to the specificity of periodontal disease. After accounting for hybridization, 110 of the samples tested positive for periodontitis-causing bacteria. After DNA preparation, PCR and hybridization were performed to detect 11 pathogens causing periodontitis. The results showed that all examined samples from the target group  showed the presence of periodontitis bacteria. The presence of all tested bacteria was demonstrated, with several types of bacteria present in each sample. In all patients, the presence of After gradient PCR to determine the binding range of the beta globin primers PC04 and GH20, we found that at all temperatures tested (51.0–60.3 °C), there was a product of size 268 nt, and only at temperatures of 51.0 °C were there non-specific bands below 200 and below 100 nt due to the lower annealing temperature of the primers. The periodontitis-positive 230 samples from the target group were also probed with consensus primers GPE6/5B/6B and primers MY09/11. After the PCR, the presence of specific fragments with a size of about 700 bp and 450 bp was confirmed, which proves the presence of HPV. The results of the conducted reaction are presented in Samples from the control group were probed with consensus primers MY09/11 and primers GPE6/5B/6B. Samples were taken from the gingival margin and buccal mucosa. The presence of specific fragments of 450 bp and about 700 bp was detected, which proved the presence of HPV. The number of HPV-positive samples taken from the gingival margin is higher, which is probably due to the structure of the oral epithelium of the gingiva, which allows the development of productive HPV infection with the assembly of functional virions, in contrast to the buccal mucosa. All HPV-positive samples from the target group were tested with an in vitro diagnostic kit for 12 high-risk HPV genotypes. After conducting PCR and visualization by agarose gel electrophoresis, the presence of fragments with a size characteristic of the respective genotypes was reported. The results of the studies are combined and presented in When examining the samples, it was found that in 110 of them, the presence of more than one HPV genotype was reported. High-risk genotypes 66, 31, and 59 were not reported in any of the samples.In a screening of 230 coronal samples taken from the control group, 100 samples tested positive for HPV with the consensus primers. When tested for 12 high-risk HPV genotypes, 9 were positive for type 45 and 9 for type 18. The results are summarized in All HPV-positive samples (100) taken from the gingival margin of patients without clinical signs of periodontitis were also positive for periodontitis bacteria. Of 130 samples negative for HPV, 10 were positive for periodontitis bacteria. The presence of HPV and the absence of periodontitis-causing bacteria were not detected in any of the samples from the gingival margin of the control group.When examining buccal mucosa samples from the control group with the consensus primers, 64 positive samples were found. Genotype 18 was proven in 16 of the samples. The results are presented in In the target group, in 170 patients or 73.9% of them, the presence of high-risk HPV genotypes was demonstrated, and all of them were positive for periodontitis bacteria. The results are presented graphically in 2 = 205; p < 0.001). The number of genotypes is also different when comparing samples taken from the coronal edge of the representatives of the target group and the control group—nine in the target group and only two genotypes in the control group for samples taken from the coronal edge.Eighteen genotypes positive for high-risk HPV, i.e., 7.8%, were found in the coronal margin samples in the control group. A statistically significant difference was found for this indicator . The results are presented in A comparison of the proportion of samples from patients with high-risk HPV genotypes from the target group with the proportion of samples from patients with high-risk HPV genotypes from the control in which bacteria were also isolated from the coronal margin (16.4%) also showed that there was statistical significance  positive for periodontitis-causing bacteria, while in patients without bacteria, there was not a single case with proven presence of HPV. The results were confirmed by the conducted McNemar’s test—χCompared with the coronal margin results in the control buccal mucosa samples, only one high-risk HPV genotype was observed in fewer patients (7%). The observed difference is implausible.From all the research performed and the statistical processing of the results, we can conclude that there is a relationship between the presence of periodontitis-causing bacteria in patients with a pronounced clinical picture and high-risk HPV genotypes.In contrast with colleagues  who founA statistically significant difference was also found between the results obtained when examining the samples from the target group and the samples taken from the gingival margin of the control group in terms of HPV infection. In contrast to previous studies, our genotyping found that the most common genotype in samples positive for periodontitis bacteria was HPV type 58, followed by types 16, 33, and 35. HPV type 58 was found to be dominant in our study and was not associated with the development of papilloma lesions in the maxillofacial area, while the other widely represented genotypes 16, 33, and 35 were associated with the development of various papilloma lesions [In the control group, a preponderance of HPV-positive samples was found in materials from the coronal margin compared to those from the buccal mucosa. This may be due to the fact that, theoretically, the structure of the oral gingival epithelium allows the development of a productive HPV infection with the assembly of functional virions, unlike the buccal mucosa. In relation to the structure of the gum and the pathological processes caused by the bacteria causing periodontitis, it is possible that there is a synergy between the bacteria and HPV. This interaction may be based on the mechanical exposure of the coronal margin (often accompanied by bleeding and pockets), which allows the virus to come into contact with one or both basement membranes as well as the junctional epithelium. We assume this is preparation for an opportunistic infection. Since the gingival mucosa bordering the tooth enamel lacks this differentiation, as does the oral epithelium or skin, i.e., keratinocytes , the absHPV localizes to the inflamed periodontal tissue and is thought to infect the basal keratinocytes in the ulcerated epithelium of the gingival sulcus. Inflammatory periodontal pockets play an important role as a reservoir for HPV. Although interactions between HPV and periodontal bacteria remain unclear, oral HPV infection may be associated with a characteristic oral microbiome. Carcinogenic HPV and periodontitis are likely to contribute to the development of oral cancer. To date, however, oral expression of HPV E6/E7  has not been fully studied in individuals with periodontitis. Overall, the available literature suggests that oral HPV may be associated with periodontitis. To clarify the relationship between oral HPV and periodontitis, the effects of clinical factors contributing to the spread of HPV DNA in the mouth must be considered . Pm (Peptostreptococcus micros), Fn (Fusobacteric nucleatum/periodo), En (Eubacterium nodatum), and Cr (Campylobacter rectus) was recorded in all patients. No statistically significant association was found between well-defined periodontitis-causative agents and well-defined high-risk HPV genotypes. We have reason to believe that it is an opportunistic infection due to the specificity of the disease and the replication cycle of HPV.In our experiments, the presence of all tested bacteria was detected, with several types of bacteria present in each sample. The presence of In the studies we conducted in Bulgaria, we found that for patients with a clinical picture of periodontitis and the presence of bacteria causing the corresponding disease, in the absence of a clinic of oropharyngeal lesions in the oral cavity, the presence of high-risk HPV was proven with a statistically reliable frequency. In control samples from the buccal mucosa and periodontal margin, the presence of high-risk HPV genotypes was significantly less. We can conclude that there is a relationship between the presence of periodontitis bacteria in patients with a pronounced clinical picture and high-risk HPV genotypes as well as a relationship between the presence of these bacteria and the presence of HPV. We could conclude that oral HPV infection is probably an opportunistic infection of the inflamed and bacterially infected gingival tissues. The presence of HPV in patients with periodontitis may influence disease severity and response to treatment. Therefore, the identification of HPV in patients with periodontitis may be essential to determine the appropriate course of treatment.


Oral cancer is the 11th most common type of cancer in the world, with established major risk factors as tobacco and alcohol, and recently included high-risk human papillomavirus types 16 and 18. HPV types 16 and 18 are the etiologic agents of cervical cancers and a proportion of oropharyngeal cancers. However, the picture of HPV and the clinical implications of oral cancers are not clear with most reports combining oral cancer data with head and neck cancers. It has been confirmed as a favorable prognostic factor in oropharyngeal cancer. However, the prognostic value of HPV in oral squamous cell carcinoma is still unclear.The main objective of this article is to present the evidence encountered following a bibliographical review of recent publications specifically related to oral cancer and its differences from oropharyngeal cancer. The secondary goals are to present the findings of a five-year retrospective observational study of the prevalence of HPV infection in oral cancer patients treated by the Oral and Maxillofacial Surgery Department at La Paz University Hospital , and finally, we to evaluate and compare our country’s HPV prevention program in comparison to other European countries.According to the review of the literature, HPV positive oral squamous cell carcinoma is associated with significantly decreased overall survival and distant control. Bibliographic review suggest HPV infection can be used as a negative prognostic factor in oral squamous cell carcinoma.As regards diagnostic testing for HPV, it should be extended to as many cases of oral cavity squamous cell carcinoma as possible, especially in those with risk factors. The current vaccination program in Spain does not have adequate coverage and is significantly under the level of other European Union countries; it should be expanded and catch-up strategies should be included. Key words:HPV, OSSC, Papillomavirus, oral carcinoma, prevention. The first bibliographic reference associating HPV infection and oral cancer is from 1983 by Dr. Syrjanen (Finland) , and finally, we to evaluate and compare our country’s HPV prevention program in comparison to other European countries.For the literature review we used the PubMed search engine for articles indexed in the US National Library of Medicine, National Institute of Health, NIH/NLM, USA. An advanced search was performed with the following query: ((((human papilloma virus) OR (vph)) AND ) NOT ). A total of 837 results were obtained.The search was then restricted to articles published between 2016 and 2022 and 339 articles were obtained. Of these, 47 were selected for their relevance, demographic criteria, and applicability to the objective of this review.These articles were analyzed to find differences between HPV-positive oral cavity epidermoid carcinoma cases and HPV-negative cases in terms of three variables: patient profile, clinical location, and prognosis, Fig. .This review is complemented by data from our own clinical experience in the form of a retrospective descriptive study of all patients treated by the Oral and Maxillofacial Surgery Service of La Paz University Hospital, between February 2017 and February 2022 determining how many cases were tested for HPV and the results. A total of 429 samples and a total of 135 p16 tests performed were included.Finally, the guidelines published by the interterritorial health council of Spain and by the different health agencies of the European Union countries were reviewed to analyze and compare the different prevention plans against HPV infection between Spain and the other European Union countries.The results obtained from the literature review are:- Patient profile,24:o It stands out that patients with epidermoid carcinoma of the oral cavity with HPV infection were less related to toxic habits of tobacco consumption (42.1% vs. 59.6%) and alcohol (5.3% vs. 22.1%).o Predominance of male sex (73.7% vs. 68.4%).o Lower age (61.9 +- 12.3 vs. 62.8 +-13.6).- Location,24,25:o There is an increased risk of oral cancer in the tongue, palate and floor of the mouth.o Specifically and separately, E7 protein positivity for HPV serotype 16 implies an OR 2.71 for oral floor cancer and OR 3.34 for oral palate cancer.- Prognostic–29:o A systematic review of 2126 studies determines that the relationship between HPV infection and oral cavity squamous cell carcinoma implies a lower overall survival (OS) HR= 1.45, (95% CI 1.10-1.93) and lower distant disease control (DC) HR = 2.16, 95% .o Furthermore HPV-16 is a worse predictor at 5 years for:p=0.075).• Overall survival in uncomplicated disease (p=0.113).• Overall survival in advanced disease  65 samples were sent, the test was performed in 31 (47%), with negative result in 29 and positive in 2. In 2018, 70 samples were sent, the test was performed in 20 (28%), with negative result in all. In 2019, 131 samples were sent, 32 (24%) were tested, with negative results in 27 and positive in 5. In 2020, 68 samples were sent, 20 (29%) were tested, with negative results in 18 and positive in 2. It should be pointed out that there was a decrease in surgical and healthcare activity during this period due to the COVID-19 pandemic.In 2021, 84 samples were sent, 30 (35%) were tested, with negative results in 21, positive in 8 and inconclusive in 1. In 2022 (up to February), 11 samples were sent, 1 was tested with a negative result, .Analyzing the positive results individually, the year with the most positive results was 2021 and the one with the least was 2018, , Fig. 3.The vaccination schedule published by the interterritorial council of the Spanish national health system includes vaccination against HPV at 12 years of age for girls only, with two doses separated by at least 5-6 months.After 12 years of age and up to 18 years of age, it includes vaccination only for women who have not been vaccinated, or have been partially vaccinated, previously.As for adult vaccination (from 18 years of age), it is only contemplated in the population at risk, both men and women who meet at least one of the following conditions:- WHIM syndrome (IDP): vaccine covering types 6 and 11.- HIV infection, up to 26 years of age.- Men who have sex with men, up to 26 years of age.- Persons in prostitution, up to 26 years of age.- Women with cervical excisional treatment (any age).If we review the vaccination program of other European countries, most of them include vaccination between 10 and 14 years of age for males and females. Many also have catch-up strategies to rescue those adults not vaccinated during childhood.According to the available literature there is high evidence that vaccination is an effective tool in protecting against the development of malignant and premalignant lesions, being especially useful in reducing transmission ,30,31.Available cost-effectiveness studies indicate that increasing vaccination coverage is cost-effective, especially in the male population ,32.There is a paradigm change in the available evidence on risk factors specifically for oral cavity cancer. While HPV infection is widely accepted as a positive prognostic factor in the prognosis of cervicofacial cancer globally, in particular in oral cavity cancer, specifically, it seems to be related to more disseminated disease and a lower overall survival, and can therefore be considered as a negative prognostic factor.This specific characteristic of oral cancer, which makes it different within the group of cervicofacial cancer, makes it advisable to conduct more studies to analyze in depth this discrepancy and its consequences.One of the first needs is to have well collected the prevalence of HPV in all possible cases. This makes it especially profitable to perform specific p16 tests on histological specimens from oral cancer resection surgeries and also on pre-surgical diagnostic biopsies.Until now the usual protocol contemplates the performance of p16 testing only in cases of high suspicion, but analyzing the data of our study we see that in the years that more tests have been performed there are more positive results.Taking into account that it is a rapid test with an affordable cost and its proven specific prognostic factor in oral cancer, it seems to be a recommendation to extend the protocol and perform this test to all the histological samples studied.As regards prevention, the unfavorable situation in Spain is notable, especially the absence of childhood vaccination in men, the absence of a “catch-up” program and the limited vaccination coverage in adults, non-existent outside cases of risk.With the proven efficacy of vaccination against malignant and premalignant disease, and especially against transmission, as well as being a cost-effective strategy, it seems advisable to urgently review the national vaccination program to expand its coverage.


There are differences in postural tachycardia syndrome (POTS) incidence and manifestations in children between the sexes. However, there is limited evidence on how the gender affects the prognosis of POTS in children. This study is aimed at exploring the differences between the sexes regarding the prognosis of children with POTS. A retrospective study was conducted on children  who were diagnosed with POTS. All the POTS patients were given health education and autonomic function training, their water and salt intake was increased , and they were administered oral metoprolol (1 mg/kg per day) for 3 months. The prognosis was defined by the head-up tilt test results after treatment. It was observed that male and female children exhibited different trends in POTS prognosis. Further, the sex showed a stable independent effect on prognostic in children with POTS. To elaborate, females had a 503% increased risk of poor prognosis compared to males. We hence hypothesize that there is an association between the sex and the POTS prognosis in children. Female patients have a significantly higher risk of poor prognosis compared to males. A slight increase in the dose of oral rehydration salt could help lower the risk of poor prognosis in children with POTS. A higher absorption of total metoprolol, lower local concentrations, and slower metabolic excretion are documented in research in female POTS patients during treatment. It is recommended that the optimal dose of metoprolol should be lowered in female children undergoing treatment, to limit the risk of poor prognosis. Neurally-mediated syncope is mainly caused by autonomic nerve-mediated reflex dysregulation or autonomic dysfunction and accounts for 70% to 80% of the total cases of syncope. Postural tachycardia syndrome (POTS) is one of the common types of neurally-mediated syncope.–6 It is characterized by an excessive increase in the heart rate (HR) after standing and chronic day-to-day orthostatic intolerance symptoms, such as dizziness, headache, chest discomfort, palpitations, blurred vision, profuse perspiration, and even syncope. A standing test or head-up tilt test (HUTT) can provide important evidence for the diagnosis of POTS. The possible potential pathophysiological mechanisms include partial autonomic neuropathy, hypovolemia, and the hyperadrenergic state. Further, patients often exhibit overlapping characteristics of one or more of these pathophysiological mechanisms. The approach in treating POTS is mainly based on treating the underlying pathophysiologic mechanism.Syncope is a clinical condition that manifests as a transient loss of consciousness caused by hypoperfusion of the whole brain and makes 1 fall to the ground due to decreased muscle tone. However, one can recover on their own in a short time.,8,9Non-pharmacological treatment is the main intervention method for POTS, which includes health education, such as avoidance of triggers, using stockings, employing abdominal binders, taking vasoconstrictors, performing appropriate physical exercises, autonomic nervous function exercises, and increasing the intake of salt and water . suggest that non-pharmacological treatment alone is not sufficient and that additional medication may be required to improve symptoms. The medication administered is usually ORS and metoprolol. ORS, which increases the patient’s blood volume by supplementing with water and electrolytes, has become the basic treatment approach for POTS in children,11 and may reduce the symptoms of syncope induced by the Bezold-Jarisch reflex. Additionally, metoprolol is listed in “2018 Chinese Pediatric Cardiology Society guidelines for the diagnosis and treatment of syncope in children and adolescents” as a drug that suppresses sympathetic excitability in children with POTS. Although the combination of non-pharmacological and pharmacological treatments of POTS in children has been clinically effective in the majority of cases, differences in prognosis have been observed among the sexes in children with POTS. This is despite being treated with the same treatment protocol with the number of females with a poor prognosis outnumbering that of males.,13 We hypothesize that various physical characteristics associated with sex may have an impact on prognosis, but this has yet to be documented in recent research. In this research paper, we propose to investigate the impact of sex on the outcome of POTS in children undergoing the same treatment protocol.While non-pharmacological treatment has significantly improved the prognosis of POTS in children, additional pharmacotherapy is necessary when the outcome of non-pharmacological treatment is not satisfactory. For example, Boris et al After 3 months of treatment follow-up, if syncope or pre-syncope symptoms improved and the HUTT results no longer met the diagnostic criteria for POTS it was considered a good prognosis. If the syncope or pre-syncope symptoms did not improve and the HUTT results still met the diagnostic criteria for POTS it was grouped as a poor prognosis.This is retrospective research to explore if the difference in sex has an impact on the prognosis of POTS in children who had the same treatment protocol. The dependent variable was the prognosis of the children treated with non-pharmacological treatment combined with pharmacological treatment. We divided them into the good prognosis group and poor prognosis group based on whether the HUTT met the diagnostic criteria for POTS after therapy and if the clinical symptoms improved.The data of participants with newly-diagnosed POTS were non-selectively and consecutively collected from the Department of Pediatric Cardiovasology, The Second Xiangya Hospital, Central South University. Our data did not include identifiable participants’ data for protecting their privacy. Data were then compiled from Hospital’s Electronic Medical Record System. Informed consent from the participants was not required in this research protocol because its nature was that of retrospective research. The Ethics Committee of The Second Xiangya Hospital, Central South University has approved this research. The inclusion and exclusion criteria are showed in Figure A total of 347 participants were screened. The dates of inclusion and completion of participant data entry were between August 2012 and July 2019, respectively. The clinical diagnosis and treatment process of each participant were completely following “2018 Chinese Pediatric Cardiology Society guidelines for the diagnosis and treatment of syncope in children and adolescents.”am to 11:00 am, the environment was quiet and the room temperature was 20°C–24°C. For the HUTT, the tilting device was the Head-up Tilt Test Monitoring System (SHUT-100) from Jiangsu Standard Medical Technology Co., Ltd. The subjects were kept supine on the tilt bed for 10 minutes, and their HR, blood pressure (BP), and electrocardiogram (ECG) were monitored and recorded. After recording the baseline ECG and BP of the subject in the prone position, the subject was tilted 60° with the head high and feet low position within 15 seconds. The HR, BP, and ECG indicators were monitored dynamically during the tilt for 10 minutes or at the time point of a positive response.The diagnosis of POTS in this research involved only the baseline head-up tilt test. Research subjects discontinued cardiovascular active drugs that might affect autonomic functions for more than 5 half-lives and related foods such as coffee before the trial. Before the experiment, 4 hours of fasting was followed and drinking was prohibited. The examination time was arranged from 8:00 The following were the criteria: children who were below 18 years old; orthostatic intolerance symptoms such as dizziness, headache, weakness, blurred vision, chest tightness, palpitations, hand tremor, limited movement in an orthostatic position, and even syncope; and HUTT: supine HR was normal, and, during the initial 10 minutes of HUTT, HR increased ≥40 bpm or is ≥130 bpm (in children 6–12 years old) or ≥125 bpm (in adolescents 12–18 years old), without orthostatic hypotension (BP decrease > 20/10 mm Hg).The variables used in this study can be divided into the following categories: continuous type variables: age, height, weight, metoprolol dose (mg/d), and the ORS dose (mL/d) and categorical variables: sex and prognosis.,16ORS III  and metoprolol (1 mg/kg per day). The follow-up was after 3 months of treatment. ORS III was manufactured by Xi’an Anjian Pharmaceutical Co Ltd . The formulation details are given here, glucose anhydrous 3.375 g, sodium chloride 0.65 g, potassium chloride 0.375 g, and sodium citrate 0.725 g. Sodium concentration 75 mmol/L, osmolality 245 mOsm/L.The dosage was 5.125 g/dose, Bid, was dissolved in water and given orally in divided doses.Regarding metoprolol, the manufacturer was AstraZeneca Pharmaceuticals Ltd. .2 test , Student t test , or the Mann–Whitney U test (skewed distribution) to analyze the differences among different prognosis groups of POTS . Univariate analysis was used to roughly evaluate the relationship between each variable and the prognosis of POTS. Further, multivariate Logistic regression was used to analyze the possible association between the sexes and the prognosis of POTS under the same treatment protocol. We constructed 3 models to illustrate the stability of this relationship. The details are given herewith. Model Iwas adjusted for none. Next, Model II adjusted for age, height, and weight. Finally, Model III was adjusted for age, height, weight, ORS dose, and metoprolol dose. To address the nonlinearity of metoprolol dose and prognosis of POTS for different sexes, a generalized additive model and smooth curve fitting  were conducted. All the analyses were performed employing the statistical software packages R (version3.4.3)  and EmpowerStats . P values less than .05 (2-sided) were considered statistically significant.Based on whether the continuous variables were normally distributed, we presented continuous variables in this study as mean ± standard deviation  or quartile  (skewed distribution). Categorical variables were expressed in frequency or as a percentage. We employed the χP > .05). While the age was greater in females (P < .05), the prognosis was better in males than females (P < .05).The basic characteristics of the study participants are presented in Table P = .04). There was a trend towards an increased risk of poor prognosis with increasing age, height, weight, and metoprolol dose . Further, a trend emerged towards a decreased risk of poor prognosis with increasing ORS dose (OR = 0.88)  hyperestrogens and progesterone were associated with increased adrenaline and possibly more fluid retention, thereby improving the orthostatic tolerance in these patients. Further, Coupal et al also found that hormonal changes in the body during adolescence could affect orthostatic intolerance. The lower orthostatic tolerance in females compared to males is associated with increased susceptibility to autonomic dysfunction. Increased leptin, insulin, thyroxine, and increased sympathetic activity or catecholamine levels during puberty can exacerbate the abnormal increase in heart rate in patients with POTS.In addition, the better prognosis for males compared to females may also be related to hormonal changes in the body. For instance, Stickford et al Therefore, a moderate increase in the dose of ORS can help to reduce the risk of poor prognosis in POTS.We scrutinized the relationship between the ORS dose and poor outcomes in a univariate analysis and found that the risk of poor outcomes reduced as the ORS dose increased. POTS is caused by cerebral hypoperfusion in the brain due to a relative lack of blood volume, thus inducing orthostatic intolerance symptoms. ORS is a liquid suitable for gastrointestinal absorption and can rapidly rehydrate blood volume and relieve orthostatic intolerance produced by cerebral hypoperfusion in the brain.–27 The rate of absorption of metoprolol is also affected by many parameters including intestinal transit time, drug hydrolysis and lipid solubility, the pH at the absorption site, the degree of ionization and molecular weight of the medication, and intestinal motility. It was documented that the baseline and maximum amounts of gastric acid and the acidity of gastric acid were higher in males than in females during drug absorption (pH = 1.92 vs. pH = 2.59) while the pH of metoprolol was 6.0-7.0. Further, the digestive tracts of males have a lower pH than that of females, resulting in lower metoprolol absorption. Additionally, the hepatic clearance of the drug is influenced by hepatic blood flow and enzyme activity. Metoprolol is predominantly metabolized by the liver, and higher levels of estrogen and progesterone in females change hepatic enzyme activity. This increases metoprolol accumulation or lowers metoprolol clearance overall resulting in higher total blood metoprolol concentrations in females than in males. However, the smooth curve fitting between different sexes show that higher metoprolol concentrations significantly increased the risk of poor prognosis in female. Further, several bodily components also have an impact on drug distribution such as plasma volume, body mass index, mean organ blood flow, total body water, plasma proteins, body fat, and cardiac output. Metoprolol is a class of ionotropic medication and is fat-soluble. As female patients have more body fat than males, given the same quantity of fat-soluble drug exposure, the higher fat content in females would increase the volume of distribution and lower the drug concentration. Additionally, the high overall concentrations and low target concentrations increase the risk of poor prognosis in females. The kidney is the primary organ for the elimination of drug metabolites, and the physiological functions of glomerular filtration, tubular secretion, and tubular reabsorption vary by sex. In males, the renal clearance is generally higher than in females and metoprolol metabolites are excreted at a slower rate in females than in males. Additionally, negative feedback reduces metoprolol metabolism in the liver to some extent and thus prolongs the duration of the high-concentration levels of metoprolol in the body.We observed different responses of the sexes to different doses of metoprolol in smooth curve fitting. It was revealed that a high dose of metoprolol was harmful to the prognosis of females with POTS. Sex differences in prognosis are mediated by multiple factors. These include differences in body composition, weight, drug absorption and distribution, metabolic enzymes, and excretion pathways between males and females. Metoprolol is effective for pediatric POTS at an initial dose of 0.5 mg/(kg·d) divided into 2 administrations. The total dose of the drug should not exceed 2 mg/(kg·d). For instance, Zhang et al,29 reported the efficacy of metoprolol [1 mg/(kg·d)] combined with ORS in the treatment of pediatric POTS and found that the efficiency at 3 months of treatment was higher in the treatment group than in the control group . Further, the degree of increase in the HR of the HUTT process was significantly lower in the treatment group at the post-treatment review compared to the pretreatment baseline values. In another report, Raj et al found in a randomized controlled crossover trial (n = 72) that while oral propranolol (20 mg) significantly reduced tachycardia symptoms in patients with POTS, oral propranolol (80 mg) did not further improve the outcome and even worsened the condition. Oral metoprolol [1 mg/(kg·d)] may improve symptoms in POTS children who do not respond well to health education and rehabilitation exercises and whose POTS symptoms remain significant. Additionally, Arnold et al reported the effect of low-dose propranolol on the maximal exercise capacity in POTS patients (n = 11) and found that low-dose propranolol (20 mg) improved maximal oxygen uptake in POTS patients. However, higher doses of propranolol (80 mg) and metoprolol (100 mg) did not the improve maximal oxygen uptake. It is believed that low-dose propranolol is beneficial in improving HR modulation and organismal exercise capacity in POTS patients. This research treatment for pediatric POTS used a moderate dose of metoprolol [1 mg/(kg·d)], which both reduced the symptoms of excessive increase in HR caused by POTS and improved the maximum oxygen uptake in POTS.Regarding the metoprolol dosage, following “2018 Chinese Pediatric Cardiology Society guideline for diagnosis and treatment of syncope in children and adolescents” is recommended.To summarize, a slight increase in the amount of ORS could help lower the risk of poor prognosis in children with POTS. Given the higher absorption of total metoprolol, lower local concentrations, and slower metabolic excretion in female children with POTS during treatment, the dose of metoprolol should be lowered in female children on treatment, if appropriate, to limit the risk of poor prognosis.Female children with POTS had a worse prognosis than males for the same treatment protocol. Further, the dose of metoprolol should be adjusted to account for sex variations as required.This research analysis investigated the possible influencing factors in the prognosis of children with POTS from the perspective of sex differences. It also analyzed the absorption, distribution, and metabolism of the drug in the body. It may offer a new way for better treatment in some female children with POTS who have a bad prognosis and also serve as recommendations for modifying sex-specific metoprolol dose protocols in children with POTS.Regarding the limitations, the modest sample size was only aimed at studying the trends in the treatment efficacy in children with POTS according to the sex. The extent of metoprolol dose reduction for female children with POTS who do not respond well to treatment is to be further validated with an expanded sample size.The authors thank all the staff members in our institution.Conceptualization: Yali Peng, Hong Cai, Yuwen Wang, Cheng Wang, Shuo Wang.Data curation: Yali Peng, Hong Cai, Juan Zhang, Yuwen Wang, Cheng Wang, Shuo Wang.Formal analysis: Yali Peng, Runmei Zou, Juan Zhang, Cheng Wang, Shuo Wang.Investigation: Yali Peng, Runmei Zou, Juan Zhang, Yuwen Wang, Cheng Wang, Shuo Wang.Methodology: Yali Peng, Runmei Zou, Hong Cai, Cheng Wang, Shuo Wang.Project administration: Runmei Zou, Shuo Wang.Resources: Yali Peng.Software: Yali Peng, Runmei Zou, Hong Cai, Yuwen Wang, Cheng Wang, Shuo Wang.Supervision: Yali Peng, Shuo Wang.Validation: Yali Peng, Shuo Wang.Visualization: Yali Peng.Writing – original draft: Yali Peng, Runmei Zou, Cheng Wang, Shuo Wang.Writing – review & editing: Yali Peng, Cheng Wang, Shuo Wang.


Vulvar and vaginal cancers are relatively rare cancers, but their incidence is increasing worldwide. Overall, 78% of vaginal cancers and 25% of vulvar cancers are associated with Human papillomavirus (HPV) infection. Immunization could be an option for the management of these cases. We researched and assessed the evidence on the efficacy of HPV vaccination administered to women previously treated with surgery, radiotherapy, or chemotherapy with respect to the recurrence of vulvovaginal disease. From 2006 to November 2022, only one study evaluated the efficacy of HPV vaccination with respect to preventing vulvovaginal recurrences in treated women and showed that a quadrivalent HPV vaccine administered after the surgical treatment of vulvar high-grade squamous intraepithelial lesion (HSIL) can reduce vulvar recurrence of the disease. Therefore, the efficacy of HPV vaccination with respect to vulvovaginal recurrence is still an unexplored field. Further studies are needed to produce stronger evidence in order to appropriately support interventions to protect women’s health. Human papillomavirus (HPV) is the most common sexually transmitted virus . It is eCervical cancer, which is the fourth most frequent female cancer, annually constitutes almost 8% of all cancer deaths among women. In fact, 604,127 new cases and more than 341,831 deaths were reported in 2020 [Since cervical cancer is a type of tumor that can be thoroughly prevented and effectively treated through vaccination, screening, and therapies, in May 2018, a global call for action was issued by the World Health Organization’s (WHO) Director-General in order to eliminate this burden. In August 2020, the World Health Assembly adopted the Global Strategy for Cervical Cancer Elimination by 2030, which identifies strategic actions necessary to achieve: 90% HPV vaccine coverage for girls by the age of 15 years, 70% coverage for screening, and 90% of women identified with cervical cancer to be treated This is the first global health strategy for eliminating a cancer that has been identified as a public health problem [Vulvar and vaginal cancers are relatively rare tumors worldwide, with 45,240 and 17,908 new cases estimated in 2020, respectively. The rates of both cancers tend to be higher in high-income countries .Squamous cell carcinoma (VSCC) represents the majority of vulvar cancers, and it can arise through two distinct pathways: HPV-associated and HPV-independent. The precursor of HPV-associated VSCC is vulvar high-grade intraepithelial neoplasia (VIN), which progresses to invasive carcinoma in fewer than 10% of cases but recurs in a higher percentage . A recenSeveral multivalent prophylactic HPV vaccines, which are based on recombinant DNA technology and prepared from purified L1 protein that self-assembles to form virus-like particles, are currently available. The bivalent vaccine includes antigens for HPV 16 and 18 . The quaThe target population includes 9–14-year-old subjects who have been administered two doses of an HPV vaccine and those ≥ 15 years of age who have received three doses. Other recommendations also cover adults who may be at higher risk . A recenEvidence shows that prophylactic HPV vaccination, as in the case of cervical cancer precursors, also prevents HPV-related vulvar and vaginal HPV-associated precancers ,21.The vaccination of people with previous HPV infection may have less effectiveness, but it prevents new infections with HPV types to which those infected have not been previously exposed. Indeed, having previously been diagnosed or treated for an HPV-related lesion is not a contraindication to vaccination . This isMany surgical interventions are available as treatment for vaginal and vulvar cancers, some examples of which include ablation, local excisions, vaginectomy, and vulvectomy. In addition, radiotherapy and chemotherapy are also used, which present a variable rate of recurrence after primary treatment . HPV vacA literature search was conducted to evaluate the efficacy of HPV vaccination with respect to preventing vulvovaginal recurrences in treated women. Scopus, Pubmed, Clinical Trials, and Embase databases were scoured for this purpose. We considered only clinical trials and observational studies investigating the efficacy of HPV vaccination administered to women after treatment for vulvovaginal disease. Reviews and meta-analyses were excluded in the analysis. The research question of the current study was as follows: “Which is the efficacy/effectiveness of HPV vaccination in preventing recurrence in women treated for HPV-related diseases of vulva and vagina?”.The keywords “HPV vaccination”, “recurrence”, “vulvo-vaginal neoplasm”, and “treatment” were used in this research and, accordingly, adapted to the format of the different literature databases. We limited our research to articles published in English from 2006 onward. Inclusion criteria for the selection of the articles were as follows: manuscripts concerning administration of HPV vaccination after diagnosis or treatment of vulvovaginal lesions and evaluation of the efficacy of HPV vaccination on vulvovaginal recurrence. Outcomes related to cervical recurrence and studies about the prophylactic administration of HPV vaccination were excluded. This review was performed in November 2022. The review was independently performed by three pairs of authors. The same pairs of authors screened the titles and abstracts and then the full texts of the papers for eligibility. Any disagreements on the inclusion/exclusion process were resolved in a consensus meeting. We extracted data on the following variables from the studies included in the final synthesis: first author, year of publication, geographical region, aim of the study, study setting, population, type of HPV vaccine administered, outcome type, and type and duration of follow-up.The search yielded 606 records from 2006 to November 2022 . After the elimination of duplicates, the remaining number of records was 345. We reviewed the titles and abstracts of all these records and, subsequently, excluded 238 papers because they were not related to the topic of the research (201 papers) or were reviews and meta-analyses (37 articles). Then, we focused on the 107 remaining records, which were reviewed as full texts, and we excluded 106 of them. Specifically 67 articles that did not report topics related to HPV vaccination, 17 articles that reported issues related to HPV vaccination solely for the prevention of cervical disease, 8 articles that reported topics related to HPV vaccination solely for the prevention of warts or anal cancer, 5 articles that did not analyze the eligible population , 4 articles that were not full texts, 3 articles that did not report clinical data, and 2 papers that were conference abstracts were excluded.The selection process of the articles to be included in the final review is provided in the following flowchart . p = 0.19), clinical effectiveness in preventing a reactivation of the previous HPV infections (relapse) was observed (78.5% of effectiveness). A smaller gap between the groups in terms of recurrence, which was due to the “reactivation” of infection provoked by the same HPV type found during the first surgery, was observed. Therefore, this study supports the impact of vaccination on influencing the natural course of HPV infection after surgery. Only one study specifically evaluated the efficacy of HPV vaccination with respect to preventing vulvovaginal recurrences in treated women . The manIn conclusion, HPV vaccination has a significant role in the prevention of HPV-related diseases. In addition, recent studies have highlighted the effect of vaccination on the management of HPV-related diseases and its impact on recurrent infection or reactivation. However, almost all research conducted until now has mainly focused on cervical disease, while only few studies concern vulvovaginal disease. A recent meta-analysis suggested that the use of adjuvant vaccination is associated with a reduced risk of Cervical Intraepithelial Neoplasia (CIN)1 and CIN2 recurrence after surgical treatment . In factOverall, vulvar cancer is more common than vaginal cancer, although it is much less common than cervical cancer. Therefore, one reason for the lack of evidence on this topic could be the low incidence of vulvovaginal cancers , which cIn addition, vulvovaginal cancer represents a burden for the patients affected and has a high psychological and sexual impact on women and their partners. Considering the causal role of HPV in a large portion of vulvar and vaginal cancers, preventive actions such as the administration of an HPV vaccine have the potential to significantly reduce the burden of HPV disease . Therefo


The human papillomavirus (HPV) vaccine reduces the burden of cervical and other cancers. In numerous countries, a slow uptakeof this vaccine persists, calling for a better understanding of the structural factors leading to vaccine acceptation. We aimed to assess the attitudes toward HPV vaccination among its intended public to explore its specific characteristics.A random cross-sectional telephone survey of the French general population provided data from a sample of 2426 respondents of the target public: the parents of young women and the young women aged 15-25 themselves. We applied cluster analysis to identify contrasting attitudinal profiles, and logistic regressions with a model averaging method to investigate and rank the factors associated with these profiles.A third of the respondents had never heard of HPV. However, most of the respondents who had heard of it agreed that it is a severe (93.8%) and frequent (65.1%) infection. Overall, 72.3% of them considered the HPV vaccine to be effective, but 54% had concerns about its side effects. We identified four contrasting profiles based on their perceptions of this vaccine: informed supporters, objectors, uninformed supporters, and those who were uncertain. In multivariate analysis, these attitudinal clusters were the strongest predictors of HPV vaccine uptake, followed by attitudes toward vaccination in general.Tailored information campaigns and programs should address the specific and contrasted concerns about HPV vaccination of both young women and of their parents. The links between Human Papilloma Virus (HPV) infections and some forms of cancer have been widely reported in the literature –4. SeverHPV vaccination is a strategic component of the battle to prevent cervical cancers caused by this virus. The World Health Organization (WHO) recommends HPV vaccination combined with screening and education strategies to reduce the impact of these infections on global public health –11. Its Over the past decade, HPV vaccination may have been affected by the rise of vaccine hesitancy (VH) in Europe . In MarcA considerable amount of literature has examined attitudes toward HPV infection and HPV vaccination, drawing mainly on surveys among parents –27 and yThe available research on HPV vaccination has identified several barriers to HPV vaccination but tended to oppose those in favor of this vaccine and those opposed to it instead of considering the various forms of reluctance. We explore this diversity and analyse whether there are socially differentiated clusters of VH toward HPV vaccination and how they influence vaccination behavior. Our aims were: 1) to explore hesitancy toward this vaccine among the young women (targeted group) and their parents (who often take the decision) specifically by considering simultaneously four different perceptions related to the disease's severity and its frequency, and the vaccine's efficacy and side effects respectively, as well as their possible combinations; 2) study the potential sociodemographic differences between profiles associated with the different types of vaccine hesitancy; 3) to test the extent to which these profiles to predict self-reported vaccination behavior.We used data from the 2016 Baromètre Santé, a national cross-sectional telephone survey addressing health issues in a representative population sample, conducted by the French Public Health Agency (Santé Publique France) , 30. DatRespondents were asked about their attitude toward vaccination in general . To capture different structural factors involved in the attitude toward HPV vaccine, we measured reported knowledge and perceptions about this vaccine. Therefore participants were asked whether or not they had ever heard of HPV vaccination and then to either agree or disagree  with four assertions related to, respectively, the severity and the frequency of HPV infections and the effectiveness and potential side effects of the vaccines against it. These questions were also asked of participants who stated that they had not heard of it, to see the extent to which people may endorse attitudes toward an unknown vaccine driven by their attitude toward vaccination in general. The questionnaire also collected data on HPV vaccine uptake: parents reported their daughters' vaccination status, and young women reported their own. Thus, the HPV vaccine uptake was evaluated by the answers "yes/no/don’t know" of both young women and parents of teenage girls.Finally, the questionnaire collected information about participants’ sociodemographic background: gender, age, educational level, and household income. The equivalized household income per month was computed taking into account household size and composition, to estimate participants’ standard of living .Data were weighted so the distribution of the main sociodemographic characteristics  matched the sample to the national census. Weights were applied to all statistics.First, we analyzed the perceptions of HPV infection and vaccination simultaneously, by conducting a cluster analysis to summarize the variety of perceptions reported by participants into contrasting attitudinal clusters toward the HPV vaccination. Items measuring agreement were coded from 1 (“Absolutely”) to 4 . These scores were transformed into Z-scores before clustering with the standard agglomerative hierarchical procedure .2 independence tests. Then we examined the factors associated with HPV vaccination status, including sociodemographic indicators, the clusters, and attitude toward vaccination in general. Using a logistic model, we used a multimodel averaging approach based on the Akaike information criterion to rank the explanatory variables by their relative importance. This approach estimates all possible models, given the explanatory variables introduced, and computes the final model as the weighted average of all parameters and standard errors from all possible models [We also investigated the sociodemographic composition of the resulting clusters, as well as their association with attitudes toward vaccination in general, by using χe models . We usede models  to quante models  and relae models , 37.The sample of the 2016 Baromètre Santé included 15,216 respondents with full interviews (participation rate: 50%). The questions about HPV vaccination concerned 2168 participants from two subgroups —young women  and parents of young women  whose attitudes and behavior toward HPV vaccination we sought to study. Among our participants, the overall HPV self-reported vaccine uptake rate was 35.2%  of respondents had not heard of HPV vaccines at the time of research , there was consensus across the four clusters regarding the severity of HPV infections (between 77.6% and 95.9% of participants considered them absolutely or somewhat severe). Second, the four clusters displayed contrasting opinions about the potential side effects of the vaccines, but in each cluster at least one third of respondents believed that HPV vaccines could cause severe side effects. Participants in the different clusters agreed that HPV infections are serious and the vaccine is effective but had divided views about the frequency of these infections and about the safety of the vaccine.Cluster 1 comprised 40.6% of participants. All of them had heard of HPV vaccines. Nearly all (95.5%) agreed that HPV infections are severe, and 79.3% perceived HPV as frequent. The vast majority considered HPV vaccines to be effective (97.7%), but more than a third were concerned about possible side effects (37%). We labeled this profile as Informed supporters.Objectors. Most (88%) had heard of the HPV vaccine, and agreed that HPV infections are severe (92.9%). Among them, only 46.1% agreed that HPV infections are frequent, and almost all were concerned about its potential side effects (94.2%).Respondents in cluster 2 (23.6% of the sample), were labeled as Uninformed supporters (29.2% of the sample) because 99.5% of respondents in this cluster reported they had not heard of the HPV vaccine. Most of them considered HPV infections to be serious (95.9%), and a large majority agreed that these infections are common (67.4%). According to 86.3% of the respondents in this cluster, HPV vaccines are effective but 49.4% thought that they might have side effects.We labeled Cluster 3 Uncertain. Among them, 77.6% agreed HPV infections are severe (17.7% didn’t know) and a third agreed they are frequent (39.5% didn’t know). The questions concerning their views of the effectiveness of the vaccines and their potential to cause side effects showed high levels of uncertainty (respectively 53.7% and 54.3% did not know).Finally, in Cluster 4, only half of the participants (who represented 6.6% of the whole sample) had heard of this vaccine. This cluster concentrated most of the "Don’t know" answers, and was labeled as Our results showed that parents were more frequently uncertain toward HPV vaccination than young women . Uninformed supporters and uncertain participants were the least educated.Results for household income were similar: objectors had an average profile for household income per consumption unit, while informed supporters were wealthier and low-income households were overrepresented among the two other clusters.Finally, the majority of objectors were nonetheless favorable to vaccination in general (58.8%), versus 87.6% of informed supporters, 84.8% of uninformed supporters, and 73.2% among the uncertain.In the bivariate analyses, self-reported HPV vaccine uptake was significantly more frequent among informed supporters  and young women aged 20–25 (52.9%)  Table . Once coIn our study, 35.2% of participants reported HPV vaccine uptake. This result was reasonably close to the actual French national coverage . CombiniBefore discussing our results, we must acknowledge several limitations of our study. First, this study shares the usual shortcomings of quantitative telephone surveys, including a moderate participation rate (50%). The announcement letter describing the survey and requesting participation did not give any details about the topics to be investigated: thus there is no reason to suspect that respondents’ answers regarding the attitudes toward the HPV vaccine and the vaccine uptake were correlated with non-participation. In addition, the data were weighted for various factors that are known to often be associated with survey participation.Second, like any data collection based on self-report, this survey is subject to social desirability and recall biases, especially regarding past vaccination of participants’ daughters.It has been frequently claimed that contemporary VH has been fueled by the very success of vaccination in controlling and eliminating diseases: severe infections that were previously common have almost disappeared, and so people stopped worrying about them . In ourThe contrasting attitudinal clusters, based on perceptions related to the specific risks and benefits of HPV vaccination, turned out to be slightly more predictive of this vaccination status than attitudes toward vaccination in general. This reflects the specificity of contemporary VH, which is often not guided by a general attitude toward vaccination, but that instead takes the specificities of each vaccine and each context into account , 30. Nev. Finally, mothers were more frequently objectors. This gender effect has already been observed for other recent vaccines in France  were more likely to report complete HPV vaccination: this may result from both the mechanical effect of age (older participants have had more opportunities to be vaccinated during their lifetime) and a more supportive attitude toward this vaccination . Among ine, see , for theine, see ), and a ine, see –49. To oine, see , 46. In ine, see . We can The relation between participants’ socioeconomic status and their attitudes toward vaccination may depend on both the vaccine considered and the national context . PreviouOverall, in 2016, a majority of French people supported HPV vaccination. However, there is still great room for improvement regarding information issues, as a third had never heard of this vaccination, while half shared concerns about its safety. Tailored information campaigns and programs should consider young women and their parents as distinct targets who may have different concerns. Vaccination uptake strongly depends on specific attitudes toward HPV vaccination, which can be enhanced by information campaigns, but also on general attitudes toward vaccination, which involve trust issues.