Lactobacillus iners coupled with the absence of Lactobacillus crispatus alongside Atopobium spp., Prevotella spp., and Gardnerella spp. could serve as defining factors for severe cervical lesions. The results also showed a significant association between microbiota diversity, HPV infection, and cervical lesion progression. (4) Conclusions: As the microbiota profile seems to vary among different populations and individuals, a deeper comprehension of its composition has the potential to develop personalized detection and treatment approaches for cervical dysplasia and cancer.(1) Background: Cervical cancer is a significant health concern, with the main cause being persistent infection with high-risk Human Papillomavirus (hrHPV). There is still no evidence for why viral persistence occurs in some women, but recent studies have revealed the interplay between cervical microbiota and hrHPV. This research aimed to characterize the cervicovaginal microbiota in cervical lesion progression and HPV infection status. (2) Methods: This study included 85 cervical specimens from women from the north-eastern region of Romania. DNA was isolated from cervical secretion for HPV genotyping and 16S ribosomal RNA gene NGS sequencing. (3) Results: Our study revealed a distinct pattern within the studied group when considering Lactobacillus species, which differs from findings reported in other populations. Specifically, the presence of  Cervical cancer is a significant global health issue, ranking as the fourth most diagnosed cancer among women. In 2020, it was estimated that there would be approximately 604,000 new cases of cervical cancer diagnosed and 342,000 deaths worldwide due to this disease. These numbers highlight the urgent need for effective prevention, early detection, and treatment strategies to reduce the burden of this pathology on women’s health globally .In Romania, despite being a preventable disease, cervical cancer ranks as the third most prevalent cancer among women, following breast and colorectal cancer . Based oThe primary cause of cervical intraepithelial neoplasia (CIN) and cervical carcinoma is persistent infection with high-risk Human Papillomavirus (hrHPV), HPV16 being the most prevalent and significant genotype . There iLactobacillus species such as Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners dominate the microbial community. These Lactobacillus species contribute to a well-balanced environment with a low pH, creating a protective barrier against viral and bacterial pathogens. In contrast, an increased risk of infection has been associated with the presence of anaerobic pathogens, including Gardnerella, Atopobium, and Dialister [Lactobacillus iners has been reported to dominate the microbial community when the vaginal microbiota transitions between normal and abnormal states and indicates susceptibility to CIN, which is also correlated with a higher frequency of HIV, HPV, and HSV-2 [The cervical microbiota is represented by the various microorganisms that colonize the reproductive tract. In a healthy cervical microbiota, ialister . Howevernd HSV-2 .Lactobacillus spp. are the most common bacteria in CSTs I, II, III, and V, while anaerobic genera like Prevotella, Atopobium, Dialister, Gardnerella, Aerococcus, Sneathia, Eggerthella, Finegoldia, Megasphaera, Peptoniphilus, and Mobiluncus are more common in CST IV [In a study from 2011, Ravel et al. classified the cervicovaginal microbiota into five community state types (CSTs). These CSTs were determined based on the presence and relative abundance of species identified through 16S rRNA gene sequencing. n CST IV ,8.2O2, and bacteriocin by Lactobacillus, disruption of mucosal integrity facilitating viral entry, and higher levels of oxidative stress induced by dysbiosis [Recent studies have indicated that the cervicovaginal microbiome plays a crucial role in HPV infection persistence and the subsequent development of cervical precancerous or cancerous lesions ,9. As thLactobacillus species, L. crispatus and L. iners both produce lactic acid, but they exhibit notable differences, especially concerning their impact on host health. The presence of L. crispatus in the vaginal environment has consistently been linked to good health, whereas communities dominated by L. iners may offer the host fewer protective benefits [L. iners frequently coexists with various bacterial species that colonize the vagina during instances of bacterial vaginosis [L. iners tends to have a higher vaginal pH compared to that dominated by L. crispatus [L. iners tends to increase in women who are HPV-positive and in those with LGSIL and HGSIL cytology. This particular increase is associated with a greater likelihood of transitioning to a dysbiotic vaginal microbiota. Furthermore, Lactobacillus iners has been linked to a higher risk of developing cervical dysplasia [Among the four predominant benefits ,11. Impoaginosis ,12. Addirispatus . A recenysplasia .Sneathia spp., may also help HPV to enter and stay in the body by affecting cellular targets, such as the expression of immunosuppressing cytokines.Some species, such as Sneathia sanguinegens, Anaerococcus tetradius, and Peptostreptococcus anaerobius and a lower abundance of Lactobacillus jensenii in comparison to women diagnosed with Low-Grade Squamous Intraepithelial Lesions (LGSILs) [Hence, considering the vaginal microbiome as a promising marker not only for HPV infection but also for cervical precancerous lesions holds significant importance . Women d(LGSILs) .HPV, as a pathogenic factor, interacts with the cervicovaginal microbiome, resulting in the proliferation of abnormal microbiota. Conversely, it is also possible that the presence of abnormal microbiota increases susceptibility to HPV acquisition. Moreover, the dysbiosis of the cervical microbiota has been found to be associated with HPV infection and its persistence .Gardnerella spp. or Prevotella spp. had the highest genital inflammatory profiles [Based on what we know so far, there may be a link between HPV that does not go away and changes in the microbiome. For example, 11% of women with persistent HPV have bacterial vaginosis, compared to only 5% of women whose HPV infection clears itself . Moreoveprofiles ,17.Recently, it was shown that abnormal vaginal microbiota plays a significant role in the development of cervical cancer. In the lower female reproductive tract, a healthy or normal microbiota is characterized by low microbial diversity, with one or a few species of Lactobacillus dominating the microbial community, whereas a higher diversity was found in HPV-infected women ,19.Moreover, Brotman et al. conducted a study that revealed a potential association between specific types of vaginal microbiota and the clearance or persistence of HPV infection .Numerous factors, such as ethnicity, way of life, location, and prior infectious history, can affect the cervical microbiota’s composition ,21.Therefore, it is crucial to characterize the diversity and composition of the microbiota in different individuals, particularly to understand its role in cervical cancer progression and persistent HPV infection, the latter being the leading risk factor in oncogenesis.Lactobacillus crispatus in women with normal cytology.Taking together these data, the main objective of this study was to investigate the role of the cervicovaginal microbiota in the progression of cervical lesions associated with HPV infection in women from the north-east region of Romania. Considering recent findings, our specific objective was to examine the presence of Lactobacillus iners could be associated with low-grade cervical dysplasia (LGSIL) or Sneathia with high-grade cervical dysplasia (HGSIL). The presence of these three microorganisms may allow us to establish an affordable PCR test in order to improve cytological and HPV screening. Moreover, this study seeks to identify microbiological markers that may be linked to the severity of cervical lesions and HPV infection and that may be added to the actual screening strategy (Pap and HPV tests). Furthermore, we planned to evaluate the functional composition of the microbiome in order to gain a comprehensive understanding of its role in these conditions.Additionally, we aimed to explore whether This study included 85 cervical specimens that fit the inclusion criteria from a total of 345 women who self-referred for gynecological examinations in “Cuza Voda” Clinical Hospital of Obstetrics and Gynecology between April 2021 and June 2022. The inclusion criteria for this study were as follows: women aged 18 and above, not currently pregnant, who had not taken any antibiotics, probiotics, or immunosuppression medication for at least one month prior to sampling. Additionally, participants were required to have abstained from vaginal contact or showers for a minimum of three days prior to sampling. Prior to inclusion in this study, all participants were fully informed about the purpose and details of the research and provided their informed consent by signing a consent form.The patients were invited to fill out a standardized questionnaire designed to gather information regarding their ethnicity, education, age, marital status, smoking and drinking habits, medical history, sexual activity, and place of residence.During this study, all patients underwent a single visit, and samples for all assays were collected at that specific time. This method makes sure that all data points and samples are collected at the same time. This reduces the chance of differences from multiple visits and allows for a more accurate analysis of the role of the cervicovaginal microbiota in the progression of cervical lesions and HPV infection in the participants.For microbiota, the cervical secretion was collected and preserved using an ESwab , a combination of a flocked swab and 1 mL of Liquid Amies. The specimens were stored at −80 °C until they were utilized. For the Papanicolaou test and HPV genotyping analysis, a cervical sample was collected using a separate Cervex Brush. The liquid-based preparation method for collecting cervicovaginal samples was performed according to the manufacturer’s instructions , and the samples were stored until further analysis.Diagnoses were made following the grading criteria of the Bethesda System, which includes the following categories: Negative for Intraepithelial Lesion or Malignancy (NILM); Atypical Squamous Cells of Undetermined Significance (ASCUS), Atypical Squamous Cells That Cannot Exclude High-Grade Squamous Intraepithelial Lesion (ASCH); Low-Grade Squamous Intraepithelial Lesion (LGSIL); High-Grade Squamous Intraepithelial Lesion (HGSIL); and Squamous Cell Carcinoma (SCC). This criterion qualified 120 women for inclusion in this study .DNA was isolated from cervical samples using a PureLink™ Microbiome DNA Purification Kit , according to manufacturer’s recommendations. Isolated DNAs were subsequently stored at −20 °C. The concentration and purity of each DNA sample were evaluated with a NanoDrop ND-1000 spectrophotometer .® HPV Genotyping Extra II  according to the manufacturer’s instructions. The kit contains 32 HPV-genotype-specific probes, SPF10 primer set, and human DNA control primers and provides ready-to-use amplification reagent HPV genotypes. The method allows the stratification of samples into high-risk (hrHPV), low-risk (lrHPV), and undetermined risk HPV types. The test includes 13 high-risk types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.Human Papillomavirus detection and genotyping was performed for all samples using INNO-LiPAFor sequencing the variable V3 and V4 regions of the 16S rRNA gene, the 16S Metagenomic Sequencing Library kit from Illumina  was utilized. The sequencing protocol was specifically designed to analyze the prokaryotic 16S ribosomal RNA gene (16S rRNA), and it was carried out following the manufacturer’s recommendations. For this, gene-specific primers that target the 16S V3 and V4 regions with overhang adapters attached were used  .For each sample, a mix was prepared that contained 12.5 µL 2X KAPA HiFi HotStart ReadyMix , 5 µL (1 μM) Forward Primer, 5 µL (1 μM) Reverse Primer, and 2.5 µL (5 ng/µL) DNA sample to a final volume of 25 µL. PCR conditions were as follows: initial denaturation at 95 °C for 3 min; 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; final extension at 72 °C for 5 min. The amplification was followed by a PCR Clean-Up step that purified the 16S V3 and V4 amplicon by removing free primers and primer dimer species. Briefly, every sample obtained was mixed with 20 µL AMPure XP beads and shaken at 1800 rpm for 2 min. The plate was placed on the magnetic stand, and the supernatant was removed. Next, the beads were washed 2 times with 200 µL freshly prepared 80% ethanol. After the beads air-dried, a volume of 52.5 µL 10 mM Tris (pH = 8.5) was used for resuspension. The plate was placed on a magnetic stand, and a volume of 50 µL of supernatant was transferred to a new plate.The purified amplicons were subjected to an Index PCR step that allows for the labelling of the samples by attaching dual indices and adapters. For this, 5 µL of sample was mixed with 25 µL of 2X KAPA HiFi HotStart ReadyMix, 5µL of Nextera XT Index 1 Primers (i5), 5 µL Nextera XT Index 2 Primers (i7), and 10 µL of PCR-grade water. PCR conditions were as follows: initial denaturation at 95 °C for 3 min; 8 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; final extension at 72 °C for 5 min. This was followed by a new cleaning step of the final library with ethanol 80%. The final step included libraries quantification using a Qubit 2.0 Fluorometer . All libraries were pooled in equal molar ratios and denaturated with 0.2 N NaOH for sequencing. Samples were sequenced using an Illumina MiSeq v2 600 cycle kit .https://yassour.rcs.huji.ac.il/easymap, accessed on 27 January 2023). This tool was developed using the shiny R package (version 1.6.0), along with the MaAsLin2 R package (version 1.4.0) for multivariate linear regression [Data were analyzed using the EasyMap pipeline  [p < 0.05) were performed and visualized using box plots. Beta diversity analysis was assessed with PERMANOVA statistical analysis (p < 0.05) using unweighted unifrac and weighted unifrac, Bray–Curtis and Jaccard, respectively.Fastq.gz paired files generated by MiSeq sequencing were uploaded along with sample metadata (defines studied group for each sample) and manifest files (identifies sample libraries) to EasyMAP portal (ry 2023) . After tPrincipal coordinates analysis (PCoA) was used to visualize microorganism composition and abundance similarity/dissimilarity between samples. For statistical analyses, we categorized the data into two groups based on HPV infections and six groups based on cervical cytology results. This decision was made according to multiple clustering analyses since it has been confirmed that organisms clustered in the same group are related to similar diagnosis and HPV infection. Pearson’s correlation and PCoA mainly sustain the clustering by disease stage compared to HPV infection status.Linear Discriminant Analysis Effect Size (LEfSe), a taxonomy differential abundance analysis, was used to identify microorganisms with significant differences between groups from KEGG level 1~level 3 and the Wilcoxon test (α < 0.05) . TaxonomFurthermore, microbiome function related to HPV infection and in different cytological types was predicted by using taxa abundance differentially expressed and the PICRUSt, which mapped Greengenes IDs to the corresponding KEGG pathway. The predicted microbiome function was plotted by circular graphics using a log10 LDA score.https://software.broadinstitute.org/morpheus/, accessed on 15 February 2023) [The heatmaps and hierarchical clustering (Euclidian distance) were performed using Morpheus Broad Institute matrix visualization and analysis software (ry 2023) .p-value of less than 0.05.Statistical analysis was conducted with GraphPad Prism 6 software . The results were reported as medians, which allowed for the comparison of differences among the investigated groups. For qualitative variables, Chi-square and Fisher’s exact tests were applied, while the analysis of variance (ANOVA) was used for quantitative variables. Statistical significance was defined as a This study was conducted according to the principles expressed in the Declaration of Helsinki. It was approved by the University of Medicine and Pharmacy Grigore T. Popa Ethic Committee (no 57/17.03.2021). Written informed consent was obtained from all participants.n = 18, age range: 21–54 years old), HGSIL , ASCUS  , ASCH   and squamous cervical carcinomas (SCCs) . Also, an NILM  group (n = 20) comprising 11 HPV-negative samples (NILM-) (age range: 29–62 years old) and 9 HPV-positive samples (NILM+) (age range: 25–47 years old) was included. All patients with an abnormal Pap smear, current or previous HPV infection, cervical lesion suspicion, or symptoms  underwent colposcopy. All cases with ASCUS, LGSIL, ASCH, HGSIL, and SCC underwent targeted biopsy for further evaluation. However, the biopsy results did not alter the classification for the mentioned cytological classes  . Therefore, we can be guided by the Pap test result regarding the severity of the cervical lesions in order to assess microbiota screening.The data analysis revealed that age plays a significant role in the development of cancer. Our results showed that the SCC+ group had a higher median age of 63.20 ± 12.260 years. Additionally, significant associations were observed between educational level, living area, marital status, and sexual frequency. A higher frequency of cervical cancer cases was observed in rural areas compared to urban areas within the SCC group .n = 53) had single HPV infections, while 18.82% (n = 16) had co-infections with multiple HPV genotypes  of the samples tested positive for HPV. Among the NILM samples, 45% (9/20) presented HPV infection. The patients displayed a variety of HPV infections, with some individuals being infected with a single HPV genotype, while others exhibited multiple genotypes. The most prevalent high-risk genotypes were HPV16 (27.90%) and HPV45 (11.70%). Other frequent HPV genotypes found in the samples included HPV51, HPV52, and HPV66, each with a prevalence of 8.70%. In 21.74% of the samples, the specific HPV genotype could not be identified. Additionally, some samples exhibited HPV infection with low-risk genotypes. Among all HPV-positive samples, 62.35% . On the other hand, the SCC group displayed the lowest diversity, with only four genotypes identified. Notably, the SCC group exclusively consisted of patients with single HPV infections, and the identified genotypes were HPV16, HPV18, HPV45, and HPV52. Furthermore, an analysis of the percentage of single infections with HPV16 revealed an increase, although not statistically significant, as the lesion progressed from NILM to SCC b.Lactobacillales representative, a higher frequency of Enterobacteriales and Bifidobacteriales orders, and a particularly notable presence of Gardnerella vaginalis. The SCC group displayed a higher complexity of microbiota, with the most dominant orders being Bacteroidales , Bifidobacteriales, Enterobacteriales, and Bacillales (including Staphyloccocus spp.).The bacterial community composition varied between the investigated groups, each group presenting a characteristic pattern a. While Atopobium spp.). In the HPV-positive group, there was a higher diversity of microbiota, with the predominant orders being Bifidobacteriales, Enterobacteriales, Bacteroidales, and Mycoplamatales  and L. iners were predominant species in cluster I, Escherichia coli along with Enteroccocus spp. Were predominant species in cluster II, and Atopobium spp. Was predominant in cluster III. The clusters IV and V are marked by Prevotella spp., Streptoccocus spp., and Staphyloccocus spp., and the most dominant species in cluster VI was represented by Gardnerella spp.  and ASCUS (66.01%) groups, with L. iners as the dominant species. In contrast, the SCC group displayed the lowest abundance of Lactobacillus (7.75%). The most prevalent Lactobacillus species within the investigated groups was Lactobacillus iners, even in the NILM- study group. The differences in abundance between the groups were statistically significant (p < 0.0001). The HPV-negative group demonstrated a higher prevalence of Lactobacillus compared to the SCC group, which exhibited the lowest prevalence.Further, we compared the diversity of cervical microbiota among the studied groups at the genus and species levels. It was observed that the L. iners displayed the highest abundance in the negative NILM group (42.84%), while L. unclassified emerged as the predominant species in the ASCUS group (41.97%). Certain species like L. psittaci or L. zaea showed lower levels of occurrence in the cervical samples, while others, such as L. crispatus, were not detected at all.Gardnerella_u_s was predominantly detected in women with HGSIL (19.78%), followed by women with positive NILM results (13.43%) and SCC (12.98%) cases (Prevotella_u_s was primarily identified in SCC-positive samples (12.36%) and HGSIL samples (5.45%), while its presence in other groups was nearly negligible. Additionally, other species were also identified: Escherichia coli cft073, which was found in ASCH and SCC samples , and Enterococcus faecalis v583 chromosome, which was detected in women with LGSIL (11.34%) and ASCUS (6.36%) results.%) cases . Prevotep < 0.0001) between HPV-positive samples based on the presence of a single genotype or co-infection status. One of the most important observations is that L. iners is more abundant in individuals with a single HPV genotype, whereas samples with co-infection are characterized by the presence of Lactobacilus_u_s in association with a high abundance of Prevotella _u_s.The taxonomic composition varies significantly (p < 0.0001) between the two categories. As can be observed, Lactobacillus_u_s is more abundant in the HPV16 co-infection group, while L. iners more abundant in the single-infection group. Individuals with HPV16 co-infection show lower abundances of L. psittaci and L. zeae. Moreover, the HPV16 co-infection group has a slightly increased abundance of Prevotella_u_s, while the HPV16 single group showed an increased abundance of Gardnerella_u_s and Escherichia coli cft073 , indicating an increasing trend with cervical lesion progression. The diversity composition of ASCUS was similar with LGSIL, while ASCH was closer to HGSIL individuals. The SCC group exhibited the highest diversity compared to NILM . Furthermore, the HPV-positive group showed significantly higher diversity compared to HPV-negative individuals , but no differences were observed between the single and co-infection groups in comparison to the HPV-negative group.Alpha diversity was assessed using Shannon, Faith’s, and OTU diversity analyses. The Shannon index revealed significantly higher microbial diversity in the HGSIL and SCC groups . Also, HPV-negative individuals presented a higher PD compared to HPV-positive individuals and those with single-genotype HPV infection.Faith’s analysis for phylogenetic diversity (PD) showed a significant difference regarding phylogenetic diversity between NILM and ASCH, LGSIL  were mainly associated with membrane transporters, biosynthesis, and other metabolic pathways associated with nutrient acquisition. The most important pathways were ABC transporters, aminoacyl_tRNA biosynthesis (NILM), and purine metabolism (LGSIL).The microbiome functions of bacteria in NILM and LGSIL  was primarily associated with the degradation of chemical compounds, synthesis of amino acids and B-group vitamins, and flagellum assembly and motility, as well as pathways related to prostate and bladder cancer , HPV-positive samples exhibited more diverse pathways. In samples with single-HPV-genotype infection, the microbiome function was characterized by pathways related to bacterial motility, flagellar assembly, and the synthesis of bioproducts. HPV co-infection samples presented pathways associated with chromosomes, mismatched repair, and translation factors .Lactobacillus spp., which indicates changes associated with the progression of lesions. The results presented match the results that Piyathilake et al. reported, which shows that the changes in the microbiota that were seen as the lesions became worse were similar [This study is the first conducted in Romania to assess alterations in the cervical microbiota related to different cytological classes and HPV infection. The results of our study revealed an elevated diversity of bacterial species, accompanied by a reduction in  similar .L.iners was found to be the most predominant species across the samples, along with Lactobacillus_u_s, independent of cytological status. A notable aspect of our study was the absence of L. crispatus in the studied samples. This finding contrasts with the study conducted by Di Paola et al., where L. crispatus was identified as the predominant Lactobacillus species in Italian female patients who exhibited better clearance of HPV infection [nfection .Regarding alpha diversity patterns, the ASCUS samples exhibited a higher similarity to the LGSIL samples, indicating a closer relationship between these two groups. On the other hand, the ASCH samples demonstrated similarities to the HGSIL samples, suggesting a resemblance between them. The diversity pattern can be used as a potential marker to aid in the classification and risk stratification of patients with ASCUS and ASCH lesions in either low-grade or high-grade cervical lesions.E. faecalis, E. coli, and Gardnerella_u_s. In the HGSIL patient group, there was a notable increase in the abundance of Gardnerella_u_s and E. coli. The SCC samples showed increased diversity; the microbiota included E. faecalis., Prevotella spp., and A. vaginae. To note, a few intraepithelial neoplasia lesion samples were presented by Sneathia spp.LGSIL samples exhibited a modified microbiota composition, featuring the presence of L. crispatus and the predominance of A. vaginae, along with the secondary presence of G. vaginalis and L. iners, were associated with an approximately six-fold increase in the risk of developing cervical LGSIL/HGSIL [According to the study conducted by Oh et al., it was proposed that the lack of IL/HGSIL .Lactobacillus sp. such as L. crispatus, L. gasseri, and L. jensenii to produce lactic acid and hydrogen peroxide (H2O2) enables them to inhibit the progression of viral and bacterial infections [Vaginal microbiota serves as the primary line of defense against infections, with its composition playing a crucial role. The potential of fections .L. iners and HPV infection, irrespective of the cervical lesion’s severity. L. iners is more abundant in the NILM group, who have either a single HPV infection or HPV co-infection. Furthermore, we noticed an increased abundance of L. iners that can be observed in patients with HPV16 single infection compared to those with HPV16 co-infection. L. iners has been identified as a transitional species that may contribute to dysbiosis. Several studies have shown an inverse relationship between HPV infection and CIN with dominant Lactobacillus spp., except for L. iners, which shows an opposite trend, correlated with a higher frequency of HIV, HPV, and HSV-2 infections [L. psittaci and L. zeae.Our study has also brought attention to another significant finding, which is the correlation between fections . In addiGardnerella_u_s was predominantly present in the HGSIL group, followed by HPV-positive NILM and those with SCC. This suggests that certain bacteria, such as Gardnerella, could potentially serve as biomarkers for detecting cervical changes and identifying women at a higher risk of developing persistent HPV infection with the potential to progress to cancer [Gardnerella is mediated by the increase in diversity of cervicovaginal bacteria, which directly precedes the progression to dysplasia. Gardnerella vaginalis and Atopobium vaginae have also been proposed as molecular markers, as both could be involved in biofilm formation that may contribute to virus persistence [In our study, we observed that o cancer . In the sistence .Prevotela_u_s was most abundant in the SCC and HGSIL samples. It was also found that samples with HPV co-infection presented an increased abundance. This finding is noteworthy considering the research conducted by Castañeda-Corzo et al., which demonstrated the potential role of Porphyromonas and Prevotella in the development of oral cancers [Porphyromonas and Prevotella have been identified as marker genera associated with cervical cancer [Prevotella and Gardnerella in women with persistent hrHPV infection and HGSIL [ cancers . Moreovel cancer . Anothernd HGSIL .Prevotella may play a crucial role in the development of cervical cancer within the context of HPV persistence [Based on the proposed mechanisms of chronic inflammation, anti-apoptotic activity, and production of carcinogenic substances, it can be expected that sistence .Prevotella_u_s, while the HPV16 single-infection group showed an increased abundance of Gardnerella_u_s and E. coli; therefore, we can hypothesize that a specific microbiota composition in this region may contribute to the persistence of HPV infection. These observations suggest that the presence and abundance of certain bacterial species could potentially influence the outcome of HPV infection and its progression.Consistent with previous research, HPV16 continues to be the most prevalent genotype when considering the distribution of HPV genotypes . Our finSneathia spp. and HPV detection and/or the development of cervical neoplasms. It was shown that a higher prevalence of Sneathia was found in patients who are HPV-positive and have dysplastic precursor lesions [Sneathia spp. as a potential biomarker for viral status [Moreover, previous studies have revealed an association between the presence of  lesions ,31,42. Fl status .Sneathia spp. only in four samples with diverse cytology results (HPV-positive), and its abundance was found to be very low. Interestingly, Liu et al. reported a decrease in the population of Sneathia spp. in the HPV infection groups, which contradicts the findings of previous studies that identified it as a risk factor [In contrast to the studies already mentioned, our study detected k factor .The pathogenic mechanisms underlying the interaction between the microbiota and HPV are still not fully understood, and it is important to identify the most effective therapeutic strategies. The analysis of the microbiota in clinical practice among HPV-positive patients holds promise in identifying women who are at an increased risk of HPV progression or persistence .Besides identifying alterations in the taxonomic landscape during the progression of cervical cancer, we also evaluated the functional composition of the microbiome.L. crispatus, suggesting that the anaerobic biodegradation of benzoate may be linked to the positive effects observed in the vaginal microbiota of healthy women [The NILM group was mainly characterized by ABC transporters and aminoacyl-tRNA biosynthesis pathways. Intestinal prokaryotes and archaea acquire amino acids via ABC transporters. These amino acids serve as building blocks for the synthesis of microbial proteins, including the production of aminoacyl-tRNA. Additionally, they contribute to microbial energy metabolism and can be converted into various physiologically active substances, such as neurotransmitters and hormones . The benhy women . The trahy women , prostathy women , and blahy women .E. coli can decrease MSH2 and MLH1 protein levels, which are vital for the mismatch repair process [Helicobacter pylori can suppress the expression of mismatch repair genes, partly through the modulation of miRNAs [Among the significantly modified KEGG pathways in the SCC group, porphyrin and chlorophyll metabolism were noted. Liu et al. found the same pathway affected by HPV infection in cervical lesions . Bacteri process . Additiof miRNAs .These results suggest that as precursor lesions become worse, the functional profile of the microbiota changes and becomes linked to certain metabolic pathways and processes. The microbiota in cancer samples may play a role in various biological processes and potentially contribute to the development or progression of cancer. The presence of HPV and the specific infection status can influence the functional profile of the microbiota.In addition, having a comprehensive understanding of the microbiota could facilitate a targeted and personalized therapeutic approach involving the use of antibiotics and probiotics.Even though more clinical studies are needed to fully understand the link between the microbiota and the progression of cervical lesions in high-risk patients, using antibiotics and probiotics at the same time may be a good way to change the microbiota. This approach has the potential to prevent the persistence and progression of HPV-related lesions towards cervical cancer .Nevertheless, it is important to acknowledge the limitations of the current study. Due to the small number of patients included in this study, the findings may not be representative of the entire Romanian female population since the samples were collected from a specific geographical region located in the north-east of the country. Given the high incidence of cervical cancer in Romania, conducting this study within our population was deemed essential. However, further research is warranted to determine if the observed microbiota pattern is consistent across broader regions and populations within Romania.The identification of these markers has the potential to serve as a foundation for future therapeutic strategies, including the targeted administration of probiotics. Bacterial markers derived from the cervicovaginal microbiota could be employed as a predictive model for conducting further research on larger sample sizes. This research could focus on identifying appropriate probiotics for women with persistent HPV infection, precancerous lesions at various stages, or invasive cancer. Implementing such strategies could potentially impact the outcomes of chemotherapy and radiotherapy.Our study revealed a distinct pattern within the studied group when considering the Lactobacillus species, which differs from findings reported in other populations.L. iners and the absence of L. crispatus together with Atopobium spp., Prevotella spp., and Gardnerella spp. could define the landscape for severe cervical lesions. The potential use of specific bacterial species as biological markers is intriguing from a clinical standpoint since women with a particular composition of the cervical microbiota may require more frequent monitoring or alternative therapeutic approaches.The presence of Lactobacillus spp. or that their cervical microenvironment may not be suitable for their growth. Notably, the distinctiveness of our study, compared to the existing literature, lies in the almost undetectable presence of L. crispatus, even among the HPV-negative NILM group. Instead, L. iners predominates in our studied groups, giving them a greater vulnerability to the persistence of HPV and the progression of lesions to invasive cancer.The present findings suggest that women included in this study may exhibit a deficiency of protective The high incidence of cervical cancer in Romania cannot be solely attributed to inadequate screening. Thus, these particularities of the microbiota could justify the persistence of HPV infection and the progression of cervical lesions.In conclusion, the composition of the cervical microbiota appears to be specific to populations and even individuals.L. crispatus and the presence of L. iners, even in the NILM HPV-negative group. In HGSIL patients, a small number of cases showed the presence of Sneathia spp. with relatively low abundances, while their microbiota was predominantly dominated by Gardnerella spp. and E. coli.The current research demonstrated a higher microbiota diversity in patients with HGSIL and SCC diagnosis compared to NILM individuals. This study also revealed the absence of Gaining a better understanding of the composition of the cervical microbiota holds the potential to revolutionize personalized diagnosis and the management of cervical dysplasia and cancer.By delving into the intricacies of the microbiota, we can unlock valuable insights that enable tailored approaches to diagnosis and treatment, ultimately improving outcomes for patients.


Dear Editor,I read with great interest the article on HPV DNA testing for cervical cancer screening in Brazil, authored by the Brazilian Association for the Lower Genital Tract Pathology and Colposcopy .An international cross-sectional study found that 38% of adenocarcinomas were not HPV-related.In summary, it is well recognized that HPV DNA detection delivers high sensibility, which is of great help, since cytologic-based screening is known to have low sensibility and high false-negative rates.


Background and Objectives: Oral squamous cell carcinoma (OSCC) accounts for about 95% of oral cancers. It represents a serious public health problem due to the high degree of morbidity and mortality, as well as multifactorial etiology. Human papillomavirus (HPV) infection is a well-documented risk factor for oropharyngeal carcinoma, but its role in oral carcinogenesis is still debatable. Our aim was to investigate the differences in the prevalence of high-risk HPV genotypes (HR-HPV) in patients with OSCC and oral potentially malignant disorders (OPMD) from that of healthy subjects. Materials and Methods: A total of 90 subjects were included in the cross-sectional study and divided into three groups of 30 patients each: (1) patients with OSCC, (2) patients with OPMD, and (3) healthy subjects. We examined the presence of 12 HR-HPV genotypes in the obtained biological material  using real-time PCR. Results: One or more of the 12 tested HR-HPV genotypes were detected in 5/30 patients with OSCC and 2/30 with OPMD, whereas no healthy subjects were positive for any of the tested genotypes. There was a statistically significant difference in nodal involvement between HPV-positive and HPV-negative patients with OSCC. Conclusions: Oral HR-HPV was detected in patients with oral premalignant and malignant lesions but not in healthy individuals, suggesting a possible role in oral carcinogenesis. Broad HR-HPV panel testing could increase the sensitivity of risk assessment and screening for OSCC. Oral squamous cell carcinoma (OSCC) is a subset of head and neck squamous cell carcinoma (HNSCC) and accounts for over 90% of all oral cancers. It is the sixth-most common type of carcinoma, with 400,000 new cases annually worldwide. In addition, its local aggressiveness, late onset of symptoms, and tendency to metastasize early to regional lymph nodes, as well as relatively poor response to treatment, with a five-year survival rate under 50%, make this disease an important global public health problem ,2,3. AccAn important group of mucosal disorders are oral potentially malignant disorders (OPMD) that may precede OSCC. OPMD is defined as any oral mucosal abnormality that is associated with a statistically significant risk of developing oral cancer . This grHuman papillomavirus (HPV) is currently the focus of many studies ,9,13. InSo far, scientists have identified over 200 HPV genotypes and classified them into low-risk (LR) and high-risk (HR) genotypes of inducing oncogenesis. Unlike most studies that examine the already well-known HR-HPV genotypes 16 and 18, we included in our study as many as twelve HR-HPV genotypes associated with carcinogenesis  that have been classified as carcinogenic to humans by the International Agency for Research on Cancer .Given that HR-HPV is recognized as one of the etiological factors not only for OSCC but for OPMD as well, we included in our study patients with a histologically confirmed diagnosis of oral lichen planus and leukoplakia. Our pilot study is one of the few studies conducted on the territory of the Republic of Serbia with the aim of investigating the prevalence of oral HR-HPV infection in OSCC and OPMD patients and comparing it with a healthy control group. Documenting an association between HPV infection and premalignant or malignant lesions of the oral cavity might advance our understanding of the etiology of such lesions and pave the way for improving screening and risk assessment of HPV-associated oral malignancies and possibly for new therapeutic approaches.This study was conducted as a cross-sectional pilot study at the Clinic for Maxillofacial Surgery of the Clinical Center of Vojvodina and the Clinic for Dentistry of Vojvodina in Novi Sad, Serbia, between December 2018 and April 2022. We enlisted 90 subjects (60 patients and 30 controls). This study was approved by the Ethics Committee of the Clinical Center of Vojvodina  and was conducted in full accordance with ethical principles and the Declaration of Helsinki. Each subject signed a written informed consent form prior to enrolment in this study.The exclusion criteria were respondents younger than eighteen years of age, malignant diseases of other localizations, systemic and autoimmune diseases, acquired immunodeficiency syndrome, acute inflammation of the oral cavity, chronic inflammation of the periodontium, previous treatment for cancer of the oral cavity, current chemotherapy and/or radiotherapy, pregnancy, and breastfeeding.The first patient group consisted of 30 patients in whom the diagnosis of OSCC was clinically and histopathologically verified. The second group consisted of 30 patients with a clinically and histopathologically verified diagnosis of OPMD, including OLK and OLP. The third group consisted of 30 people without pathological changes in the oral cavity who came to the Clinic for Dentistry of Vojvodina for a regular dental check-up. The subjects were given instructions not to consume food or drinks (except water) and not to smoke for at least 30 min before sampling. Material was sampled using swabs  from the lesions  or buccal mucosa (control subjects). The swabs were immediately transferred into transport medium and stored at −20 °C until extraction. The presence of 12 high-risk HPV genotypes  was detected by real-time quantitative polymerase chain reaction (qPCR).DNA was isolated from clinical specimens using the RealBest DNA-Express CE IVD extraction kit C-8899  according to the manufacturer’s instructions. Cells from oral swabs were stripped in a tube with a transport medium. Then, 100 μL of transport medium with cells was transferred into a lysis buffer tube that was incubated at 98 °C for 10 min. When cooled, samples were centrifuged at 8000 rpm for 15 min, and supernatant rich with DNA was used for subsequent PCR reactions.Genotyping was performed using RealBest DNA HPV HR genotype CE IVD diagnostic kit D-8444 in strip format  following the manufacturer’s instructions and on CFX96 IVD thermal cycler . The RealBest DNA HPV HR Genotype assay kit is designed for the differential determination of DNA of HR-HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. The genotyping kit analyzes 12 HPV genotypes in 4 separate reactions, each containing 3 HPV genotypes and internal control (IC) or human beta-actin gene in the FAM, HEX, ROX, and Cy5 channels.For the PCR reaction, 50 μL of DNA samples of either specimen, negative control (NC), or positive control (PC) were pipetted into 4 separate reaction tubes on the PCR strip and left to incubate for 20 min in a thermal cycler protected from light.The PCR genotyping reaction was performed by applying the following thermal conditions: Stage 1: 50 °C, 2 min; Stage 2: 95 °C, 2 min; Stage 3: 50 cycles of step 1: 94 °C, 10 s, and step 2: 60 °C, 25 s (fluorescence recording).The PCR run was valid only if all reactions had positive IC signals. For NC reactions, IC signals were present in 3 reactions, while the signal from the human beta-actin gene was absent. In the PC reactions, signals from the IC and all analyzed genotypes were recorded, while for each patient, signals from the beta-actin gene and signals from the 3 IC were monitored. In the absence of a signal from the human beta-actin gene, the reaction was discarded as invalid.2 test (with Yates’ correction) was applied for attributive characteristics. The p-value < 0.05 was considered statistically significant.For the statistical processing of data, the commercial statistical package SPSS 23 for Windows was used. For attributive characteristics, the data were shown in the form of absolute and relative numbers and for numerical characteristics, as mean values and measures of variability. ANOVA was applied for significance testing of differences between the three groups for numerical parameters, and Fisher’s exact probability test or the χDemographic characteristics and habits of patients and control subjects enrolled in this study are presented in Real-time PCR analysis revealed the presence of HR-HPV DNA in oral swab specimens of some OSCC and OPMD patients but none in the healthy subjects. We found HPV DNA in 5/30 (16.7%) of the OSCC samples and 2/30 (6.7%) of OPMD samples .Of the 12 high-risk HPV genotypes that were analyzed, we detected the following five types: 33, 39, 51, 52, and 59. The detected HPV genotypes and their distribution among the three groups are shown in p = 0.047) in nodal involvement among HPV-positive OSCC  cervical cancer is the most common , followed by oral (men 5.96 and women 2.28) and oropharyngeal cancer (men 1.79 and women 0.40) . In geneThe geographical component has a significant impact since the highest prevalence of HPV was recorded in Africa and Asia, especially in Chinese studies conducted in regions with a high incidence of OSCC . KatiracOur study’s population was mostly composed of elderly (median age 65) residents of the Autonomous Province of Vojvodina, Republic of Serbia. Among the usual risk factors, smoking was more ubiquitous in OSCC patients, while alcohol consumption and genetic background did not present as significant risks in our study. Other intriguing findings were differences in education and employment status among the studied groups. Namely, OSCC patients were predominantly retirees of lower educational level. Interestingly, Lins et al., in their socio-demographic study, observed that the advanced stage of OSCC is more common in males (78.4%) with completed primary education (59.1%) and under single marital status (46%) . AlthougIn this cross-sectional pilot study, we analyzed 12 high-risk HPV types in OSCC and OPMD patients, as well as healthy control subjects. There have been few studies on HPV infection in OSCC or OPMD patients in Serbia. Kozomara et al. found 64% of HPV-positive samples in 50 patients with squamous cell carcinoma of the tongue or floor of the mouth . FurtherHowever, very similar results were reported by Božić et al., who aimed to determine the major risk factors for the development of OSCC in Serbia. Among 33 patients treated for OSCC and 30 patients with benign lesions, they found 5 (15.2%) and 2 (6.7%) HPV-positive samples, respectively . InvestiOut of the 12 HR-HPV genotypes analyzed in this study, we detected five: 33, 39, 51, 52, and 59, in OSCC patients and 33, 52, and 59 in OPMD patients. Although those genotypes are associated with oral carcinogenesis , it is iA series of molecular, metabolic, and histologic changes accompany the oral cancer progression of OPMD and can be used as indicators of malignant transformation. Oral epithelial dysplasia represents a transitional phase in the malignant transformation of OPMD, as dysplastic OPMDs exhibit a higher risk of progression to OSCC . Some caSome research groups have tested the hypothesis that HR-HPV plays a role in carcinogenesis with OPMD background. Syrjänen et al. hypothesized that at a very early stage of carcinogenesis, the ability of epithelial cells to duplicate is increased when apoptotic pathways are blocked by oncogenic proteins E6 and E7 . They stNumerous studies have evaluated HPV status as a prognostic factor for head and neck cancer patients. The prognostic relevance of biologically active HPV infection is clearly established in oropharyngeal squamous cell carcinoma, showing a better prognosis for HPV-infected patients . HoweverThis pilot study demonstrated a difference in HR-HPV prevalence between OSCC patients and healthy individuals that merits further investigations, on a larger cohort, to properly assess the role of these oncogenic viruses in the process of carcinogenesis at this localization. If such a link is confirmed, the analysis of HR-HPV could, in the future, become part of a panel of biomarkers that would increase the sensitivity of cancer screening by detecting pathological alterations in biological material  and combining it with molecular-level data. Early diagnosis of oral cancer is crucial for improving patient survival rates and the detection of inapparent HR-HPV infection in otherwise healthy individuals or, in patients with OPMD, may forewarn a higher risk of malignant transformation. Moreover, the prevention of such infections, e.g., through the use of vaccines that have already demonstrated effectiveness in preventing other HPV-associated carcinomas, could offer an additional layer of prevention of this life-threatening disease.


Cervical cancer due to human papillomavirus (HPV) infection is a leading cause of mortality among women in low-resource settings. Many Sub-Saharan African countries have introduced HPV vaccination programs at the national level in the last few years. However, countries are struggling to maintain sustainable coverage. This study focuses on the introduction and sustainability challenges, context-specific key lessons learned, and mechanisms of action to achieve high sustainable coverage from low and lower-middle-income countries (LLMICs) that have introduced HPV vaccination programs by collating evidence from a literature review and key informant interviews. Local data availability was a challenge across countries, with the lack or absence of registries, data collection and reporting mechanisms. Multi-sectoral coordination and early involvement of key stakeholders were cited as an integral part of HPV programs and facilitators for sustainable coverage. Key informants identified periodic sensitization and training as critical due to high staff turnover. Health workforce mobilization was fundamental to ensure that the health workforce is aware of the disease etiology, eligibility requirements, and can dispel misinformation. Schools were reported to be an ideal sustainable platform for vaccination. However, this required teachers to be trained, which was often not considered in the programs. District-level staff were often poorly informed and lacked the technical and logistic capacity to support vaccination rounds and data collection. To improve the sustainability of HPV vaccination programs, there is a need for timely microplanning, efficient preparedness assessment, assessing training approaches, periodic training, finding innovative ways to achieve equity and adoption of a bottom-up approach to ensure that processes between districts and central level are well-connected and resources are distributed efficiently. LicensuCountries have employed various strategies in introducing HPV vaccines, some implemented pilot demonstration programs or phased roll-out prior to national introduction, while others immediately introduced HPV vaccines into their national immunization plan . HPV vacSince HPV vaccination programs are relatively new in LLMICs, national decision-makers have faced several introductory challenges in addition to the global shortage of HPV vaccine including selection of optimal delivery strategy, community level communication, targeting hard to reach populations , tailoring delivery and communication approaches to sub-national level, and determining how to maximize coverage , 8. ReceDrawing on published data and key informant interviews (KIIs), this study aimed to summarize lessons learned from recent HPV vaccination program introductions, develop a deeper understanding of facilitators and barriers to HPV vaccination programs, and identify key areas for achieving sustainable coverage.2.We conducted a qualitative, multi-method study design comprising a systematic literature review and semi-structured KIIs to determine key barriers and identify what works and why in HPV vaccination programs. After collating our results, we drew on concepts from theory-based evaluation to deepen our analysis of service delivery experiences in LLMICs by extracting and synthesizing findings on how contextual elements trigger specific barriers and mechanisms of action that could improve outcomes. The service delivery experience analysis was focused on HPV introduction and post-introduction experiences.2.1.We performed a systematic literature search in August 2021 using a combination of different keywords, including individual names of LLMICs that had introduced or planned to introduce HPV vaccination programs . The detFour databases were searched in a systematic way: Ovid, PubMed, Web of Science, and Science Direct. Of 4,910 publications retrieved, we discarded duplicates in Endnote and Covidence and shortlisted 2,844 articles for abstract/title screening. Two team members (AB and DNW) screened and shortlisted articles using a screening tool based on inclusion/exclusion criteria, leaving 213 articles for full-text screening as per PRISMA guidelines . A detaiFollowing full-text screening, we shortlisted 43 articles that included the following themes: decision-making, planning and implementation, social mobilization and communication, sustainability and lessons learned from HPV vaccination programs. To keep findings context-specific, we focused only on LLMICs.2.2.via Zoom to gain insight into HPV vaccine introduction experiences and associated challenges in their respective countries (Ten LLMICs based in Asia and Africa were identified purposefully to ensure diversity in lessons learned based on: (1) introduction status, i.e., introduced or planned introduction, (2) programmatic diversity, i.e., delivery strategy, dosing schedule, target age cohort etc., (3) vaccine coverage, and (4) geographical diversity. HPV-related publications, documents and country evaluation documents were used to prepare the initial national and sub-national level stakeholders’ list, and a snowball approach was used to identify additional experts. Key stakeholders were defined as members of the National Immunization Technical Advisory Group (NITAG), senior officials from national departments of health, and representatives from international organizations, e.g., WHO and the United Nations Children’s Fund (UNICEF). Ethical approval for the study was obtained, and participants were asked to provide an informed consent. We interviewed 18 national HPV experts from February 2022 to April 2022 (until saturation) ountries . Country2.3.2.3.1.a-priori and revised twice to allow for emerging themes and was reviewed independently by the third author (AV). The matrix included domains of decision-making, planning, social mobilization and communication, monitoring and evaluation, sustainability, associated challenges and lessons learned. Data were grouped by year of introduction, country, and primary delivery strategy to study different experiences and draw best practices.Two authors  independently extracted data from the peer-reviewed and grey literature search from December 2021 –January 2022 using an Excel-based matrix comprising key themes informed by WHO guidelines for new vaccine introduction and introducing HPV vaccine into national immunization program guide , 15. TheWe used the Joanna Briggs Institute Critical Appraisal checklist tools to evaluate the risk of bias in the articles included in the study.2.3.2.a-priori from the semi-structured interview guide [S2]. Two authors randomly selected two transcripts and conducted open coding and after discussing any discordance and disagreements, developed a codebook to use to analyze the remaining transcripts using ATLAS.ti v22  independently using thematic analysis with inductive and deductive coding developed S.ti v22 . We used3.n = 4), PATH country presentation documents (n = 2), JSI country reports (n = 2), country briefs (n = 2) and a viewpoint (n = 1), WHO Bulletin (n = 1) and CHAI resource document (n = 1)  . Fourtee (n = 1) . Experie (n = 1) .We categorized results by drawing on the domains outlined in the WHO guide on introducing HPV vaccines in national immunization programs. We categorize results using the following themes: preparation and planning, implementation, social mobilization and communication. As we included countries with existing national HPV immunization programs in our analysis, we added two additional themes: sustainability, and impact of Covid-19 on HPV vaccination programs.4.4.1.Health information systems such as databases or registries to record and report data on vaccination are essential for decision-making , optimalPaper based registries were the most common reporting tool used during vaccination sessions at schools and data registries were often not integrated with the routine immunization data collection. In some countries where reporting, data storage and tracking systems were not optimized, accessing data during school breaks and determining accuracy of collected data was a challenge . Key-infAvailability of data for enumerating eligible girls is also crucial for planning the vaccination sessions and was often inaccurate or inconsistent across sectors and programs. Countries have adopted various approaches in enumerating lists of in and out of school girls which often involved consolidating these lists from district to national level through various meetings , 47. ForDefining the target age cohort was critical to enumerate schoolgirls such as in Senegal and Zimbabwe where the target cohort was of an age that transitioned between grades and/or schools during the vaccination window. This complicated registration and tracking processes. Furthermore, access to registers at school during school breaks, accuracy of data collected, and discrepancies between records at schools and health facilities were reported as barriers for data reporting and management , 46.4.2.per diem allowances for conducting vaccination sessions in schools.Global shortage of HPV vaccine was cited as a major challenge at the national level across all programs, prompting many countries  to switch from vaccinating multi-age cohorts to single-age cohorts  (KIIs). Key informants emphasized previously reported staff shortage  for cond4.3.HPV vaccination programs require not only the involvement of coordinating bodies and the health workforce but also different governmental and non-governmental organizations to facilitate planning, introduction and implementation activities at the national, regional, and district levels. One critical partner is the Ministry of Education (MoE) , as LMICOften, countries did not have a pre-existing adolescent health program at the time of HPV vaccine introduction; thus, lack of experience with nontraditional partners raised implementation challenges. However, Rwanda’s program experience demonstrated that early inclusion of the MoE, Ministry of Gender and Family Promotion, research institutes, health workforce, and additional stakeholders is possible even without an adolescent health intervention at the EPI level , 59. Thevia different national stakeholders created lack of ownership. Barriers such as changes in staff and leadership ..via diff5.5.1.Stakeholders cited training as a critical factor to ensure that the health workforce and related stakeholders are fully aware of the vaccine, the disease etiology, target age cohort, delivery strategies, data collection and can mitigate misinformation. Duration and content of training , 34, 47 Another barrier to training was budget. To cut costs, training was often combined with other vaccination programs, conducted virtually, or packaged with non-vaccine interventions as in Tanzania , 40. HowHPV training program needs extend beyond the usual EPI target age cohort adding to the need for content-focused interactive training approaches. Healthcare professionals (HCPs) are mainly responsible for vaccination, mobilization and communication activities, including responding to rumors about HPV vaccines. However, HCPs were sometimes found to have little knowledge of disease etiology and vaccine, with some reports indicating that HCPs were unaware of the disease they were vaccinating against or the MoH-recommended target age group eligible for vaccination. In addition, in Uganda and Tanzania, only a few HCPs reported attending a condensed one-day training vaccine a few weeks before the introduction , 41. In 5.2.Countries need to select a delivery approach and primary delivery site for HPV vaccination depending on vaccine supply, feasibility of access to sites, distance between health facility and schools, school attendance and cost-effectiveness of each parameter. The two main alternatives in timing of HPV vaccine delivery approaches include campaign and regular routine. In routine delivery, vaccine is available all year round. In contrast, in campaign delivery, the vaccine is administered in campaigns lasting a few days, a week or month; usually at six-or twelve-month intervals.In many countries, schools are the primary delivery site while a smaller number opted for healthcare facilities, depending on the ease of access to the target population. We analyzed different delivery approaches and the relevant facilitators and barriers per country . Eleven In terms of different delivery approaches, Ethiopia, Zimbabwe, Rwanda , and ZamIntegration of HPV vaccination and adolescent health programs in schools was recommended by WHO as a promising practice . However5.2.1.Healthcare workers are crucial for an efficient delivery strategy provided the vaccination sessions logistics are well planned. Recent literature reports that lack of remuneration for healthcare workers in Uganda resulted in demotivated or unsatisfied staff which disrupted delivery . A criti5.2.2.Microplanning is necessary to ensure that national and district level processes are well prepared, connected, and sustainable , with a 6.Social mobilization and communication for HPV vaccination require active engagement and commitment beyond the usual coordinating bodies. A variety of key stakeholders and communications channels are needed, given the target age group and the need for prior sensitization within communities and professional organizations. It also requires a well thought-out plan and funding for sustainability beyond the initial introduction. Some countries  had a communication plan in place with experts from local and international organizations to reach different target audiences across national, regional and district levels. Communication plans included guidelines for advocacy and sensitization meetings, mass media involvement, launching ceremonies, identification of target groups, and the activities and materials meant for each target group as well as a crisis management plan. Printed products containing key messages were developed and distributed to schools, communities, and healthcare facilities through mass and social media. Senegal incorporated a module on cervical cancer in the school curriculum with collaboration between MoE and MoH .Only few publications in the systematic review provided detailed data on social mobilization and communication in countries. While these experiences intensively discussed the structure of social mobilization and communication plans prior to introduction, very few programs reported on the timing and duration of these activities.In terms of message content, the Ugandan experience demonstrated that simple messages to girls, especially regarding vaccine safety and efficacy, are useful for vaccine uptake. Similarly, the need for periodic sensitization and circulation of communication material was emphasized by KIIs , 36.Senegal and Zimbabwe conducted radio broadcasts for communication and social mobilization activities which proved to be very useful in remote areas with limited or no access to television, and communication was tailored to local languages. Kenya supplemented these activities with talk shows and mass social media outreach . Novel aNot all the planned communication activities translated into high uptake of HPV vaccine. Therefore, to develop an in-depth understanding through consolidating literature review findings and KIIs, some critical facilitators included the need for early mobilization of stakeholders at all levels , involvi7.Sustainability is commonly conceptualized as the ability to sustain high coverage within a smoothly running program, and, where programs are mature enough to enable this, to integrate HPV vaccination with other adolescent health programs. Our findings suggest further that, in some cases, sustainability also requires strengthening existing infrastructure for vaccine management and capacity building of the healthcare workforce to ensure a committed workforce  and incrTransitioning out of Gavi support often presents LLMICs with a challenge, threatening sustainability if the country cannot sustain finances for running a stable HPV vaccination program (KIIs). In such scenarios, lessons learned from other countries, such as Bhutan, that collaborated with partners for long term sustainability planning are critical .8.The covid-19 pandemic disrupted immunization activities widely but more so for HPV vaccination programs as most countries deliver HPV primarily through schools which still remain closed (April 2022) in some countries . Key-informants indicated that HPV vaccination programs have very low priority across programs, compounded by parents being hesitant to bring their daughters to healthcare facilities  especially during the covid-19 pandemic, since it was difficult to conduct outreach activities. Hence, Cote d’Ivoire adopted a Periodic Intensification Routine Immunization (PIRI) approach and switched to campaign mode to increase coverage and use available vaccine supply. There is an increasing need for a consolidated contingency plan to ensure the continuation of HPV vaccination as with other routine immunization services. Programs such as Myanmar that launched during covid-19 had to switch to a community-based delivery approach instead of a planned school-based delivery approach. Zambia reported that integrating HPV vaccination in child health week allowed minimal sustainability of the program compared with other country experiences during the covid-19 pandemic (KIIs). The coverage from year 2021 is included in Covid-19 overstretched the system leaving less room for HPV-related activities in low-resource countries, including resources diverted from routine immunization services to covid-19 and overworked healthcare workforces. Similarly, small private health facilities were closed in some countries, adding further pressure to overstretched immunization activities .9.Successful elements of pre-introduction planning and implementation must work in concert to achieve sustainable HPV vaccination programs. To capture these interactions in our synthesis of findings, we considered how success factors worked together in different domains, overcoming barriers discussed in results through different mechanisms, to achieve sustainable program outcomes .Planning requires reliable local data, efficient vaccine management and strong multi-sectoral coordination, While not explicitly found in our results, it also requires adequate time, sensitization including creating awareness among parents and adolescents . HPV proLLMICs in Africa have struggled to accurately estimate doses and assess storage space. In countries with centralized equipment management coupled with lack of preparedness assessment results in challenges with cold chain capacity and insufficient vaccine storage packs, this could be improved by timely preparedness assessment and controlled temperature chain  70). Ti. Ti70). All key informants reported lack of or inadequate training of HCPs as a challenge. Training is critical component and as countries experience ongoing competing priorities, emerging misinformation and budget constraints, it is imperative to adequately budget, evaluate and adopt innovative strategies, conduct healthcare workforce mobilization, followed by training for HPV vaccination and invest in capacity building of healthcare workforce . TraininRecently, the WHO Strategic Advisory Group of Experts on Immunization (SAGE) concluded that a single dose of HPV vaccine is comparable to two-dose schedules in protecting against cervical cancer . Switchi10.This study excluded publications and topics relating to data costing models, or potential cost-effectiveness that focused only on evidence to support vaccine introduction decisions. We did not include papers that focused on vaccine sentiment or hesitancy (given the breadth of that field) if they did not address issues related to planning or implementation of HPV vaccine programs. Using this criteria enabled us to focus on themes related to planning and implementation once the vaccine was introduced, and allowed us to have an in-depth discussion on key parameters of HPV vaccination programs. Similarly, the authors focused on LLMICs to glean context-specific lessons learned. The authors relied on information generated from peer-reviewed literature, available grey literature, and KIIs with a limited and purposefully selected group of country stakeholders; therefore, the authors acknowledge that some information could have been missed. The KIIs covered HPV stakeholders at the national level, with findings covering eight LLMICs in Africa and Asia.11.Our study documented the gaps in data collection and reporting mechanisms, the importance of multi-sectoral coordination, and the need to proactively engage stakeholders (such as the Ministry of Education) with a defined framework. Furthermore, it covered HPV delivery and training approaches, highlighting the need for context-specific implementation research through a series of recommendation in The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethics committee of Antwerp University Hospital. The patients/participants provided their informed consent to participate in this study.DNW, CM, EK, and AV worked on the study’s protocol. DNW, AB, and AV conducted the systematic literature review. DNW and DG conducted the qualitative key-informant interview study. DNW: conceptualization, writing original draft, reviewing, and editing. AV: conceptualization, reviewing, and editing. AB, AS, CM, EK, DG, MH, RL, and PVD –reviewing and editing. All authors contributed to the article and approved the submitted version.The key-informant interviews study was supported by funding from the Bill and Melinda Gates Foundation under INV-006006 . The funders had no role in the design and conduct of the study nor the decision to prepare and submit the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Purpose: Melanoma is a highly malignant tumor, which metastasizes and has poor prognosis in late-stage cancer patients. α-Mangostin possesses pharmacological properties, including antioxidant, anti-infective, and anticarcinogenic activities. We investigated α-Mangostin effect on melanoma growth, migration, and invasion and its possible molecular mechanism.Methods: Melanoma cells growth inhibition was determined by the colorimetric 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. Morphological changes of α-Mangostin-treated melanoma cells were evaluated by transmission electron microscopy and JC-1 staining. Cell apoptosis and cell cycle arrest were assessed by flow cytometry. The effect of α-Mangostin on tumor cells migration and invasion was observed by migration and invasion in vitro assay. Furthermore, the nude and C57BL/6 mouse subcutaneous melanoma models were used to evaluate the in vivo anti-tumor effect of α-Mangostin. Western blot and real time-PCR were performed to analyze the influence of α-Mangostin on some of the common signaling pathways in melanoma cell lines. Signaling pathways were further verified in dissected tumor tissues.Results: α-Mangostin inhibited in vitro melanoma cells proliferation, migration, and invasion of melanoma cells, induced cell cycle arrest in G0/G1 phase, and caused mitochondrial swelling and membrane depolarization, whereas it effectively suppressed melanoma growth in xenografted mice. In addition, α-Mangostin potentiated the in vitro and in vivo anti-tumor effects of cisplatin both in vitro and in vivo. Mechanistically, α-Mangostin down-regulated expression of RAS protein and mRNA, as well as phosphorylation of PI3K in A375, B16F10, M14 and SK-MEL-2 cells. MITF protein and mRNA were inhibited only in M14 cells.Conclusion: α-Mangostin suppresses melanoma cells growth, migration and invasion, and synergistically enhances the anti-tumor effect of chemotherapy, whose mechanism may be mediated through inhibiting Ras, PI3K and MITF. BRAF and MEK inhibitors represent the standard first-line therapy for BRAF-mutated advanced melanoma, but BRAF gene mutation rate in Chinese population is less than 26%.BRAF and MEK inhibitors, or have experienced serious drug-related adverse reactions.Melanoma is a highly malignant tumor with multiple organ metastasis, highly increasing incidence, high mortality and poor prognosis.Mangosteen is a traditional medicine in Southeast Asia used to treat skin infections, wounds, fever and diarrhea.20in vivo and in vitro melanoma cells growth, apoptosis, invasion, and migration and determine its synergistic effect with chemotherapeutic drugs and potential mechanism of anti-tumor effect. Results will provide a theoretical basis for new drug development and clinical application in melanoma.Taken together, the aim of the present study was to evaluate the effects of α-Mangostin on 2. α-Mangostin  was dissolved in DMSO in the concentration of 10 mmol/L and stored at 4°C, whereas Cisplatin (Sigma-Aldrich) and dacarbazine (Sigma-Aldrich) were dissolved in phosphate buffered saline (PBS) at 10 mmol/L and stored at -20°C until use. Paclitaxel  and vincristine  were dissolved in DMSO at 10mmol/L and stored at -80°C.The human melanoma cell lines A375 and M14 were cultured in DMEM culture medium , containing 10% fetal bovine serum (FBS), where the murine cell line B16F10 and the human melanoma cell line SK-Mel-2 were cultured in RMPI1640 culture medium (Gibco BRL), containing 10% FBS in a humidified incubator at 37 °C and 5% COCell proliferation was measured by the colorimetric 3- -2,5-diphenyl tetrazolium bromide assay . We incubated 5×10³ cells/well in 96-well plates and different drug concentrations for 24 h, 48 h and 72 h. To evaluate the synergistic inhibitory effect of α-Mangostin combined with chemotherapeutic drugs on cell proliferation, we used 10 nmol/L α-Mangostin plus different concentrations of chemotherapeutic drugs against melanoma cells. After treatments, MTT 4 h at 37°C, after which supernatant fluids were discarded, and formazan crystal were dissolved in DMSO (150 μL / well). Optical density (OD) values were then read in a microplate spectrophotometer  at 540 nm. The cell proliferation index was calculated according to the following formula: experimental OD value/ control OD value.Cells were incubated in 6-well plates and treated with 0, 5, 10 μmol/L α-Mangostin for 24 h, after which a single cell suspension was prepared using 0.25% trypsin. As a control group, 0 μmol/L α-Mangostin was composed of 0.05 % of DMSO and PBS. After washing in PBS three times, cells were fixed with 70% ethyl alcohol at -20 °C, overnight. Next, we washed the cells and incubated them with RNase for 30 min at 37 °C, after which 1mg/mL propidium iodide  was added to cells and incubated for 30 min in darkness. We then determined the percentage of each cell cycle phase by flow cytometry .For this assay, we incubated tumor cells in 6-well plates with 0 (0.2 % of DMSO in PBS), 5, 10 μmol/L concentrations of α-Mangostin for 24 h after which a single cell suspension was prepared using 0.25% trypsin. After washing in PBS three times, cells were suspended in 250 μL of binding buffer and stained with PI and Annexin V-FITC in darkness. Cell apoptosis percentages were determined by flow cytometry.Cells were treated with 0 μmol/L (0.05 % of DMSO in PBS), 5 μmol/L α-Mangostin for 24 h, then cells were lysed with cell lysis buffer (RIPA buffer and 1% PMSF) and whole-cell lysates, and protein concentrations were quantified using the BCA reagent . Proteins were separated in 10% sodium dodecyl sulfate-polyacrylamide gels and electro transferred to PVDF membranes. Membranes were then blocked with 5% bovine serum albumin (BSA) in PBS plus 0.1% Tween-20 for 1 h at room temperature (RT), probed with primary antibodies overnight at 4 °C, incubated with secondary antibodies for 1 h at RT, and developed with the ECL Advance Western blotting detection kit , using the following antibodies: anti-β-actin , HRP-conjugated affinipure goat anti-mouse IgG  and anti-rabbit IgG , anti-MITF , anti-RAS , anti-RI3K , and anti-pRI3K .Supplementary Table 1.Total RNA from tumor cells was isolated using the Trizol reagent . cDNA was reverse transcripted by the RevertAid kit (Invitrogen) and amplified by the AceQ® Universal SYBR qPCR Master Mix  on a StepOnePlus Real-Time PCR System , using GAPDH gene expression as an internal control. The primer sequences we used are shown in 5 cells/mL with serum free culture medium, and 200 μL cell suspension were added into the upper chamber of Transwell culture system  with or without pre-coated Matrigel (Sigma Aldrich) for cell invasion or migration assay, respectively, whereas 500 μL of culture medium were added to the lower chamber. After incubation for 8 h at 37 oC in an atmosphere of 5% CO2 in 95% air, Transwell chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Non-migrated cells on the upper side of the membrane were wiped off with a damp cotton swab and chambers were dried. Cells migrating to the lower surface were photographed under a microscope and counted in random three field. Experiments were repeated three times.A375, M14, SK-MEL-2 and B16F10 cells were treated with α-Mangostin for 24 h, after which cell density was adjusted to 2×10oC for 24 h, samples were rinsed with 0.1 M phosphate buffer three times, and immersed in 1% osmic acid-0.1M phosphate buffer solution for 2 h at RT. Samples were then rinsed with 0.1 M phosphate buffer three times and dehydrated in 50%, 70%, 80%, 90%, 95% and 100% ethanol. After dehydration, samples were soaked in a mixture of acetone and embedding agent (1:1) overnight, followed by soaking in pure embedding agent overnight. Specimens were polymerized in an oven at 60 oC for 48 h, and cut into 60 nm ultra-thin slices, after which they were immersed in a 2% uranium acetate saturated aqueous solution and lead citrate for 15 min, and dried at RT overnight. Cellular ultra-morphological structure was observed and photographed by electron microscopy .Cells treated with α-Mangostin were precipitated in 1.5 mL centrifuge tubes. After which 1 mL of 2.5% glutaraldehyde-0.1M phosphate buffer (pH 7.4) solution was carefully added along the tube wall. After fixing at 4 Cells were incubated in 6-well plates and treated with 0 (0.1 % of DMSO in PBS), 5, and 10 μmol/L α-Mangostin for 24 h, after which a single cell suspension was prepared using 0.25% trypsin and suspended in 500μL of cell culture medium, stained with 500 μL of JC-1 working solution  and incubated for 20 min at 37 °C. Cells were then washed with PBS twice and suspended with 1 mL PBS before evaluation by fluorospectro photometer .6 cells/100 μL in 1× PBS) was injected into the mouse right flank. In addition, B16F10 cells were trypsinized and washed with PBS twice before suspended in PBS at 5×106/mL, after which 100 μL of this cell suspension were subcutaneously injected into the right flank of six-weeks-old female C57/BL mice. Seven days after implantation, α-Mangostin, cisplatin, and dacarbazine were intraperitoneally administered every two days. α-Mangostin was dissolved in a mixture of 20% anhydrous ethanol and 80% castor oil at a concentration of 2.4 mg/mL. Cisplatin and dacarbazine were dissolved in PBS at 0.9 mg/mL and 1.35 mg/mL, respectively. The following doses were applied per treatment group: 10 mg/kg α-Mangostin, 3 mg/kg cisplatin, 4.5 mg/kg dacarbazine. Mice in control group 1 received 0.1 mL PBS and 0.1 mL anhydrous ethanol-castor oil mix injections. Because DMSO may be toxic, mice in control group 2 (PBS + DMSO) were intraperitoneally administered with 0.1 mL of 2.5% DMSO. Mice weight and tumor volume were measured every two days. At the end of the treatment, mice were euthanized and entire tumors were removed and fixed with 10% paraformaldehyde for further experiments. Nude mice blood was obtained for biochemical analysis, and normal tissues such as heart, liver, spleen, lung and kidney were dissected for histopathological analysis. Animal experiments were accomplished under the protocols of the Hubei Provincial Animal Care and Use Committee, following the guidelines and ethical standard of the Animal Experimentation Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology .The animal study was approved by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. The A375 subcutaneous melanoma model was established using eight-week-old female nude mice. A375 cells suspension .Data represent mean ± SEM of triplicate determinations from three independent experiments. Statistical differences were determined by the student Fig. ). We showed that viability of α-Mangostin-treated A375, M14, SK-MEL-2, UACC257 and B16F10 cells was significantly (p < 0.05) reduced in a time- and concentration-dependent manner. The IC50 of cells treated with α-Mangostin in each group is shown in Table 1. To evaluate the synergistic antitumor effect of α-Mangostin and chemotherapeutic drugs in vitro, cells were incubated with α-Mangostin or/and commonly used clinical chemotherapeutic drugs  for 24 h. Viability of cells incubated with α-Mangostin and chemotherapeutic drug alone or in combination was determined by colorimetric MTT reduction assay, after which the proliferation inhibition rate (IR), the synergistic index (CI50) and addition inhibition rate (Ebliss) were calculated. CI50 < 1 and Ebliss < IR of combination indicates that the chemotherapeutic drug has a synergistic effect when combined with α-Mangostin. As shown in Figure 2, CI50 of α-Mangostin + cisplatin and α-Mangostin + dacarbazine were lower than 1, and the Ebliss of the combined treatment groups were lower than IR, which suggested that α-Mangostin potentiated the anti-tumor effect of cisplatin and dacarbazine. The CI50 of paclitaxel and vincristine combined with α-Mangostin was > 1 and IR was higher than that of Ebliss. Thus, these results indicated that α-Mangostin had strong anti-tumor effect in vitro and had synergisticy effect with cisplatin and dacarbazine.Human melanoma cells A375, M14, SK-MEL-2, UACC257 and mouse melanoma cell line B16F10 were treated with increasing concentrations of α-Mangostin for 24 h, 48 h and 72 h, after which cell viability was evaluated by the colorimetric MTT reduction assay . In addition, 2.5 μmol/L α-Mangostin caused accumulation in G0/G1 phase in A375 , B16F10 , M14 (47.45 ±9.31% vs 58.52 ±3.54% cognate p < 0.05), and SK-MEL-2  cells , whereas 5 μmol/L α-Mangostin induced percentages of G0/G1 of 58.35 ±0.50%, 75.00 ±0.28%, 63.46 ±3.61% and 74.43 ±4.06%, respectively. Thus, α-Mangostin induced the apoptosis and G0/G1 arrest to inhibit in vitro melanoma cells.To demonstrate α-Mangostin anti-tumor mechanism, we evaluated apoptosis and cell cycle distribution in α-Mangostin-treated melanoma cells by flow cytometry. As shown in  Fig. A. Results showed that overall morphology and cell membrane of M14 and B16F10 cells were not affected by α-Mangostin. However, cell nuclei showed regional dense granular material and mitochondria evidenced obvious swelling, a vacuole-like structure, and disappearance or discontinuity of their crest structure in the α-Mangostin group, whereas mitochondria membrane was altered in the control group. We further examined the effect of α-Mangostin on mitochondrial membrane potential of melanoma cells. As shown in Figure , the F540/F590 ratio of A375, B16F10, M14 and SK-MEL-2 cells treated with 10 μmol/L α-Mangostin significantly  increased , which showed that α-Mangostin significantly decreased the cellular mitochondrial membrane potential. Thus, these results suggest that α-Mangostin induced mitochondrial swelling and membrane depolarization of melanoma cells.After incubation of M14 and B16F10 cells with α-Mangostin for 3 h, they were fixed, embedded and stained, and cellular ultrastructural changes were observed by transmission electron microscopyFigs. ).The migration and invasion potential of tumor cells is the key factor for distant metastasis and local invasion of melanoma. Transwell migration and invasion assays showed that the number of A375, M14, SK-MEL-2 and B16F10 cells entering the lower chamber significantly (p < 0.001) decreased after α-Mangostin treatment. This may indicate that α-Mangostin suppressed the invasion and migration of melanoma cells  reduced the tumor volume, as compared with the control group . In addition, α-Mangostin in combination with chemotherapy (dacarbazine or cisplatin) significantly (p < 0.05) reduced tumor size, as compared with the control group. We did not observe differences in contraction between the combined treatment group  and the monotherapy group . However, α-Mangostin plus cisplatin treatment the lowest average tumor volume.To determine the effect of α-Mangostin on melanoma cells growth and its potential synergistic effect in combination with chemotherapeutic drugs p < 0.01) inhibited tumor volume . Furthermore, tumor volume in the α-Mangostin + cisplatin group was significantly (p < 0.01) lower than that of the α-Mangostin and cisplatin groups. Tumor volume of the α-Mangostin + dacarbazine group was also lower than that of the dacarbazine group but we did not observe significant differences as compared with the α-Mangostin group. These results indicated that α-Mangostin exerted synergistic anti-tumor effect in combination with cisplatin in vivo.To investigate the anti-tumor effect of α-Mangostin alone or in combination with chemotherapy in C57/BL mice with complete immune system, we established the B16F10 melanoma mouse model. Results showed that α-Mangostin significantly , indicating that α-Mangostin injection did not alter mice body weight. Furthermore, on the 8th day after drug treatment, all drug-treated groups, and the PBS or the DMSO groups showed similar numbers of white blood cells, lymphocytes, and monocytes . Biochemical analysis results did not show significant differences in blood glutamic pyruvic transaminase (GPT), aspartate aminotransferase (AST), and urea nitrogen (BUN) levels between the treatment group and the PBS group or the DMSO group .We recorded changes of body weight during treatments to evaluate their safety. Results showed no statistical differences between the α-Mangostin group vs the control group, the α-Mangostin + dacarbazine group vs the dacarbazine group, and the α-Mangostin + cisplatin group vs the cisplatin group . Taken together these results demonstrated that α-Mangostin alone or in combination with chemotherapy is biocompatible and safe.HYPE staining sections of heart, liver, spleen, lung, and kidney did not show organic pathological damages in each group . Real time-PCR assay also showed that mRNA expression of RAS was down-regulated, whereas mRNA expression of PI3K was not altered. These results indicated that α-Mangostin suppressed the expression of RAS and the phosphorylation of PI3K. We have previously reported that α-Mangostin significantly down-regulated MITF expression in melanocytes. It well known that MITF may regulate apoptosis, proliferation, and migration of melanoma cells. We detected the expression of MITF in A375, B16F10, M14, and SK-MEL-2 cells. However, our results evidenced that α-Mangostin only down-regulated the expression of MITF protein and mRNA in the human melanoma cells M14. We then extracted total protein from tumor tissues, as mentioned in the in vivo experiment, and detected the expression of RAS, MITF, and phosphorylation of PI3K. RAS expression and PI3K phosphorylation in the α-Mangostin group were significantly lower than those in the control group, whereas MITF and PI3K expression was not significantly alter . It was further demonstrated that α-Mangostin may down-regulate the expression of RAS and PI3K phosphorylation in vivo.We performed Western blotting and real time-PCR to explore the signal pathway related to the anti-proliferation effect induced by α-Mangostin. MITF is the key gene that controls the production of the tyrosinase family, which induces melanin synthesis. Studies have shown that RAS/PI3K/Akt/mTOR signal transduction axis plays a core role in promoting tumor cell growth, proliferation and survival by inhibiting apoptosis. RAS/PI3K/Akt/mTOR signal pathway is one of the important cellular signaling pathways that control the expression and nuclear export of MITF. BRAF and MEK inhibitors have improved the survival rate of melanoma patients., However, a marginal number of patients have completely recovered but many patients have primary or acquired drug resistance.in vitro and in vivo, and its potentiating effect in combination with cisplatin.Immunotherapy and In vivo studies also confirmed that α-Mangostin inhibited the growth and tumor volume of subcutaneously transplanted melanoma in nude and C57BL/6 mice.Uncontrolled cell proliferation and blocked apoptosis are the basic biological characteristics of malignant melanoma cells.50 of α-Mangostin and dacarbazine in A375, B16F10, M14, and SK-MEL- 2 cells was < 1, whereas the addition inhibition rate Ebliss was lower than the IR of the combination, which confirmed the synergistic anti-tumor effect of α-Mangostin and dacarbazine in vitro. However, we did not find a statistical difference in tumor volume between the α-Mangostin + dacarbazine group and the α-Mangostin group, which might be related to the lower dose gradient of dacarbazine injection shown in previous studies, the therapeutic effect of α-Mangostin in combination with dacarbazine in vivo needs further investigation.We further studied the synergistic effect of α-Mangostin in combination with the chemotherapeutic drugs, dacarbazine and cisplatin. Dacarbazine is the earliest chemotherapeutic drug approved by FDA for the treatment of advanced melanoma, however, a number of clinical trials have shown that its overall effective rate is 13.4%, and the complete remission rate is very low.in vitro. In the immune-deficient athymic nude mouse model of subcutaneous tumor transplantation, cisplatin in combination with α-Mangostin significantly inhibited melanoma growth, as compared with the control group, but there was no statistically significant difference between the combination treatment and cisplatin alone or α-Mangostin treatment. In the immunocompetent mouse C57 model, cisplatin in combination with α-Mangostin significantly inhibited melanoma growth, as compared with the monotherapy group. Studies reported that cisplatin recruited anti-tumor CD8+ cytotoxic T cells causing direct tumor cells totoxicity or growth inhibition.et al. found that α-Mangostin has a protective effect on cisplatin-induced nephrotoxicity.The effective rate of single cisplatin injection in melanoma is only 10% to 20%. In addition, it may cause severe side effects, including nephrotoxicity, ototoxicity, neurotoxicity, vomiting and myelosuppression.Furthermore, we investigated the possible anti-tumor molecular mechanism of α-Mangostin against melanoma. A previous drug screening showed that α-Mangostin significantly down-regulate MITF expression.in vivo α-Mangostin down-regulated the expression of RAS and the phosphorylation of PI3K. However, the specific molecular mechanisms of the upstream and downstream still need more assays. We will study the mechanism underlying the synergistic effect of α-Mangostin with chemotherapies, and clarify whether α-Mangostin mediates down-regulation of MITF for anti-tumor effect.In addition to MITF, we screened several potential molecules that mainly affect certain melanoma characteristics, including RAS, MITF, PI3K, NF-κB, and STAT3. RT-PCR and Western blot analysis indicated that α-Mangostin down-regulated protein and mRNA expression of RAS and PI3K phosphorylation in melanoma cells. It has been previously reported that RAS/PI3K/AKT/mTOR pathway plays an important role in tumorigenesis, regulating cell cycle distribution and angiogenesis, which is a potential target in melanoma therapy.in vivo. In order to overcome its poor solubility, α-Mangostin may be stably loaded into nanomaterials or liposomes to achieve a more significant bioavailability. In addition, the results of this study found that α-Mangositin combined with chemotherapy drugs has a synergistic anticancer effect. Constructing a dual delivery system loaded with α-Mangostin and chemotherapy drugs would have better anti-cancer effects while reducing drug dosage and toxicity.As a fat-soluble and small molecular weight drug, α-Mangostin is easy to obtain and develop as an agent with low toxicity. However, α-Mangostin has been observed to toxicity in high doses, and is poor water solubility, resulting in low bioavailability and hindering the use of α-Mangostin in vitro and in vivo effect in combination with cisplatin. Regarding α-Mangostin anti-tumor mechanism, we found that this xanthone down-regulated RAS, p-PI3K and MITF expression. As a natural extract, α-Mangostin is easy to obtain and develop as an oral agent with low toxicity. Therefore, α-Mangostin is a potential anti-melanoma drug and chemotherapeutic sensitizer, which provides a theoretical basis for the development and clinical application of new drugs on melanoma patients.We comprehensively investigated the anti-tumor effects of α-Mangostin on melanoma cells. It suppressed melanoma cell proliferation, migration, and invasion. and possessed synergistic Supplementary figure and table.Click here for additional data file.