Lactobacillus species may not necessarily be the sine qua non of healthy cervicovaginal microbiota (CVM), especially among reproductive-age African women. A majority of African women have high-diversity non-Lactobacillus-dominated CVM whose bacterial functions remain poorly characterized. Functional profiling of the CVM is vital for investigating human host-microbiota interactions in health and disease. Here, we investigated the functional potential of L. iners-dominated and high-diversity non-Lactobacillus-dominated CVM of 75 African women with and without bacterial vaginosis (BV) and high-risk human papillomavirus (HR-HPV) infection. Functional contents were predicted using PICRUSt. Microbial taxonomic diversity, BV, and HR-HPV infection statuses were correlated with the inferred functional composition of the CVM. Differentially abundant inferred functional categories were identified using linear discriminant analysis (LDA) effect size (LEfSe) . Of the 75 women, 56 (74.7%), 35 (46.7%), and 29 (38.7%) had high-diversity non-Lactobacillus-dominated CVM, BV, and HR-HPV infection, respectively. Alpha diversity of the inferred functional contents (as measured by Shannon diversity index) was significantly higher in women with high-diversity non-Lactobacillus-dominated CVM and BV than their respective counterparts . Ordination of the predicted functional metagenome content (using Bray-Curtis distances) showed that the samples segregated according to the extent of microbial taxonomic diversity and BV  but not HR-HPV status . LEfSe analysis of the inferred functional categories revealed that transport systems (including ABC transporters) and transcription factors were enriched in high-diversity CVM. Interestingly, transcription factors and sporulation functional categories were uniquely associated with high-diversity CVM, BV, and HR-HPV infection. Our predictive functional analysis reveals features unique to high-diversity CVM, BV and HR-HPV infections. Such features may represent important biomarkers of BV and HR-HPV infection. Our findings require proof-of-concept functional studies to examine the relevance of these potential biomarkers in women’s reproductive health and disease.Mounting evidence suggests that  Lactobacillus species, notably L. crispatus, L. gasseri, L. iners, or L. jensenii [Lactobacillus-dominated CVM, often with high bacterial diversity, among some healthy women. A series of studies have observed that the patterns of CVM vary markedly among women from different ethnic/racial background, with high-diversity CVM being not uncommon among women of African descent [Lactobacillus-dominated CVM, CVM with L. iners dominance is often the most prevalent [Lactobacillus spp., L. iners has been pointed out to be the least stable and least protective; hence, can facilitate transition between bacterial vaginosis (BV) and non-BV (other Lactobacillus-dominated) states [L. iners are more likely to be predisposed to BV relative to women colonized with L. crispatus [A healthy cervicovaginal microbiota (CVM) of reproductive-age women is regarded as one that is colonized predominantly by a single or multiple jensenii . However descent ,3, inclu descent , Kenyan  descent , Rwandan descent , and Sou descent ,8. Moreorevalent ,3,5,8. A) states . Women crispatus .Lactobacillus spp. does not necessarily reflect an unhealthy CVM [The differences in CVM composition within African women and between African women and women from other ethnicities/races could be attributed to human host genetic background , sociodelthy CVM  and has lthy CVM , such CVlthy CVM ,13, cervlthy CVM , and sexlthy CVM . BV, STIlthy CVM –17.Gardnerella vaginalis-dominated CVM, can consequently alter the cervicovaginal epithelial barrier [Most studies have focussed on exploring the structure (composition and diversity) of the CVM of women with and without genital syndromes, infections, and diseases. These studies have shown that the structure of the CVM of such women varies according to cervicovaginal conditions ,14. A ca barrier , thereby barrier ,20,23–25 barrier , and com barrier  and Tax4 barrier  using 16PICRUSt is a validated bioinformatics tool that has been widely used for predicting the functional contents of microbiota of different environmental sites. However, it is worth noting that there are less than a handful CVM studies that have used PICRUSt to infer the functional potential of bacteria ,21,28. AL. iners and high-diversity non-Lactobacillus-dominated CVM are the most common CVM among South African women  [L. iners concomitant with higher relative abundances of BV-associated bacteria, notably G. vaginalis, Atopobium vaginae, Prevotella sp., and Sneathia sp. [The CVM of women in sub-Saharan Africa is becoming more known ,6–8,30. ctively)  and HPV ctively) ,16 infecthia sp.  to inferG. vaginalis) and/or noticeable absence of lactobacilli on wet microscopy were regarded as an indication of BV. The second swab, which was for HPV genotyping and CVM study, was stored in Digene specimen transport medium  at -80°C until nucleic acid extraction. HPV genotyping was performed using the Roche Linear Array HPV genotyping test  that detects 37 HPV genotypes: 12 oncogenic high-risk (HR), 8 probable oncogenic HR, and 17 non-oncogenic low-risk HPV types as documented elsewhere [Endocervical samples for the present functional study of the CVM were obtained from the HPV Couples Cohort Study that investigated the concordance of genital HPV infection in heterosexually active South African couples and the impact of HIV coinfection . Ethicallsewhere . The datThe inclusion and exclusion criteria and the study population characteristics of the 87 reproductive-age (18–44 years) HIV-seronegative women included in this study are detailed elsewhere . All theComparison of baseline characteristics of the women according to HR-HPV infection status indicated that younger age was associated with HR-HPV positivity (28.0 (24.5–35.0) versus 35.0 (27.8–41.0) years; p-value = 0.015). Cervical cytology was statistically different between HR-HPV-negative versus HR-HPV positive group , with HR-HPV-positive women being less likely to have normal cervical cytology compared to HR-HPV-negative women ; p-value = 0.015). None of the variables statistically differed between women with and without BV.CCTACGGGNGGCWGCAG-3’) and 806R (5’-GACTACHVGGGTATCTAATCC-3’). PCR and sequencing controls were run concomitantly with the 87 samples. These included i) nuclease free water as a negative control, ii) Digene specimen transport medium as an extraction control to check for possible contaminants, and iii) two mock bacterial communities, HM-782D and HM-783D , comprising of genomic DNA from 20 bacterial strains, as positive controls. HM-782D contained equimolar concentrations  of each of the 20 bacteria. In HM-783D, the concentrations of these bacteria were staggered . A dual-indexing approach amplification approach was used to prepare 16S rRNA gene amplicon libraries, which were later purified using Agencourt AMPure XP System . Libraries were then pooled in equimolar ratios and sequenced on an Illumina MiSeq by Macrogen Inc. .Genomic DNA extraction, amplification, metabarcoding library preparation, paired-end sequencing, and data analysis were performed as previously described . The hypde novo OTU picking using a greedy clustering algorithm [QIIME  v1.8.0  and UPARlgorithm . Beta diL. iners: 0.0222%) in the even mock community control (HM-782D). Here, we assumed that any amplification and sequencing error in our dataset was homogeneous across the samples. So, using QIIME’s “filter_otus_from_otu_table.py” script, we used the argument “—min_count_fraction” to filter OTUs from the OTU table based on the 0.0222% threshold. The argument “—min_count_fraction” is defined as the fraction of the total observation (sequence) count to apply as the minimum total observation count of an OTU for it to be retained in the OTU table (http://qiime.org/scripts/filter_otus_from_otu_table.html).All our analyses were based on high-quality filtered sequence data. This was confirmed by the observations of minimal to absent false positives and negatives (in terms of reads) in our dataset, including controls (results not shown), as reported elsewhere . MoreoveLactobacillus spp. and bacteria with ≥0.33% relative abundance. Alpha diversity estimations of the CSTs were re-examined using rank abundance curves of the OTUs.For the identification of the CSTs, we relied on an unrarefied OTU table (unlike in beta diversity estimations). Average neighbour linkage unsupervised clustering of the samples was performed (based on Bray-Curtis dissimilarity index) as previously documented  in orderFurther analyses to detect differentially abundant bacterial taxa (in the unrarefied OTU table) between the most prevalent CSTs were performed by statistical analyses of metagenomic (and other) profiles (STAMP) v2.1.3 . The WhiPICRUSt v1.1.0  was used2-transformed raw count matrix data of the top 50 inferred functional categories with the greatest variance. These results were displayed on a heatmap generated using R package pheatmap v1.0.12 (https://CRAN.R-project.org/package=pheatmap). Associations of the identified functional clusters with BV, HR-HPV, and CST (taxonomic clusters) were computed according to Chi-square/Fisher’s exact tests  using GraphPad Prism v6.01 .In order to determine whether the CVM could be grouped by their predominant inferred functional categories at level 3 KEGG Orthology (KO), we used DESeq2 v1.26.0  in R v3.Alpha diversity estimates of the predicted functional contents  were computed using Shannon diversity and Bray-Curtis dissimilarity indices, respectively, within QIIME. For beta analyses, the number of predicted functional contents in each sample was normalized to 5,921,112 across all the samples. Next, the correlations between the participant information  and inferred functional categories were studied using principal coordinate analysis (PCoA). Differences in the abundances of the predicted functional categories were analyzed and visualized using linear discriminant analysis (LDA) effect size (LEfSe) v1.0  and STAMG. vaginalis, A. vaginae, Dialister sp., Clostridium sp., Megasphaera sp., and Sneathia sp. The other CVM were dominated by L. crispatus (CST-1: 2.3%), L. jensenii (CST-2: 2.3%), L. iners (CST-3: 21.8%), Aerococcus sp. (CST-4: 1.1%), Streptococcus sp. (CST-5: 4.6%), Chlamydia trachomatis (CST-6: 2.3%), or Corynebacterium sp. (CST-7: 1.1%). Most of the downstream analyses focussed on the most prevalent CSTs (CST-3 and CTS-8).The heatmap in L. iners and L. crispatus were in greater relative abundance than in CST-8 . In CST-8, BV-associated bacteria were more abundant than in CST-3 . Comparison of the demographic, sociobehavioural, and clinical characteristics of the women in CST-3 and CST-8 are presented in The alpha diversities of the CSTs were determined by rank abundance of the bacterial community populations . For theTo predict the functional metagenomic capacity of the CVM, we used PICRUSt ; a softwAt level 3 of the KEGG database, 264 predictive functional categories were identified. The most predominant ones included transporters (relative abundance: 6.0%), general function prediction only (3.5%), ATP-binding cassette (ABC) transporters (3.0%), DNA repair and recombination proteins (2.7%), and ribosome (2.3%). We represented top 50 predicted functional categories with the greatest variance using a heatmap . Based oLactobacillus-dominated CVM  and BV  than their respective counterparts. Women with and without HR-HPV infection did not have statistically different in alpha diversity . We corroborated the associations of the functional clusters with participant variables  using PCoA of the inferred functional categories. We found that the inferred functional categories separated according to the taxonomic diversity of the CVM  and BV-associated CVM (CST-8) have differentially abundant bacterial functions. A total of 169 inferred KEGG functional categories significantly differed between CST-3 and CST-8 . At a logarithmic LDA score of >3.0, 36 inferred functional categories differentially abundant as indicated in Next, we performed LEfSe analysis to test whether the CVM of women with In the context of BV, there were 78 predictive functional categories between the CVM of women with and without BV. Thirty five inferred KEGG functional categories differed significantly  between the groups , with 14Lastly, we compared the differences in relative abundances of the inferred functional categories between samples of women with and without HR-HPV infection. Samples from women with HR-HPV infection had 11 predictive functional categories  while those from women without HR-HPV infection had 20 categories , p-value <0.05, logarithmic LDA score >2.0 .L. iners dominance were enriched with membrane and intracellular structural molecules, LPS biosynthesis, amino acid metabolism, citrate cycle, and protein folding modules.It is interesting to point out that several inferred functional categories were significantly differentially abundant in all three comparisons . Serving as examples are sporulation and transcription factors, which were enriched in the high diversity CVM (CST-8) as well as CVM of women with BV and HR-HPV infection. In contrast, the CVM of women without BV and HR-HPV infection and with Lactobacillus spp. that promote cervicovaginal health by reducing the risk of STIs through intrinsic mechanisms [This study was undertaken to predict the bacterial functions in the CVM of South African women with and without BV and HR-HPV infection. The structure of the female genital tract provides a unique milieu for growth of endogenous chanisms . HoweverLactobacillus spp. , four by less frequently reported non-lactobacilli , and one by a mixture of BV-associated bacteria (CST-8). These CSTs are discussed in detail elsewhere [Lactobacillus spp. from the cervicovaginal milieu, L. crispatus and L. iners are thought to have the highest and lowest protective functional values, correspondingly [Lactobacillus CVM may facilitate transition to dysbiotic CVM or BV state [L. iners (mean proportion: 82.4% versus 2.6%), L. crispatus, albeit in low proportion (1.2% versus 0.002%), was more abundant in CST-3 compared to CST-8. Higher proportions of BV associated bacteria such as G. vaginalis (12.7% versus 3.1%), A. vaginae (4.4% versus 0.2%), and Megasphaera sp. (12.3% versus 0.7%) differentiated CST-8 from CST-3. Although not investigated, we hypothesize that such differentially abundant bacteria and their multipartite relationships with one another could be responsible for the differences in the inferred metagenomic functional profiles between the two CSTs.We identified eight CVM clusters (CSTs) in reproductive-age South African women. Three of these CSTs were dominated by common lsewhere . Of the ondingly . Analogoeria CST-. These Ceria CST-. These Condingly  and thatBV state . BecauseL. crispatus [G. vaginalis and Sneathia sp. strains [We compared the relative abundances of the predicted metagenome functions of CVM in CST-3 and CST-8, and CVM of women with and without BV and HR-HPV infection. Several differences in inferred KEGG functional categories that have been previously identified in cervicovaginal and uterine microbiota ,23–25, wrispatus . In our rispatus . In the rispatus  and drugrispatus . ABC trarispatus . Genes e strains ,48. In o strains  and its  strains .G. vaginalis is thought to be the driver of community diversity and biofilm development [G. vaginalis co-occurred with BV-associated bacteria. Interestingly, it has been reported that G. vaginalis associated with BV and biofilms uniquely encode genes for ABC transporters that are absent in the genomes of G. vaginalis strains not associated with BV [G. vaginalis biofilm cells compared to planktonic cells [elopment . In CST- with BV . Furtheric cells . Cells iic cells  stated tic cells , that isic cells . Moreoveic cells –55. ABC ic cells ,57.G. vaginalis, a predominant member of CVM in CST-8, BV-positive and HR-HPV-positive women in our cohort [L. iners, the predominant member of CST-3 CVM, in contrast, does not have the genetic capacity to ferment fructose [G. vaginalis. These findings are supported by a metaproteomic study that noted that proteins involved in catabolism and membrane transport functions were more abundant in CVM dominated with G. vaginalis than L. iners [G. vaginalis to outmatch lactobacilli competency for the uptake of extracellular saccharide. It is necessary to underscore that, differences in the inferred functional contents of CVM of women with and without HR-HPV infection could be due to significant differences in the women’s age and cervical cytology. This is because compositional and/or functional differences in CVM may be impacted by reproductive aging [We further noted that fructose and mannose metabolic functionalities were enriched in CST-8, and CVM of women with BV and HR-HPV infection. Starch and sucrose metabolisms as well as PTS were additionally enriched in the CVM of women with BV. Specific PTS involved in pathway of starch and sucrose metabolism have been identified in cervical samples . Fructosr cohort , has ther cohort . L. inerfructose . In a cofructose . This maL. iners . Exampleve aging  and cervve aging ,25.Lactobacillus have been associated with BV, cervicitis, obesity [G. vaginalis and other BV-associated bacteria, which were predominant in CST-8, with HR-HPV infection [G. vaginalis from women without BV have been associated with proteomic signatures of disrupted cervicovaginal epithelial integrity [Lactobacillus-dominated CVM with BV-associated bacteria have altered bacterial functions that may subsequently disrupt the host protective mechanisms against invasion by pathogens and pathobionts.CVM composition and function have a profound effect on women’s reproductive health. Lower relative abundances of  obesity , and HPV obesity ,10. We pnfection . Communintegrity . It is tntegrity . Subsequntegrity . In the ntegrity . Thus, oWe acknowledge that 16S rRNA gene metabarcoding approach can result in selection biases of certain bacterial taxa, this stemming from amplification and sequencing biases. Our results might not be fully reliable since computational gene annotations may be inaccurate . It has In our cohort, the functional potential of CVM was impacted by microbial diversity and BV, but not HR-HPV infection. Our analysis revealed functional potential signatures of high-diversity CVM, BV and HR-HPV infections. Such differentially abundant functional categories in CVM of women with and without microbial diversity, BV, and HR-HPV infection may have diagnostic, therapeutic, and prognostic applications. Predicted functional categories that were common in women with high-diversity CVM, BV, and HR-HPV infection suggest interconnectivity between CVM and these reproductive outcomes (BV and HR-HPV infection). Our findings are hypothesis-generating and warrant confirmation using functional studies.S1 FigNSTI value describes the average branch length that separates each OTU in a sample, weighted by the relative abundance of that OTU in the sample. NSTI values range from 0 to 1, with a high value depicting greater distance to the closest sequenced relatives of the OTUs in each sample. A higher value could be as a result of unexplored diversity. A lower NSTI depicts a higher similarity to the closest sequenced taxon. From the bar charts, most of the samples had weighted NSTI values of between 0.07 and 0.15; thus, reflecting availability of reference genomes and relatively good quality of the PICRUSt predictions.(TIF)Click here for additional data file.S2 FigPCoA according to high-risk human papillomavirus (HR-HPV) status (Maya blue: HR-HPV-negative and Indian red: HR-HPV-positive). Each solid dot represents one cervicovaginal sample. The first three PCoA axes and the percentage variation explained by each indicated are shown .(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Thermotoga maritima in the production of gardenia blue. The enzyme exhibited a maximum activity of 10.60 U/mL at 90 °C. Single-factor and orthogonal analyses showed that exogenously expressed heat-resistant glucosidase reacted with 470.3 μg/mL geniposide and 13.5 μg/mL glycine at 94.2 °C, producing a maximum yield of 26.2857 μg/mL of gardenia blue after 156.6 min. When applied to the dyeing of denim, gardenia blue produced by this method yielded excellent results; the best color-fastness was achieved when an iron ion mordant was used. This study revealed the feasibility and application potential of microbial production of gardenia blue.Gardenia blue is a natural blue pigment that is environmentally friendly, non-toxic, and stable. The hydrolysis of geniposide, catalyzed by β-glucosidase, is a critical step in the production process of gardenia blue. However, β-glucosidase is not resistant to high temperatures, limiting the production of gardenia blue. In this study, we investigated the effectiveness of a heat-resistant glucosidase obtained from  In contrast to synthetic dyes, which can cause serious adverse effects in humans, they are generally environmentally friendly and non-toxic. Moreover, numerous harmful substances are generated in the production processes of synthetic dyes. Blue dyes are the most challenging to obtain because there are few natural sources . Most naGardenia jasminoides  and [100 480 14 180]. The initial population was set at 20, double-precision was selected as the population type, Roulette was selected as the population selection method, single-point crossover was selected as the crossover method, the crossover probability was set at 0.75, the variation probability was set at 0.05, the evolutionary generation was set at 100, and the other parameters were kept as default.2.7After obtaining highly concentrated gardenia blue pigment, we verified its dyeing effect on denim textiles  in different conditions.The direct dyeing scheme was as follows: a water bath was heated to 40 °C, and a white denim cloth and the gardenia blue pigment were added to the water bath at bath ratios of 10:1, 30:1, and 50:1. Then, the water bath was heated to 60 °C at a rate of 2 °C/min, and this temperature was maintained for 10 min. Then, NaCl (2.3%) was added to the water bath according to the volume of the reaction system. The reaction system was maintained at 60 °C for 30 min for continuation of the dyeing process. Finally, the textile was washed with deionized water at room temperature (20–25 °C) and dried.For the mordant dyeing method, iron ions, aluminum ions, cerium ions, or lanthanum ions were used to prepare different dyeing media. Dyeing periods of 10, 20, 30, 40, 50, and 60 min and dyeing temperatures of 30, 40, 50, 60, and 70 °C, and 80 °C were tested.33.1E. coli BL21, a plasmid harboring bglA was constructed and transformed into E. coli BL21 cells. The expressed protein was subjected to SDS-PAGE. As expected, strong expression of a protein of approximately 80 kDa was observed  was defined as 1 nmol of p-nitrophenol produced per min per unit volume. First, a standard curve was constructed, as shown in 3.3T. maritima I (1w3j.1.A), family 1A bglA from Thermotoga neapolitana, mutant E349A (5idi.1.A), bglB (4hz6.1.A), Halothermothrix orenii bglA A (4ptv.2.A), M3-mutant bgl6 (5gnz.1.A), and heat-resistant bglA. The degrees of sequence similarity between bglA and these proteins were 73% (1w3j.1.A), 71% (5idi.1.A), 61% (4hz6.1.A), 61% (4ptv.2.A), and 61% (5gnz.1.A). A series of heat-resistant bglA models were obtained using each homologous protein as a template, and the model with the highest degree of sequence identity (73%) was selected as the final template.A homology-based model of the heat-resistant bglA was generated using the SWISS-MODEL program. To this end, five proteins of known structure with significant amino acid sequence similarity to bglA were selected, including family 1 bglA from Molecular docking of bglA and geniposide was performed using Biovia Discovery Studio , and the positions of the vital active sites of the enzyme were determined. The top five amino acid residues that generated hydrogen bonds were ASN246, GLU166, ASN223, HIS298, and SER296, and the top five amino acid residues that generated hydrophobic interactions were TRP324, VAL173, TRP406, HIS180, and HIS298 .Fig. 4A.3.4595 measurement for subsequent experiments. Glycine exhibited the highest OD595 absorption peak and was therefore selected for use in subsequent experiments . Color-fastness was tested according to national standards  and graded from 1 (worst) to 5 (best). The results are shown in 4E. coli, and the enzyme was found to exhibit optimal activity (10.60 U/mL) at 90 °C. Recombinant bglA is a typical thermophilic enzyme with high thermal stability and optimal enzyme activity above 70 °C. However, as thermophilic enzyme-coding genes are mostly found in archaea, their codon usage rates are significantly different from that of the model bacterium E. coli. Therefore, the exogenous expression and purification of thermophilic enzymes are challenging. In this study, bglA was codon-optimized for efficient expression in E. coli [In this study, thermotolerant bglA was successfully produced in  E. coli . However E. coli .Homology modeling of the heat-resistant bglA clearly showed that it displayed a typical TIM barrel structure, consisting of a peptide backbone with eight outer α-helices and eight inner parallel β-chains. The TIM barrel structure is common in known proteins, and the active sites of enzymes with this structure are generally located at the C-terminus of the β-chain. The salt bridge formed inside the β-chain is thought to contribute to the stability of the whole protein structure . CompariWith respect to gardenia blue preparation, many studies have reported the production of genipin by using bglA to hydrolyze geniposide. These studies have led to improvements in the hydrolysis efficiency of bglA by discovering new enzymes , mutatioGiven the numerous applications of dyes in the daily lives of humans, the pollution caused by the synthetic dye chemical industry is becoming increasingly serious. The demand for natural pigments that are inherently non-toxic and harmless and have environmentally friendly production processes is increasing . One of The current study had a few limitations. Only glycine was selected for the experiments to optimize the synthesis of gardenia blue; optimal reaction conditions for other amino acids remain to be explored. The blue pigments produced when different amino acids are used as substrates vary (from indigo to purple colors).A previous study 5T. maritima was transformed into E. coli and expressed efficiently. The exogenously expressed enzyme exhibited an optimal activity of 10.60 U/mL at 90 °C. When the exogenously expressed enzyme was reacted with 470.3 μg/mL geniposide and 13.5 μg/mL glycine at 94.2 °C, a maximum yield of 26.2857 μg/mL of gardenia blue was obtained after 156.6 min. The gardenia blue pigment produced by this method showed excellent performance in the dyeing of denim, with the best color-fastness obtained when iron ions were used as a mordant.A heat-resistant glucosidase-encoding gene from Wenxi Li: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – original draft, Supervision, Project administration. Jielin Li: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – original draft, Visualization. Ying Xu: Methodology, Modeling, Formal analysis, Data curation, Visualization. Shuqi Xu: Validation, Investigation, Formal analysis. Zirui Ou: Validation, Investigation, Formal analysis. Xiaoli Long: Supervision, Project administration, Visualization. Xinyu Li: Methodology, Validation, Formal analysis, Investigation. Xinyu Liu: Methodology, Investigation. Zening Xiao: Investigation. Jiaqi Huang: Investigation. Weizhao Chen: Conceptualization, Methodology, Writing – review & editing, Supervision, Project administration, Funding acquisition.The authors have no known competing interests to declare.


This paper describes the influence of the presence of an obstacle near the flue pipe’s mouth on the air jet, which directly affects the parameters of the sound generated by the flue pipe. Labial pipes of the most common types of mouth were tested. The method of interval calculus was used instead of invasive measuring instruments. The obtained results prove that the proximity of an obstacle affects the sound’s fundamental frequency, as the airflow speed coming out of the flue pipe’s mouth changes. The relationship between the airflow speed, the value of the Reynolds number, and the Strouhal number was also established. The thesis of the influence of the proximity of an obstacle on the fundamental frequency of the sound of a flue pipe was generalized, and formulas for calculating the untuning of the sound of the pipe were presented for various types of mouth. Pipe organ tuning is a tedious process. Some of them require many series of tweaks during tuning. Finally, it is possible to tune the instrument. This is one of the reasons why probably no one has analyzed the problems accompanying the tuning process so far.Another problem is the undoubted lack of time to undertake tests and the lack of willingness to break the routine in the activities performed. Knowledge in the field of organ building is handed down from generation to generation. The next problem is the lack of a sufficient explanation in the scientific papers regarding the everyday work of organbuilders. There are conferences or meetings about organ building but from the musicians’ point of view, not the organbuilders. There are no conferences where the organbuilders could exchange their experiences or discuss together the problems bothering them.The impetus for this research on the optimization of organ tuning are the problems that have been mentioned by organbuilders for many generations ,2. The uThe problem of untuning is an issue worthy of attention. It is a popular issue as it concerns the work of every organbuilder. Optimizing the tuning process will significantly shorten the time of tuning pipes. This entails savings also in expenditure on the construction or renovation of the instrument, which in the case of the pipe organ is usually one of the key elements of investment. In addition, it will facilitate work with small organs.In a previous publication  the authThe state-of-the-art research in the area of sound generation in organ pipes raises various aspects, e.g., airflow analysis ,5,6, or This paper contributes to the research area of sound generated by flue pipes. First of all, it aims to facilitate the process of pipe organ tuning. The results of this research apply to the pipe organ, in which the flue pipes are close to an obstacle. Such an obstacle may be the organ case or the adjacent pipes, which are the obstacles for each other. A good example is a chest organ, where the space on the windchest is limited. We propose an equation that allows determining the fundamental frequency of a pipe’s sound depending on the proximity of an obstacle. This issue is practical and will facilitate the daily work of organbuilders. The novelty is the use of a non-invasive method, with calculations based on the interval calculus.The paper is organized as follows. The measurements of the pipes—both geometric dimensions and acoustic measurements—were carried out on the intonation table in the organbuilder’s workshop. We used two measurement microphones Behringer ECM8000 with omnidirectional characteristics, 1.0 mV/Pa sensitivity, and 20 dBA equivalent noise level. The first of them was used for recording the sound at the top of the pipe (distance from the pipe top about 5 cm), and the second one near the pipe lip (distance from the lip about 5 cm). The sampling rate was 48 kHz. The recordings were repeated at least 5 times. The positioning of the microphones is shown in The recordings were made after calibration using an acoustic sound level calibrator Sonopan KA-50 . The analyzed types of pipes with their dimensions, construction and photos are presented in f0) of the sound is located. To determine the precise frequency values, Discrete-time Fourier Transform (DTFT) [jωe) for the discrete sequence of the signal x[n] is given by Equation (1):In the first step, we selected the central part of each of the audio recordings, representing the steady state. Next, these fragments were transformed into spectra using the Fast Fourier Transform (FFT) with a Hanning window of 65,536 samples. The spectrum calculated via the FFT was used to determine the range in which the fundamental frequency   was calcMσ of the obtained results. We used the standard deviation Mσ for individual values of the fundamental frequency.Mµ is the average of the set M = {M[1], …, M[n]}, M[k] is the k-th value of the M set, n is a number of measurements.Equation (2) determines the measurement uncertainty f0 for each recording with an accuracy of 0.01 Hz, as we calculated DTFT for the frame length equal to the entire sound (about 5 s). The sound level for f0 was also calculated in relation to the sound level generated by the calibrator and the measurement uncertainty Mσ from Equation (2).The use of DTFT allowed us to determine the fundamental frequency f0 were calculated, which are expressed in cents and are the musical distances between two sounds. Equation (3) was used to calculate each of the intervals, where x is the distance of the obstacle from the pipe lip and x0 is the distance, at which the sound frequency does not depend on the obstacle. f0 and x.To describe the changes in sound frequency in the situation of the presence of an obstacle, the intervals WThe fundamental frequency of the pipe decreases as the obstacle is closer to the lip. The changes of the pipe sound frequency depending on the distance of the obstacle to the mouth of the pipe are presented in The spectrum of the sound recorded at the mouth of the pipe may differ significantly from the spectrum of the sound at the top of the resonator (i.a. significant difference in the number of harmonics may be observed);The fundamental frequency of the pipe, measured at the top of the pipe resonator, is sometimes different from the fundamental frequency measured at the lip of the pipe. It can be seen in the spectrum, especially on the logarithmic frequency scale, as the harmonics of the sound do not overlap, as shown in In the spectrum of some pipes, there are additional components that are not harmonics of the fundamental frequency of the pipe, see Based on the measurements of the behavior of flue pipes, generating sound in the condition of the presence of an obstacle near the labia, we observed the following effects:Inserting any measuring instrument inside the pipe disturbs the basic parameters of the sound. The measurement is disturbed also during inserting a measuring instrument between the lip and the obstacle. The same applies to the doping of coloring substances to the air jet in the pipe, as this changes the gas density and its thermal parameters, and thus also distorts the results of the measurements. Consequently, there is a problem with carrying out a direct flow measurement. To solve the impossibility of making the measurement, various mathematical methods to estimate calculations, such as interpolation or approximation, were reviewed.In this paper, the interval calculus is used to approximate the flue pipe airflow parameters. The use of this method makes it possible to find the data intervals that depend on other known ranges. Interval operations are performed on numerical intervals, and the operation result is also in the interval form. This method began to be developed in the 1960s  and it ia, b are sets, min is the minimum value of the set, max is the maximum value of the set.This paper uses interval arithmetic based on the operations on intervals , presentg of g. It is equivalent to the real-valued function g, satisfying the relationship (9).y is an element belonging to the interval, y is the interval.In the case of interval arithmetic, the result of operations on numbers is contained in the result of operations on intervals. In addition, we used an interval extension of a function, the so-called inclusion function z as the centered form of the inclusion function of h was used , c is the speed of sound [m·s−1] and λ is the wavelength [m].The frequency of the sound is related to the wavelength through the speed of sound propagation c in a gas depends primarily on temperature, as shown in Equation (14) , μ is the molar mass of the gas. For dry air: κ = 1.401, μ = 0.029 kg·mol−1. For the measurement temperature of 19 °C, the speed of sound in the air is c ≈ 343 m·s−1.The speed of sound ion (14) . TherefoThe air jet in a flue pipe is turbulent already in the case of sound initiation, in the so-called initial transient. The vortices are noticeable in the first stages of air jet formation, in the foot of the pipe . At the D , l is the characteristic dimension or length [m], and ν is the kinematic viscosity [m2·s−1].The drag coefficient neration . This vaconstant .(16)Re l concerns the distance, which directly influences the stability of the fluid movement. When the flow is turbulent, the drag coefficient depends only on the Reynolds number, and when it does not change, the drag coefficient does not change either [DC can be assumed to be constant. For air at a temperature of about 20 °C, the kinematic viscosity is ν = 1.461·10−5 m2·s−1 [The characteristic dimension or length e either ,32. In t5 m2·s−1 .eR = 2300 is the critical Reynolds number for flows in circular tubes. A flow is always turbulent above that value. In non-circular tube flow systems, the critical Reynolds numbers are different. Moreover, no constant values of critical Reynolds number can be used as they depend on the characteristic dimension or length of various measurable objects. In our case, the characteristic dimension l was the distance of the wedge cutting off the air jet in the form of the upper lip from the flue, from which the air is coming out. This selection was based on the papers [Previous measurements and calculations ,35 have e papers ,8,36.u (in liquid or gas), the characteristic dimension l, and the fundamental frequency of the sound f0 generated by turbulent flow. This relationship is called the Strouhal number rS and is described by Equation (17):Our work is based on sound generation model taking flow into account ,5,27,29,l can be distinguished [f0, and the relationship between them is described by the Strouhal number [f0. Therefore, f0 can be expressed as a function of the Strouhal number.The conditions of the phenomenon described by V. Strouhal are present during the generation of sound in the organ pipe. These conditions are: turbulent flow and the presence of a resonator, in which a certain characteristic dimension nguished . As the l number , see EquThe relationship between the Reynolds number and the Strouhal number can be determined using Equations (16) and (17), as shown in Equation (18):α and τ are constants depending on various intervals of the Reynolds number.The research on the relationship between the Reynolds number and the Strouhal number  shows theR > 2300 [f0 >> 1, the Reynolds number is very large. Using Equation (19) and the interval calculus [rS for the fundamental frequency of the pipe f0 will fall.An organ pipe generates a sound only when the airflow is turbulent. Thus, the Reynolds number in this situation exceeds its critical value, hence e > 2300 . Additiocalculus , it is prS intervals, depending on the eR intervals, are presented in r1S interval, depending on the interval e1R = [2300, 5000), is obtained as shown below. Using the interval arithmetic for Equation (19) and the values of the constants α and τ :The calculated  α and τ  for a corS is approximately constant and the mean for this range is rS ≈ 0.2. Since the characteristic dimension l , and then the Reynolds number eR can be determined from Equation (19), as shown in The results of calculations presented in k be an integer multiple of 5 mm. We will analyze the influence of an obstacle, placed at distances being integer multiples of 5 mm to the pipe’s mouth, on the fundamental frequency of the generated sound. Based on the Strouhal number analysis in rS is constant. Let kf be the fundamental frequency of the pipe, calculated by DTFT for the k-th distance, and fk+1 for k + 1 distance. We denote the airflow velocities in the pipe’s mouth for the k-th measurement as ku, and similarly uk+1 for k + 1. Then we have the following dependencies, presented in Equations (21) and (22):Let eR from Equation (18) will depend only on the fundamental frequency f0, which will depend on the flow velocity u (Equation (17)).In this case, the change of the Reynolds number eR > 4000, the aerodynamic drag coefficient is very small, close to zero [k · 5 mm. Therefore, if for each pair  the value of the fk+1/kf ratio will be similar, then in the flue pipe the velocity of the airflow is directly proportional to the fundamental frequency f0 of the generated sound.According to the Kozena-Carman formula , for lar to zero ,39. Therfk+1/kf calculated for a single pipe is almost constant and is approximately 1.003. The measurement uncertainty, i.e., the divergence between the calculated and the measured frequency, does not exceed 1% for five-fold measurements of the fundamental frequency of the pipe sound. The smallest dispersion of these proportions is 0.1%. Moreover, calculations from the interval calculus prove to be a good representation of this phenomenon, since the Strouhal number is approximately constant.The performed calculations show that the ratio f0 of the pipe sound as the obstacle is located closer to the mouth of the pipe looks similar for all analyzed types of mouth. The characteristics of this phenomenon are very close to the logarithmic curve , connecting the b coefficient with the fundamental frequency of the pipe f0. The calculated values of b2 are presented in Equation (23) was modified by introducing the b2 coefficient does not change much. The measurement uncertainty Mσ for different mouth types is 0.001, which is 0.1% of the mean. Thus, it can be assumed that b2 is constant for all pipes and equals 0.998. This generalization allows using Equation (25) for all pipes in stops, within a specific type of mouth.a is the logarithmic regression coefficient depending on pipe’s mouth type.The One of the assumptions of the presented research was the use of methods that do not interfere with the sound generated in a labial pipe. Research carried out by other authors, to investigate the laws of physics for turbulent air flow, used techniques to color the air, e.g., doping with smoke  or inseru to the product of the frequency f0 and the distance from the upper lip to the flue l is constant. Although the authors did not explicitly specify that, this value is the Strouhal number. Hruška and Dlask [rS ≈ 0.2 was determined. The stability of the Strouhal number is also confirmed by other researchers [eR > 2000, the value of the Strouhal number is approximately constant. The authors examining the whistling effect in the tubular system came to similar conclusions [Czyżewski et al.  noted thnd Dlask  proved tnd Dlask , as wellnd Dlask , confirmearchers , analyziclusions .The influence of the obstacle proximity on the sound parameters is often omitted in research. e.g., Odya et al. , while cThis paper quantitatively determines the phenomenon of changes in the sound parameters of a labial pipe. Such measurement is difficult due to disturbances caused by measuring instruments when they are used close to the pipe’s mouth. This approach has not been used in another study so far. It is worth mentioning that the obtained results are directly dependent on the basic principles of turbulent flow or on the Strouhal phenomenon, which occurs in items similar to the pipes, such as in tubes or slits.r2 for proposed Equation (25) is 91% on average. It is not an ideal match but this indicates that the function model is sufficiently fitted to the data obtained from measurements and DTFT, which is confirmed by the plots of DTFT values and the values calculated from Equation (25), as shown in f0(x), presented in Equation (25).It is worth noting that the coefficient of determination The fundamental frequency of the sound is directly proportional to the speed of the airflow in the pipe’s mouth;The speed of the airflow in the pipe’s mouth increases with the distance of the obstacle from the pipe’s mouth;The value of the Reynolds number in the pipe increases with the distance of the obstacle from the pipe’s mouth;The value of the Strouhal number for a labial pipe does not change significantly and can be approximated by a constant value.The results of the presented measurements and analyzes of the behavior of the basic parameters of the sound generated by the labial pipe in the presence of an obstacle prove that:The thesis about the dependence of the fundamental frequency of the sound on the distance between the obstacle and the pipe’s mouth was also generalized. The formula describing the untuning of the pipe sound is presented for different types of mouth. The obtained logarithmic regression formulas have a high coefficient of determination, which proves that the models fit the data sufficiently. The proposed Equation (25) can be used in organbuilders’ practice of pipe tuning. The authors believe that this formula is suitable for use in organ building.The research was carried out in a non-invasive way that does not influence sound parameters, with the use of interval calculus, which has not been used so far in the area of pipe acoustics. Thanks to the use of this method, the obtained conclusions are quantitative.The conducted research is innovative due to the analysis of the sound parameters of the pipes depending on the obstacles  in their surroundings. This is the first study that accurately describes the dependence of the Strouhal number on the fundamental frequency of a sound in the proximity of other pipes or obstacles. Moreover, the use of the method of interval calculus instead of the use of invasive measuring instruments is new. The introduction of measuring equipment significantly disturbs the results because it becomes an additional obstacle for the emitted air jet.Future work will focus on improving the proposed model for determining the fundamental frequency of sound depending on the proximity of an obstacle. Additionally, interval calculus can be used as a method of determining the ranges for the data inquired for in other research, where invasive measurement methods have been used so far.


The instruments' cyclic fatigue resistance at both temperatures differed significantly for each instrument type (P < 0.001). The RB instruments displayed greater cyclic fatigue resistance at the tested temperatures compared with the VB and XB instruments (P < 0.001). Reciprocating kinematics positively influenced cyclic fatigue resistance. Blue heat-treated instruments showed decreased cyclic fatigue resistance as the temperature increased (P < 0.001).The main aim is to evaluate the cyclic fatigue resistance of blue heat-treated instruments with different kinematics. Twenty-four endodontic instruments of the same brand were used for each of three experimental groups: VB (Vortex Blue 40/0.04), RB (RECIPROC Blue 40/0.06), and XB (X1 Blue 40/0.06). The instruments were randomly distributed and subjected to temperatures of 20°C and 37°C. The fatigue test was performed using a stainless steel device. Data were analysed using the Shapiro–Wilk test, Student's  Cyclic fatigue is one of the main mechanisms leading to nickel titanium (NiTi) instrument fracture in root canals –4. This These treatments are methods to transform shape memory properties and influence the mechanical behavior of the alloy, exhibiting better performance when comparing similar instruments made of conventional superelastic NiTi wires , 11. TheAutomated instruments tested at different temperatures have considerably different fatigue behaviours , 17. An Manufacturers have developed a special thermal process to increase the flexibility of NiTi alloy, which primarily contains a stable martensitic phase under clinical conditions , 24. Thein vitro cyclic fatigue tests show that reciprocating instruments exhibit better fatigue resistance. However, the mean time to fracture is not directly proportional to the increase in the number of reciprocations required for a complete rotation (360°) probably because the resulting speeds are not directly proportional [Regarding motion kinematics, ortional .Per their manufacturers, when subjected to blue thermal treatment, the instruments Vortex Blue , RECIPROC Blue , and X1 Blue  are indicated when better cyclic fatigue resistance is desired. Thus, the present study evaluated the cyclic fatigue resistance of instruments with blue thermal treatment and different kinematics, continuous, and reciprocal rotation  at 20°C (±0.5°C) and 37°C (±0.5°C) simulating room temperature and the temperature inside the root canal, respectively. Considering that body temperature (around 37°C) can significantly affect the transformation temperatures of the NiTi crystalline phase, the cyclic fatigue behavior of NiTi files.The null hypotheses tested were the absence of significant differences in cyclic fatigue resistance between blue heat-treated instruments with different kinematics at 20°C (±0.5°C) and 37°C (±0.5°C).http://www.random.org) into two groups (n = 12) and subjected to the temperatures of 20°C and 37°C. The endodontic instruments were used with a 6 : 1 reduction handpiece  coupled to the VDW.Silver® RECIPROC® motor . The VB group instruments (Vortex Blue 40/0.04) were used at a speed of 500 rpm and a torque of 1.3 Nmm. In the RB (RECIPROC Blue 40/0.06) and XB (X1 Blue 40/0.06) groups, the instruments were used in the “RECIPROC All” mode, with the same kinematics.Three brands of blue heat-treated NiTi endodontic instruments with different kinematics were tested. Twenty-four new endodontic instruments were used for each of the three experimental groups: VB (Vortex Blue 40/0.04), RB (RECIPROC Blue 40/0.06), and XB (X1 Blue 40/0.06), all with size ISO 40 and a length of 25 mm and commercially available in the country of origin of the study. Prior to use, all instruments were checked by a single operator under a stereomicroscope  at 16x to qualitatively standardize the instruments in terms of lack of defect or deformation, such as distortions or coarse burrs on the cutting blades. No signs of change were detected, and no instrument was discarded. Twenty-four instruments of the same brand, all from the same production batch, were randomly distributed ) test with Tukey's or Tamhane posttests. Tukey's multiple comparisons test was used in cases where the equality of variances hypothesis was confirmed, and the Tamhane comparisons test was used when the hypothesis of equality of variances was rejected. The significance level was set at P=0.05.The data were descriptively analysed using SPSS 23.0  for Windows and Excel 2010. The normality of the data for each file type and temperature combination was tested using the Shapiro–Wilk test. The analysis was inferential, using Student's P < 0.001). The RB instruments displayed greater cyclic fatigue resistance at the tested temperatures compared with the VB and XB instruments (P < 0.001). The multiple comparison tests showed that NCF significantly differed among the three instrument types at 20°C (P=0.004) and between the RB instruments and the other two brands at 37°C (P < 0.001). The mean fragment length (4.62–5.40 mm) did not differ significantly among the instruments tested at 20°C or 37°C (P > 0.05).The mean and standard deviation of the NCF values are shown in P < 0.001), for groups XB and RB. The NCF values among the three instrument types tested differed significantly at 20°C (P=0.004). At 37°C, this difference occurred for the RB instruments relative to the VB and XB instruments (P < 0.001). Thus, the null hypotheses were rejected (P < 0.05).The results of the present study showed that decreased temperatures increased the cyclic fatigue resistance of blue heat-treated instruments. These instruments are the result of the oxidation that heating and cooling induce on their surface, making the NiTi alloy more martensite, hence giving it better fatigue life . To dateIn the past, most cyclic fatigue resistance studies were performed at room temperature, but this did not reflect clinical conditions, as the root canal had a different temperature . The envAn ideal experimental model for testing cyclic fatigue would involve instrumentation in natural teeth. However, sample standardization is difficult and restricts the sample size because of the large variation in length, radius, and degree of curvature within the same dental group . In addiReciprocating motion kinematics extend the useful life of the instruments because they expose the instruments to lower voltage values compared to continuous rotation, thus increasing NCF –35. ThroA smaller radius of curvature and an increased instrument diameter result in fewer cycles to fracture . The insP < 0.05). At 37°C, the XB instruments differed only from the RB instruments (P < 0.001), presenting a lower NCF. When compared between the temperatures tested, the NCFs of the XB instruments differed significantly.The blue heat-treated XB reciprocating instruments, produced with controlled memory (CM) NiTi alloy, were recently launched on the market. They have an inactive tip and a triangular cross section per the manufacturer's instryctions. No studies have reported XB file behaviour (40/0.06) in cyclic fatigue tests. In our study, the NCFs of these instruments at 20°C were significantly smaller and larger, respectively, than those of the RB and VB instruments (The VB rotary instruments manufactured with CM NiTi alloy and blue heat treatment are characterized by a constant taper, a triangular cross section, and the absence of radial lands , 40. In Some studies have suggested that cooling down to low temperatures may be an interesting strategy to improve the fatigue resistance of rotary NiTi files although cooling down to a low temperature during the instrumentation may be difficult to achieve . This exWithin the limitation of the study, the RECIPROC Blue instruments showed greater cyclic fatigue resistance compared with the X1 Blue and Vortex Blue instruments at 20°C and 37°C. The results indicate that the fatigue test should be conducted at body temperature instead of room temperature.


Objective: In this prospective, population-based study, we evaluated the utility of high-risk human papillomavirus (HR-HPV) genotyping for triaging women with atypical squamous cells of undetermined significance (ASC-US) in the Chinese rural area.Methods: A total of 40,000 women were recruited from rural areas of Shanxi Province, China, between June 2014 and December 2014. Women with Pap results of ASC-US underwent HPV genotyping, colposcopy and histopathological examination. For those with normal cervixes or cervical intraepithelial neoplasia (CIN) 1 on the initial evaluation, a 2-year follow-up study was performed.Results: The reporting rate of ASC-US was 5.76%  in the study population. The detection rates of CIN 2 or above (CIN2+) and CIN 3 or above (CIN3+) in women with ASC-US were 7.28% and 1.75%, respectively. HPV 16 (39.53%), HPV 58 (17.83%), and HPV 52 (15.50%) were the three most prevalent HR-HPV genotypes among all women with ASC-US cytology. The five most common HR-HPV genotypes in CIN3+ lesions were HPV16, HPV58, HPV33, HPV31 and HPV18. Compared with the 15 HR-HPV testing, genotyping for a combination of HPV16/18/31/33/58 increased specificity significantly with virtually no loss of sensitivity for detecting CIN2+ and CIN3+ lesions, as well as significantly reduced colposcopy referral rate . In addition, in the 2-year follow-up period, women with infection of HPV16, 18, 31, 33 or 58 genotypes were the most likely population  to develop CIN2 lesion.Conclusion: Our results demonstrate that genotyping for a combination of HPV16/18/31/33/58 provides a more efficient and cost-effective model to risk-stratify women with ASC-US in the Chinese rural population. Atypical squamous cells of undetermined significance (ASC-US) cytology is the most frequent abnormal interpretation on cervical Papanicolaou (Pap) test, accounting for ~5% of all cervical cytology in the western population Based on the published evidence, the American Society for Colposcopy and Cervical Pathology (ASCCP) Guidelines have recommended women with an ASC-US cytology result should have a reflex HPV testing, and an immediate colposcopy referral or conservative follow-up based on patients' previous screening history is recommended for women with high-risk (HR) HPV-positive ASC-US It is well-known that the HR-HPV prevalence and distribution varies geographically worldwide. There are some distinctive characteristics in the Chinese population, including more frequent HPV52 and HPV58 genotypes, and less HPV45 and HPV18 genotypes in cervical cancer and precancerous lesions The study design and methods have been described previously Figure 1).In Yangqu County, 1,034 women with ASC-US cytology were identified and 1,026 women underwent HPV baseline genotyping and immediate histopathologic examination. 77 women were diagnosed with CIN2 or worse including 4 squamous cell carcinomas on the histologic examination and all these patients received clinical treatment. 949 women were found to have normal cervixes or CIN1 and were followed-up for additional 2 years . All inspections and detections were implemented under double-blind conditions. The study was registered in the Chinese Clinical Trial Register (ChiCTR), registration number: ChiCTR-ROC-15006479. The study protocol and informed consent form were reviewed and approved by the Ethics Committee of Second Hospital of Shanxi Medical University.Among 1,270 women with ASC-US cytology in Jiexiu County, 382 patients with normal cervix refused to have HPV genotyping test. 90 women with CIN2 or worse lesions received clinical treatment, while 798 women with normal or CIN1 lesion were followed-up for additional 2 years (http://www.lituo.com. cn). The cytological evaluation was performed by two cytopathologists at The Second Hospital of Shanxi Medical University. Cytology results were classified using the 2001 TBS criteria for reporting cervical cytology The liquid-based cytology (LBC) method was used in this study. The LBC preparation was produced by Lituo Biotechnology Corp. . HybriBio GenoArray test is a commercially-available HPV genotyping diagnostic assay, and was approved by Conformitè Europèenne In Vitro Diagnostic (CE-IVD). Earlier studies have demonstrated the overall agreement in HR-HPV detection between HybriBio GenoArray genotyping assay and HC2 was 92.5-92.9% with a kappa value of 0.814 and 0.80 The colposcopy procedure and biopsy were performed by experienced gynecological specialists from The Second Hospital of Shanxi Medical University. The average time from Pap sampling to colposcopy was 15 days (range: 3-62 days). Colposcopy examinations were performed using the Preventive Oncology International microbiopsy protocol. During the colposcopy, the cervix was divided into quadrants and each quadrant was examined independently. All visually abnormal areas were biopsied. Quadrants with normal colposcopic impressions were biopsied at the squamocolumnar junction (“random biopsy”). Women with abnormal cytology results and negative or inadequate colposcopic findings underwent endocervical curettage (ECC). All histological slides were reviewed by two gynecological pathologists, who were blinded to the cytology results at The Second Hospital of Shanxi Medical University. Immunohistochemistry for p16INK4A (p16)  was utilized in adjudicating the diagnosis. If the two pathologists disagreed on the diagnosis, a third senior pathologist reviewed the slides, and some difficult or equivocal cases were considered for the pathology panel consensus diagnosis.The statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 22.0 software for windows . Chi-squared test or Fisher's exact test were used for the categorical variables. Statistical tests were two-sided, and P <0.05 was considered statistically significant.Among 40,000 women in this study population, the overall ASC-US rate was 5.76% , including 5.17%  in Yangqu County and 6.35%  in Jiexiu County. Of 2,304 women with ASC-US cytology, 2,296 women underwent colposcopy and immediate histopathologic examination. Histology confirmed that 1,667 patients (72.60%) had normal cervixes, 462 (20.12%) had CIN1, 127 (5.53%) had CIN2, 27 (1.18%) had CIN3, and 13 (0.57%) had squamous cell carcinoma (SCC). The detection rates of CIN2+ and CIN3+ in women with Pap results of ASC-US were 7.28% and 1.75%, respectively.Table 1 shows the relative frequencies of HPV infections in women with ASC-US Pap test according to histological diagnosis. The overall rates of HR-HPV and LR-HPV infections were 33.70%  and 1.83% . Among 645 women with HR-HPV infection, 445 patients were infected with a single HPV genotype and 200 patients were infected with multiple HPV genotypes including 20 patients co-infected with low-risk HPV genotypes. The prevalence of HR-HPV genotypes in women diagnosed with <CIN2, CIN2, CIN3 and SCC was 29.36%, 73.23%, 96.30% and 100%, respectively. LR-HPV infections alone, without HR-HPV infections, were not detected in any of the CIN3 and above cases.Of 2,296 women with histologic examination, 1,914 women had HPV genotyping test. Table 2 presents age-stratified HR-HPV prevalence in women with ASC-US cytology. Among all age groups, women aged between 30 and 39 years had peak rate of HR-HPV infection in Yangqu  and Jiexiu  Counties. There was a dramatic decline in the HR-HPV infection rates after age 50 in both Counties.The median age of women with ASC-US was 51 years (range: 20-65 years) in Yangqu County and 50 years (range: 20-65 years) in Jiexiu County. Table  HPV16 (35.57%) was the most common HR-HPV genotype, followed, in order of decreasing frequency, by HPV52 (19.65%), HPV58 (17.91%), HPV53 (8.21%), HPV56 (6.97%), and HPV33 (6.97%). Additionally, HPV18 (4.48%) was in the 11th position. The distribution of HR-HPV genotypes by histologic outcome was different. HPV16 showed an increasing prevalence with the severity of the histological diagnosis . HPV58 was the second-most prevalent genotype in CIN2+ lesions and the third-most prevalent genotype in <CIN2 lesions.In women with multiple infections, HPV genotypes were reported separately. The prevalence of HR-HPV genotypes in HR-HPV-positive cervical lesions in Yangqu County are presented in Table 3B). The six most common HR-HPV genotypes in women with ASC-US Pap test, in decreasing order, were HPV16 (46.09%), HPV58 (17.70%), HPV51 (9.47%), HPV52 (8.64%), HPV33 (7.41%) and HPV31 (6.58%). HPV16 was still the most common genotypes in different histologic types. HPV58 and HPV33 were tied for the second-most prevalent genotypes in CIN2+ lesions.Interestingly, the prevalence of specific HR-HPV genotypes in HR-HPV-positive cervical lesions in Jiexiu County showed some difference from those seen in Yangqu county , HPV 58 (17.83%), and HPV 52 (15.50%) were the three most prevalent HR-HPV genotypes, while HPV 18 was less prevalent (4.34%). The five most common HR-HPV genotypes in women with CIN3+ lesions were HPV16 (82.05%), HPV58 (12.82%), HPV33 (10.26%), HPV31 (10.26%) and HPV18 (5.13%). The rest of 10 HR-HPV genotypes were found in ~17.94% of women with CIN3+ on the immediate histologic examination. In women with CIN2 lesions, the five most common HR-HPV genotypes were HPV16, HPV58, HPV31, HPV33 and HPV52. HPV18 was the seventh-most prevalent genotype in CIN2 lesions and the fifth-most prevalent genotype in CIN3+ lesions.Table 4 displays the sensitivity, specificity, PPV and NPV of the combination of 5 highest-risk HPV genotypes (HPV16/18/31/33/58) and 15 HR-HPV genotypes (HPV16/18/31/33/35/39/45/51/52/53/56/58/59/66/68) in detecting CIN2+ and CIN3+ lesions in women with ASC-US. The sensitivity of the 5 highest-risk HPV genotypes (HPV16/18/31/33/58) for detecting CIN2+ and CIN3+ lesions was 72.46 % and 95.00%, respectively. The specificity for detecting CIN2+ and CIN3+ lesions was 81.57% and 78.39%, respectively. The NPV for detecting CIN2+ and CIN3+ was 96.87% and 99.86%, respectively. The sensitivity and NPV of the 5 highest-risk HPV genotypes were similar to those of the 15 HR-HPV genotypes for detecting CIN2+ and CIN3+ lesions. However, compared with the 15 HR-HPV genotypes, the 5 highest-risk HPV genotypes had significantly higher specificity for detecting both CIN2+ and CIN3+ lesions. In addition, the 5 highest-risk HPV genotypes had significant higher PPV for detecting CIN2+ lesions. Moreover, the colposcopy referral rate using the 5 highest-risk HPV genotypes  was significantly lower than that using 15 HR-HPV genotypes .Of 1,747 women with normal cervix or CIN1 at baseline colposcopic examination, 1,385 patients (79.28%) including 990 HR-HPV negative and 395 HR-HPV positive women underwent both HPV genotyping test and colposcopic examination at the 2-year follow-up. Among 990 women with initial negative HPV testing, 130 cases were found to have HR-HPV positive (13.13%) and 8 cases developed CIN2 at the 2-year follow-up. Among 395 women with baseline positive HPV testing, 50 cases had new HR-HPV infection (12.66%), and 52 cases had persistent infection (13.16%). 14  women with persistent HR-HPV infection developed CIN2.Table 5 demonstrates the prevalence of HR-HPV genotypes of women who underwent 2-year follow-up testing including 180 women with new HR-HPV infection and 52 women with persistent HR-HPV infection. Among them, the most common genotypes were HPV16 (28.02%), HPV58 (20.26%) and HPV52 (17.67%). Of the 16 CIN2 cases with positive HR-HPV test, the 5 highest-risk HPV genotypes were HPV16 (68.75%), HPV58 (37.5%), HPV18 (12.5%), HPV31 (12.5%) and HPV33 (12.5%) (Table 6).Cervical cancer remains an important public health issue in China, especially in the large rural areas where the health resources are limited, and the cervical cancer screening coverage is low. In order to develop a more cost-effective and efficient cervical cancer screening program in the Chinese rural population, we initiated a large population-based cervical cancer screening program and a prospective cohort in the rural areas of Shanxi province, China in 2014. We herein report the results of HPV genotyping, immediate histologic correlation and 2-year follow-up of 2,304 women with ASC-US cytology. To the best of our knowledge, this is the largest prospective study on ASC-US women with genotyping and histological correlation in the Chinese rural population.In this study, the detection rate of ASC-US among 40,000 women in two rural counties was 5.76% , which is within the range of 3.7-10% of ASC-US reporting rate in the Chinese population after the introduction of LBC preparation method According to the principle of “equal management of equal risks”, clinical management should be treated differently for ASC-US patients with different HPV genotypes. Previous studies have confirmed that HR-HPV genotyping could help identify women at highest risk for high-grade cervical lesions In this study, we also followed-up the women with ASC-US cytology who did not receive any treatment for a 2-year period of time. Our results found that the incidence and risk of progression to CIN 2 were the highest in women with persistent HR-HPV infection during the follow-up period. These findings are in line with those of previous studies indicating that persistent HR-HPV infection is the most important risk factor for the development of cervical cancer and precancerous lesions The current study has some limitations. First, this study population had not previously been vaccinated for HPV. As a result, the prevalence of HR-HPV genotypes in HPV-Vaccinated population may be different. Second, in this study, the selection of 5 HPV genotypes (HPV16/18/31/33/58) was based on a relatively small sample size, and larger sample sizes and study in different geographic regions are needed to validate this novel HPV genotyping assay.In conclusion, in this large-scaled prospective study with cross-sectional analysis as well as 2-year follow-up analysis, the genotyping for a combination of HPV16/18/31/33/58 provides a more efficient and cost-effective risk-stratification model in women with ASC-US in our study population. Women who tested negative for the 5 highest-risk HPV genotypes (HPV16/18/31/33/58) may not require immediate colposcopy. Further prospective studies with larger sample sizes, as well as from different geographic regions are needed to validate this novel HPV genotyping assay in the triage of women with ASC-US in the Chinese rural population.


Cartilage oligomeric matrix protein (COMP) is an emerging independent prognostic marker for breast cancer patients. COMP expression by cancer cells affects their metabolism, metastases, and the abundance of cancer stem cell populations. This study assessed the levels of COMP in the sera of metastatic breast cancer patients. Further, matched tumor tissues from the primary tumor and metastases were stained for COMP expression with immunohistochemistry. The levels of serum COMP were highest in the blood of metastatic ER-positive and HER2-positive patients. The expression of COMP in primary tumors correlated with COMP expression in the metastatic loci. Lymph node metastases (LNM) with COMP expression were associated with reduced survival. The expression of COMP in LNM at the time of primary diagnosis could indicate later development of visceral and lung metastases.Cartilage oligomeric matrix protein (COMP) is a regulator of the extracellular matrix and is expressed primarily in the cartilage. Recently, COMP expression was also documented in breast cancer patients both in sera and tumor biopsies, in both of which it could serve as an independent prognostic marker. This study aimed to assess COMP as a potential biomarker in the group of metastatic breast cancer patients. Levels of COMP were measured by ELISA in serum samples of 141 metastatic breast cancer patients. Biopsies from primary tumors, synchronous lymph node metastases, and distant metastases were stained for COMP expression. The levels of serum COMP were higher in patients with ER- and HER2-positive tumors when compared to triple-negative tumors and correlated with the presence of bone and lung metastases, circulating tumor cell count, and clusters. Most of the primary tumors expressing COMP (70%) retained the expression also in the lymph node metastases, which correlated with visceral metastases and reduced survival. In conclusion, COMP appears as a valuable biomarker in metastatic breast cancer patients indicating a more severe stage of the disease. Serum COMP levels were associated with specific types of metastases in patients with metastatic breast cancer emphasizing that further studies are warranted to elucidate its potential role as a monitoring marker. Cartilage oligomeric matrix protein (COMP) has been traditionally viewed as an extracellular matrix (ECM) protein that is expressed by chondrocytes of cartilage  as well Moreover, COMP has been found in several cancer types in which it is expressed by epithelial cancer cells and in the surrounding matrix. This has so far been observed in breast- , prostatThe mechanisms underlying the effects of COMP in breast cancer progression are under investigation. It has been already established that COMP expression in cancer cells increases their invasive potential, alters metabolism (Warburg effect), and renders them resistant to apoptosis . FurtherThe goal of the current study was to further evaluate COMP as a potential biomarker in metastatic breast cancer patients by assessing the serum COMP levels and COMP expression in relation to the severity of the disease and outcome.Patients with newly diagnosed MBC were included in a prospective monitoring trial  before the start of systemic therapy. The inclusion criteria were MBC diagnosis, age > 18 years, Eastern Cooperative Oncology Group (ECOG) performance status 0–2, and predicted life expectancy > 2 months. Serum samples were taken at a baseline, before the start of systemic therapy and stored at a biobank. Circulating tumor cells (CTCs) were enumerated using the CellSearch system. Clinicopathological data and monitoring of patients were prospectively collected in case report forms. Included patients received systemic therapy according to national clinical guidelines. A response evaluation was performed approximately every third month and the progression versus non-progression was defined according to a modified RECIST criteria .The S-COMP serum levels were measured using an IVD approved ELISA  following manufacturer instructions. In brief, serum was diluted (1/10) in the provided sample buffer, added to a precoated 96-well plate together with an enzyme conjugated antibody, and incubated for 2 h at room temperature. The plates were then washed 6 times, developed with TMB substrate, and measured at 450 nm using Cytation 5 .Breast cancer tissues were mounted in a tissue microarray. Antigen retrieval was performed with the Envision Flex high pH kit  using a PT-link module (Agilent-Dako). The tissues were stained with 0.47 μg/mL rabbit polyclonal affinity purified anti-COMP in-house antibody that was previously evaluated for its specificity , utiliziFor comparison of clinicopathological variables, a Pearson’s chi-squared test or a Pearson’s chi-squared test for trend was used. The comparison of COMP expression status between primary tumors, LNM and DM, was performed using the McNemar’s test. Kaplan–Meier curves and log rank tests were used to illustrate and compare survival. Time to progression or death of any cause was calculated from the baseline and patients without events were censored at the last follow-up. The median follow-up time was 49 (27–93) months. All of the statistical analyses were performed using SPSS statistics .Serum samples that were taken before the start of the systemic therapy were available for 141 patients, as illustrated in the flowchart . Of thesWhen analyzing progression-free (PFS) and overall survival (OS) in relation to S-COMP levels there were no significant differences in patients with high versus low S-COMP levels, illustrated by Kaplan–Meier curves .p = 0.01; p = 0.13; Comparisons of the S-COMP levels and COMP levels that were detected using immunohistochemistry (IHC expression) in cancer cells and stroma in distant metastases (DM) revealed a positive correlation of high S-COMP levels in patients that had a strong IHC COMP expression in DM stroma (p = 0.049), revealing that in 70% of the pairs the expression was stable but there were more pairs with a loss of COMP expression from the primary tumor to LNM than with a gain. Similarly, a trend was seen in shifts regarding COMP IHC cancer cell expression from the primary tumor to LNM, however this was not significant (p = 0.077). Regarding shifts from the primary tumor to DM and from LNM to DM, there were no significant shifts.To evaluate shifts in COMP expression during tumor progression, matched pairs of primary tumors, synchronous LNM and DM, were analyzed by IHC. McNemar’s analyses were used for the evaluation of concordance and discordance of IHC COMP expression in cancer cells and stroma. As illustrated in p = 0.005) and in primary tumor stroma with MFI (p = 0.007) (p = 0.013) as well as in the primary tumor stroma (p = 0.013) and the presence of liver metastases. When analyzing possible correlations between LNM IHC COMP expression and clinicopathological variables there were significant correlations to performance status (p = 0.023) and furthermore a high COMP IHC expression in stroma correlated to visceral metastases (p = 0.03) and presence of lung metastases (p = 0.039). Associations of cancer cell and stroma COMP expression in DM showed no significant associations to the respective clinicopathological variables  . Furtherariables .p = 0.040 and OS, p = 0.041; p = 0.019; For survival analyses, a combined variable was used that assessed the COMP IHC expression in both the cancer cells and stroma. When analyzing PFS and OS in relation to COMP IHC expression, no significant correlations were seen for primary tumors and DM COMP expression in cancer cells and stroma A,B. HoweWe recently revealed that COMP levels in the serum of breast cancer patients could serve as an independent prognostic marker. Patients with metastatic disease had higher serum levels of COMP compared with those in the early stages . In the The serum level of COMP is a well-established marker of cartilage turnover . Studies+ or HER2+) are treated nowadays with targeted systemic therapy as well as chemotherapy and have a better prognosis compared to patients with triple-negative disease [In previous studies, COMP expression by the tumor cells but not in stroma was associated with a worse prognosis of patients with early breast cancer ,16. COMP disease . Thus, l disease ,16 showiThe levels of COMP in serum correlated with the deposition of COMP in the stroma of DM. A similar trend was observed between the levels of serum COMP and the expression of COMP by the cancer cells of DM. This demonstrates a correlation between the blood levels of COMP and the local expression of COMP in the distant metastasis. Furthermore, high S-COMP levels correlated with the presence of bone metastases, indicating a possible role of COMP in bone metastatic progression of breast cancer.A significant association between serum COMP and the number and clusters of CTCs was found, supporting the hypothesis that serum COMP can be a marker of a more severe stage of metastatic disease. The presence of ≥5 CTCs carries a strong prognostic value in MBC and the presence of CTC-clusters indicates an even worse prognosis . CTC-cluOne advantage of the current study is that it includes matched tumor tissue that was collected from the same patient from both the primary tumor, synchronous LNM and DM. Analysis of this material revealed that most of the LNM cells retained the expression of COMP as identified in the primary tumor in both the cancer 73%) and stromal cells (70%). From the remaining paired samples, more LNM lost the expression of COMP  than gained it . Despite this loss, COMP expression in cancer and stromal cells in LNM was associated with progression-free and overall survival from the date of metastasis as well as from the primary breast cancer diagnosis. These findings suggest that COMP may have a role in the intravasation of the tumor cells from the primary tumor already at the time of initial breast cancer diagnosis. These data are in accordance with published studies showing that the expression of COMP by cancer cells induced the epithelial to mesenchymal transition . In addi% and strHigh expression of COMP in the stroma of LNM correlated with visceral metastases and the presence of lung metastases. LNM in breast cancer patients are regional metastases that are commonly identified at the time of initial cancer diagnosis. In contrast, DM are found in later, more advanced stages of the disease. One may hypothesize that COMP expression in LNM contributes to the development of distant metastases either by affecting the intravasation of the cancer cells to the bloodstream or the lymphatic system  or by prIt is clear that breast cancer cells can express COMP since we detected it in an intracellular location when using IHC. Further, COMP is deposited in the stroma of the tumors, but future studies must be designed to address the source of COMP in the stroma; this could be fibroblasts as these were previously shown to express COMP under certain conditions . The iniOne weakness of the current study lies in the limited number of patients that were included, and, therefore, further studies including larger cohorts will be needed to fine-tune conclusions on how to best use COMP as biomarker in the management of breast cancer. The main limitation of the study is the lack of a group of non-metastatic patients. Moreover, a cohort including all patients with metastatic breast cancer is heterogeneous in the sense that the underlying biology is different between the subtypes, and patients vary regarding the extent of the metastatic disease and the therapy that was received. Lastly, for some patients, tissue material was not collected from all of the metastatic sites.Taken together, COMP emerges as a biomarker of advanced breast cancer progression, especially in ER-positive and HER2-positive patients.High COMP expression in lymph node metastases in breast cancer has prognostic implications suggesting that COMP is involved in the progression to a more aggressive metastatic disease. The serum level of COMP appears to be a valuable marker of breast cancer progression as it could distinguish ER or HER2 positive patients with advanced disease. Patients with ER- and HERs-positive MBC and an increased serum level of COMP could potentially benefit from applying more aggressive therapeutic strategies.


Breast cancer (BC) is the most frequent tumor in women worldwide. A minority of BC patients have a family history of the disease, suggesting the importance of environmental and lifestyle factors. Human papillomavirus (HPV) infection has been detected in a subset of tumors, suggesting a potential role in BC. In this review, we summarized relevant information in respect to this topic and we propose a model of HPV-mediated breast carcinogenesis. Evidence suggests that breast tissue is accessible to HPV, which may be a causal agent of BC in a subset of cases.Breast cancer (BC) is the most commonly diagnosed malignancy in women worldwide as well as the leading cause of cancer-related death in this gender. Studies have identified that human papillomavirus (HPV) is a potential risk factor for BC development. While vaccines that protect against oncogenic HPVs infection have been commercially available, global disparities persist due to their high cost. Interestingly, numerous authors have detected an increased high risk (HR)-HPV infection in BC specimens when compared with non-tumor tissues. Therefore, it was suggested that HR-HPV infection could play a role in breast carcinogenesis in a subset of cases. Additional epidemiological and experimental evidence is still needed regarding the role of HR-HPV infection in the development and progression of BC. Breast cancer (BC) is the most commonly diagnosed malignancy in women worldwide as well as the leading cause of cancer-related death in this gender. It was estimated that 2,261,419 new cases of BC and 684,996 deaths worldwide were attributable to this malignant tumor in 2020 ,4.Approximately 90–95% of BCs are adenocarcinomas, which arise from the cells lining the milk ducts or lobular (milk-producing) glands. Of these, 75–80% are invasive ductal carcinomas (IDC) and 5–10% are invasive lobular carcinomas (ILC). Special types of invasive BC include mixed , inflammatory, metaplastic, papillary, tubular, adenoid cystic, cribriform, medullary, and mucinous carcinomas . Based oAge, family history, use of exogenous estrogen, and lifestyle  are important risk factors of BC . A cohorSome viral infections have been proposed as risk factors for sporadic BC. For instance, the mouse mammary tumor virus (MMTV) is almost absent in hereditary BC compared with 30% of sporadic BC . The lowHPV is a non-enveloped virus which belongs to the Papillomaviridae family and exclusively infects epithelial cells. The HPV genome comprises 8 kb double-stranded circular DNA containing 8 protein-coding genes. Its genome is functionally divided into three main regions: early (E), late (L), and the long control region (LCR). The E region contains open reading frames (ORFs) which encode the E1 to E7 non-structural proteins. These proteins are implicated in both viral replication and the transformation of host cells. Additionally, the L region encodes for two structural proteins, the major capsid protein (L1) and the minor capsid protein (L2) (reviewed in ) Figure. Based oThe biological functions of each HPV oncoprotein are summarized in The possible route of HPV infection to the breast is yet to be fully known. Lawson et al. found the same HR-HPV genotype in both BC specimens and squamous intraepithelial lesions of the cervix (SILs) counterparts from the same patients . AdditioThe prevalence of HPV in BC ranges 1.6–86.2% worldwide ,85, althp = 0.01), while both episomal and integrated forms (mixed) were evident in 8.3% of BCs from Indian patients [As koilocytes have been detected in normal breast skin and BC tissue, it was suggested that HPV virions can reach the breast . HR-HPV patients . It is wpatients , althougpatients . Other mpatients ,118. Regpatients ,119. Thupatients . Howeverpatients ,122. It patients . Moreovepatients . HPV infpatients . Additiopatients ,120. A lpatients . Neverthpatients . Similarpatients . The exppatients . BC cellpatients . HPV16 Epatients . Moreovepatients . It was patients . APOBEC3patients . HPV16 Epatients . In addipatients . Converspatients ,133. Recpatients . Howeverpatients  or weaklpatients , thus adpatients ,138.Exposure to estrogens has been widely shown to increase the risk of cervical, endometrial, and BC ,140,141.Koilocytes presence and molecular approaches confirm that HPV virions can reach the breast, most likely through epithelium or mucosal contact during sexual intercourse. This transmission route requires microlesions in the areola for HPV entry. Importantly, HPV components such as nucleic acids can reach the breast from infected sites through the bloodstream by extracellular vesicles. The extremely low viral load detected in BC specimens indicates that HPV genomes are not clonally distributed in the tumor, although HPV is frequently integrated into the host genome. Taken together, epidemiological and experimental evidence suggests that HR-HPV may be involved in BC development in a proportion of cases. On the other hand, it is well known that HR-HPV is a necessary condition for the development of cervical cancer, though the infection itself is not sufficient to establish cell transformation, suggesting that other factors are potentially involved in HPV-driven carcinogenesis. Thus, interaction models between HPV and additional host  or environmental factors  offer a plausible way to explain the frequency of HR-HPV infection in BC. The identification of HR-HPV as a causal agent in a subset of BC patients with increased risk of disease progression will lead to prevention or therapeutic alternatives. Additional epidemiological and experimental approaches are warranted to dissect the specific molecular alterations involved in HPV-driven BC.


Human papillomavirus (HPV) is one of the most important viral agents associated with several classes of cancers in humans. The aim of this study was to investigate HPV in esophageal cancer in the East Azerbaijan province, northwest of Iran. n = 35) and ESCC (n = 35) as the case group and those without tumour in esophagus tissue as a control (n = 70).  140 paraffin-embedded specimens of esophageal tissues were investigated using nested-polymerase chain reaction (nested-PCR) with primer designing for the L1 region of HPV genome. According to the pathological diagnosis, the samples were divided into two groups: 70 patients with esophageal cancer EADC ( HPV DNA was isolated from 20 (28.57%) of the 70 paraffin-embedded tissue specimens of esophagus cancer. Of these, 6 cases (17.14%) of EADC and 14 cases (40%) of ESCC were positive. In contrast, all cases of the control group were negative for the HPV genome. Sequence analysis revealed that HPV types 16 and 18 are the most frequent ones identified in this study.  The prevalence of HPV in esophageal cancer can vary depending on the geographical location and other factors. Based on the findings of this study, HPV infection may possibly have contributed to an increased risk of esophageal cancer in a group of patients in Tabriz. The esophageal carcinoma is the sixth leading cause of cancer death and also the seventh most common cancer in the world . EsophagIn the present study, 140 paraffin-embedded specimens of esophageal tissues were collected from the Pathology Department of Imam Reza Hospital in Tabriz (2017–2019). Of these, 70 samples were related to patients with esophageal cancer (as the case group) and 70 samples to noncancerous patients (as the control group), both of which were diagnosed and confirmed by the pathologist.The obtained samples were transferred under appropriate conditions to Tabriz University of Medical Sciences. Medical and demographic information relevant to each patient were obtained from archived medical records of the pathology department. The age range of patients with esophageal cancer and the control group was (26–98) and (28–97) years, respectively.Thin sections were prepared from paraffin-embedded esophageal tissue specimens. To prevent cross-contamination between samples, new scalpel razors and disposable gloves were used for each sample. Additionally, all steps were performed under the biological hood. Then, the samples were collected in a sterile microtube. Subsequently, sections were deparaffinized by xylene and subsequently rinsed using ethanol. After ensuring complete evaporation of the ethanol from the samples, DNA extraction was carried out according to Yekta-Tajhiz kit protocol .β-globin gene [To ensure the quality and control of the extracted DNA and rejection of the presence of PCR inhibitors, the OD of the samples was read using a spectrophotometer, and then, the PCR test was performed on all samples for bin gene .µl), primer MY09 (10 µM), primer MY11 (10 µM), and 500 ng of DNA template in a ﬁnal volume of 50 µl. At first, DNA was denatured for 5 min at 94°C, followed by 38 amplification cycles at 94°C for one minute, 55°C for one minute, and 72°C for one minute, and the ﬁnal extension step was 7 minutes at 72°C.After confirming the quality of the extracted DNA, the HPV genome was detected by nested-PCR assay. The first stage of nested-PCR was performed using MY09/MY11 primer pairs that amplified the L1 gene [µl), primer GP5+ (10 µM), primer GP6+ (10 µM), and 500 ng of DNA template in a ﬁnal volume of 50 µl. At first, DNA was denatured for 5 min at 94°C, followed by 38 amplification cycles at 94°C for one minute, 40°C for 1.5 minute, and 72°C for 1.5 minute, and the ﬁnal extension step was 7 minutes at 72°C. In both PCR reactions, extracted DNA from HeLa cell line were used as positive control, and distilled water was used as negative control. The PCR product at this stage was 150 bp.The GP5+/GP6+ primer pairs were used in the second step of nested-PCR. The ampliﬁcation mixture consisted of 10x PCR buffer, Mgcl2 (25 mM), dNTps (10 mM each), Taq DNA polymerase .Direct sequencing of L1 genes was carried out  using 0.5 Genotyping was performed by phylogenetic analysis with reference sequences of HPV genotypes. Phylogenetic tree generation was conducted using the MEGA software (version X), with the maximum likelihood (ML) method in the Kimura two-parameter substitution matrix B . Bootstrt-test was performed to compare the mean age between case and control groups. Furthermore, the chi-square test was used to compare sex, age groups, and pathology sample for HPV between case and control groups. P values of less than 0.05 were considered as statistically significant.The data analyses were conducted using SPSS software (version 25). Descriptive statistical tools such as mean and standard deviation were used to display quantitative data. Frequency and percentage were used to display qualitative data. The independent n = 35) and ESCC (n = 35) as case and those without esophageal tumour as control (n = 70). In general, the HPV genome was found in 20 (28.57%) out of 70 paraffin-embedded tissue specimens of esophagus cancer (case group). Six cases (17.14%) of EADC and 14 cases (40%) of ESCC were positive for the presence of HPV genome. Nevertheless, none of the control samples was positive for HPV DVA. The clinical characteristics of the patients are given in In the current study, 150 paraffin-embedded esophagus tissue blocks were collected. The quality of the extracted DNA was found unsuitable in 10 samples; therefore, they were excluded from the study. Subsequently, we investigated 140 paraffin-embedded tissue blocks that were investigated in two groups based on the pathological diagnosis: 70 patients with esophageal cancer EADC  in the case and control groups. The results showed that 28.57% of all esophageal cancer cases were positive for the HPV L1 gene. In esophageal adenocarcinoma, six cases (17.14%) and in esophageal squamous cell carcinoma, 14 cases (40%) were positive for the HPV genome. No positive samples were observed in the control group.In China, based on the National Central Cancer Registry report, the fourth leading cause of death from cancer was related to esophageal cancer. The prevalence of HPV in the ESCC varied from 6.7% to 83.3% , 20.In a study in the high-risk area of South Africa (Transkei), the prevalence of HPV in esophageal cancer samples was estimated to be 46% . In TurkAccording to the reports from the International Agency for Research on Cancer (IARC) in 2018, age-standardized incidence rate (ASR) per 100,000 for esophagus cancer in Iran in both males and females was 6.6 and 5.2, respectively. Additionally, age-standardized mortality rate (ASMR) per 100,000 for esophagus cancer in Iran was 2.9 . The preIn a study in the high-risk area of Iran (Turkmen Sahra), HPV DNA was recognized in 49.4% of patients with ESCC, and the positive cases encompassed HPV-16 (54.7%), HPV-18 (4.8%), HPV-6 (14.3%), HPV-66 (7.1%), and HPV-52 (4.8%) .In a case-control study in Iran (Tehran), the HPV L1 gene was detected in 36.8% of ESCC samples and 13.2% of control samples. Among positive samples, HPV-16 and HPV-18 were observed using type-specific E6/E7 primers in 13.2% and 7.9% of ESCC samples, respectively. While, 13.2% of the control samples were positive for the HPV-18 E6/E7 gene .In another study in Mazandaran, Northern Iran, 177 cases of ESCC were investigated. Of these, 49 cases 27.68%) were positive for HPV DNA [% were poP < 0.0001). Nevertheless, the values obtained in this study were lower than the high-frequency areas elsewhere in the world and in Iran. Differences in sample size, research design, the sensitivity of the diagnostic methods, statistical analysis, environmental factors contributing to esophageal cancer incidence, nutritional habits, and race may be responsible for the differences in the results of the studies in various regions.In the present study, the prevalence of HPV in EADC (17.14%) and in ESCC (40.0%) was lower than previously conducted studies in the high-risk areas for ESCC. Therefore, based on the results of the chi-square test, significant differences in the prevalence of HPV were observed in the case group than in the control group  and esophageal cancer , 29–32.In this study, the high-risk genotypes of 18 and 16 were detected in HPV-positive samples by analysis of sequencing, as reported in most other high-risk areas.The results of this study revealed significant differences in prevalence of HPV between the case group and control group which is in consistent with previous studies. Another finding was that the presence of high-risk genotypes (16 and 18) implicates HPV as one of the possible etiological factors in esophagus cancer. As different rates of prevalence of HPV in esophageal cancer have been reported from Iran and other parts of the world, a further extensive investigation with larger numbers of subjects over wider geographical areas is needed to strongly support the role of human papillomavirus in esophageal cancer.