To determine the association of human papillomavirus infection (HPV) and transitional cell carcinoma (TCC).Using polymerase chain reaction, fifty-nine bladder tissue specimens of patients with transitional cell carcinoma of bladder compared with 20 bladder samples of cases with non-neoplastic disorders.P = 0.62). Mean age was 67 ± 10.8 years and 52 ± 20.3 years in the case and control groups, respectively (P = 0.6). Of the 59 tissue specimens with diagnosis of transitional cell carcinoma, HPV DNA was detected in 21 (35.6%) samples, while it was present in only one sample (5%) in the control group (P = 0.008). HPV18 was the most common type of virus with the incidence rate of 17/21(81%).Male to female ratio was similar in the two groups (50/9 vs. 16/4, HPV might play a causative role in transitional cell carcinoma of bladder in our geographic area. The etiology of transitional cell carcinoma (TCC), which represents 90 percent of bladder malignancies, is not quite clear, while squamous cell carcinoma 5%) of the bladder is well associated with some factors like urinary stones and prolonged infections. Some genital malignancies like vulvar or cervical neoplasms are associated with previous infection with human papillomavirus (HPV). Recently, conflicting findings have been reported on association of HPV infection and TCC -5.% of the Using PCR, the overall prevalence of human papillomavirus (HPV) DNA in penile carcinoma is about 40–45%, which is similar to the detection rate of HPV-DNA in vulvar carcinoma . Males mWhile Kamel et al found HPV DNA in 57% of cases of TCC , Tekin'sThe medical records and bladder tissue specimens of 59 consecutive patients with transitional cell carcinoma of bladder who underwent transurethral resection of bladder tissue from October1999 to May 2002 were reviewed and compared with those of 20 consecutive patients with non-neoplastic disorders who were served as controls. So, a total of 79 biopsy tissues of urinary bladder were re-evaluated and analyzed by polymerase chain reaction (PCR). The designing and conducting of this study was in accordance with the ethical standards of UNRC ethics committee. Table Specimens were taken from paraffin-embeded bladder tissues stored in laboratory of our hospital- Tajrish Shohada medical center. All specimens were collected and transported to molecular biology research center of shaheed Beheshti University of Medical Sciences under a well-controlled condition. Biopsy specimens were digested in lyses buffer  at 37°C for 4 hours. Phenol-chloroform extraction and ethanol precipitation were performed. The precipitated DNA was suspended in distilled water and used for amplification.2, 0.1 mM of each dNTP, 20 pico mol of each primer, 0.1 μg DNA, 1.25 unit of Taq DAN polymerase and 1× PCR buffer. PCR conditions are: 30 cycles of denaturing at 94°C for 30 sec., annealing temperature at 45°C for 30 sec. and extension at 72°C for 30 sec. PCR reaction was performed using automated thermal cycler machine .Two primer pairs were designed of HPV L1 gene (encodes the major capsid protein): one primer pair amplify a 269-bp fragment of HPV 6, 11, 31 and 33, and another primer pair amplify a 564-bp fragment of HPV 16, 18 and 35; and HPV types were defined by PCR products restriction analysis. PCR: Amplifications were carried out in 50-μl volumes. The reaction mixture contained 1.5 mM MgClPCR products were electrophoresis on 2% agarose gel. Gels were stained by ethidium bromide and photographed by UV transilluminator at 254 nm.P < 0.05.Statistical analysis was performed using Mann-Whitney, chi-square and fisher's exact tests as appropriate with significance considered at P = 0.62). Mean age was 67 ± 10.8 (range 38 to 85) years and 52 ± 20.3 (range18 to 81) years in the case and control groups, respectively (P = 0.6).The study population comprised 59 cases and 20 controls whose demographic and clinical characteristics are given in table P = 0.008) that was histolologically diagnosed as severe chronic cystitis with some degrees of atypical hyperplasia, that might be considered as a pre-malignant condition.Of the 59 tissue specimens with diagnosis of transitional cell carcinoma, HPV DNA was detected in 21 (35.6%) samples. Human papillomavirus DNA was positive in only one sample (5%) in the control group , one male and 1 female; HPV 18 was found in 14/21 patients (66.7%),13 males and 1 female ; and HPV 33 sequences were detected in2/21(9.5%), 1 male and 1 female. In 3 HPV-positive male cases (14.3%) more than one type was found; 2 patients with HPV6/HPV18, and 1 witht HPV18/HPV33. Thus, HPV18 was the most common type of virus with the incidence rate of 17/21(81%). HPV18 was also found in one male patient in the control group. HPV types 11, 16, 31 were present in no tissue specimens. Table P = 0.59).The histopathological stages and grades of tissue samples with diagnosis of transitional cell carcinoma considering infection with HPV are given in Table P = 0.00). In the case group, 12 out of 21 (57%) infected patients and 20 out of 38 (52.6%) non-infected were cigarette smokers, respectively (P = 0.7). Ten cases (16.9%) but no patient in the control group were addicted to opium (P = 0.05). On the other hand, four infected (18.2%) and 6 non-infected (10.5%) subjects were addicted to opium (P = 0.36).Smoking rate in the case group was significantly higher than control group  oncoprotein E7 is a mechanism by which HPV promotes cell growth . Human pHPV may have a great role in progression of TCCs toward higher stages and/or grades by inactivation of the tumor suppressors or other unknown mechanisms.Larue et al. reported that sensitivity of detection of HPV is largely dependent on a series of technical factors such as tissue fixation, DNA preparation and amplification conditions. In their study, presence of HPV correlated with grade but not stage of the tumors. . Our speHigh rate of HPV positivity in present study suggests that other sexually transmissible viruses may play some roles in development and progression of transitional cell carcinoma of the bladder. As Gazzaniga et al demonstrated, there may be a high synergism between Human Papillomaviruses type 16 and 18 , Epstein-Barr virus (EBV), cytomegalovirus (CMV) and herpes simplex virus type 2 (HSV-2) and bladder carcinogenesis. In thisOn the basis of high rate of HPV positivity in TCC cases (35.6%), comparing with control group(5%), the present report supports an etiologic role of HPV in bladder carcinogenesis.This quasi-experimental study is the first report from Iran on HPV infection and transitional cell carcinoma. Our findings, in spite of some previous reports, suggest that HPV may play a causative role in transitional cell carcinoma of bladder in our geographic area. Public education regarding HPV transmission and high-risk behavior may be helpful for decreasing the incidence of urogenital malignancies.HPV: Human Papilloma VirusThe author(s) declare that they have no competing interest.M.R.B. and A.H. introduced the cases and performed surgical operations. S.M.M.H.M. designed the study and performed statistical analyses. B. K. performed laboratory examinations and PCR. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The Early-7 (E7) protein of HPV binds to the underphosphorelated form of the tumor suppressor protein – pRb and displaces the E2F transcription factor that is normally bound by pRb. The latent membrane protein-1 (LMP-1) of EBV prevents apoptosis of B cells by up regulating the expression of bcl-2, and it activates growth promoting pathway that are normally triggered by T cell – derivate signal. The aims of this study to know that in cervical cancer stay HPV and EBV.DNA was isolated from nineteen sample cervical cancer tissues frozen section. Diagnose related with HPV and EBV was made by Polymerase Chains Reaction (PCR).The result of this experiment showed that from 19 samples diagnosed as cervical cancer, 17 samples are positive HPV and 13 samples had HPV and EBV positive. The conclusion of this experiment is 89% of cervical cancers are infected with HPV and 68% also infected with HPV and EBV. The traditional way to diagnose tumors is by histopathology stains and analysis. The diagnosis of cancer relies primarily on invasive tissue biopsy . The conCancer of the uterine cervix is the most common form of cancer in women developing countries as leading cause of cancer-related deaths in women in the world as a whole . CervicaThe E7 protein binds to the underphosphorelated form of the tumor suppressor protein pRb and displaces the E2F transcription factor that is normally bound by pRb ,5,6.The latent membrane protein-1 (LMP-1) of EBV prevents apoptosis of B cells by up regulating the expression of bcl-2, and it activates growth promoting pathway that are normally triggered by T cell-derivated signal ,5,6.Nineteen frozen section tissues are collected from obstetric and gynecologic part of doctor Muwardi Hospital Surakarta patient with cervical cancer from January 2001 to January 2002. DNA isolation was made by Henk Schmits method with some modifications. Cut up to 25 mgr of tissue into small pieces, place in 1.5 ml a microfuge tube volume, and add 200 ul of DNA extraction buffer. Add 20 ul of Proteinase K stock solution, mix by vortexing, and incubate at 55°C overnight .5/Ebna 3C3 primers (CYBERGENE AB). PCR protocol for both amplifications are 94°C for 50 seconds, 59°C for 50 seconds, 72°C for 50 seconds and 4°C soak. The Amplification of HPV-L-1 gene produced 450 bp long. The amplified EBV-Ebna 3C product is 153 bp long as EBV1 and 246 bp as EBV2.Diagnose related with HPV infections are made by Henk Schmits and/or Nigel McMillan and Nina Fowler PCR-method with some modifications ,8. DiagnThe traditional way of classifying tumors is by histopathology; the staining and analysis of tissue samples. Now, the ability to analyze change in the levels of the transcripts and/or protein products for literally thousands of genes promises interesting possibilities as a research tool – for understanding the underlying molecular mechanisms, but also for automated tissue diagnosis ,10. The The result of this experiment is from 19 cervical cancer samples found 17 samples (89%) with HPV positive.Neoplasm of the cervical uterine is the most common form neoplasm in women ,13-20. TMore than 85% of invasive squamous cell cancer infected by HPV types 16 and 18 and, less commonly, types 31, 33, 35, and 51. The affinity of these viral proteins for the products of tumor suppressor genes differs depending on the oncogenic potential of HPV. Early-6 and E7 proteins derived from high-risk HPV bind to pRb and p53 with high affinity. The Early-6 protein of HPV binds to and facilitates the degradation of the p53 gene product. The Early-7 protein binds to the underphosphorelated form of the tumor suppressor protein pRb and displaces the E2F transcription factor that are normally bound by pRb [Human papilloma viruses as an oncogenic virus are well characterized viruses that play role in cervical cancer. However there are possible that other viruses might also involve in this cancer incidence ,13-21.Other result of this experiment is from 17 samples with HPV positive found 13 samples had also EBV positive.Epstein-Barr viruses known involve in many malignancy cases such as nasopharyngeal carcinoma, limphoma maligna, Burkitts limphoma, Hodgkins disease and gastric carcinoma. It is known that EBV also infected squamous cells ,13,22-25There is increasing evidence that among the concequences of viral infection are influence on cell survival which result from inference, by viral elements, with the normal regulation of apoptosis. Epstein-Barr virus enhancing the survival of B cells via the number of mechanism. Of these, conversion of cell from bcl-2 negative to bcl-2 positive through to transactivating ability of the latent EBV gene LMP-1 and EBNA-2, provides the virus with a strategy for survival during the germinal centre stage of B cell development when expression of bcl-2 protein is normally down regulated ,5.From this experiment we found 13 samples (68%) had HPV and EBV positive. Our conclusion is expected HPV may be EBV also involve in carcinogenesis of cervical cancer.


Human papillomaviruses (HPVs) are the primary etiological agents of cervical cancer and are also involved in the development of other tumours . Serological survey of the HPV infections is important to better elucidate their natural history and to disclose antigen determinants useful for vaccine development. At present, the analysis of the HPV-specific antibodies has not diagnostic value for the viral infections, and new approaches are needed to correlate the antibody response to the disease outcome. The aim of this study is to develop a novel ELISA, based on five denatured recombinant HPV16 proteins, to be used for detection HPV-specific antibodies.The HPV16 L1, L2, E4, E6 and E7 genes were cloned in a prokaryotic expression vector and expressed as histidine-tagged proteins. These proteins, in a denatured form, were used in ELISA as coating antigens. Human sera were collected from women with abnormal PAP smear enrolled during an ongoing multicenter HPV-PathogenISS study in Italy, assessing the HPV-related pathogenetic mechanisms of progression of cervical cancer precursor lesions. Negative human sera were collected from patients affected by other infectious agents. All the HPV-positive sera were also subjected to an avidity test to assess the binding strength in the antigen-antibody complexes.Most of the sera showed a positive reactivity to the denatured HPV16 proteins: 82% of the sera from HPV16 infected women and 89% of the sera from women infected by other HPV genotypes recognised at least one of the HPV16 proteins. The percentages of samples showing reactivity to L1, L2 and E7 were similar, but only a few serum samples reacted to E6 and E4. Most sera bound the antigens with medium and high avidity index, suggesting specific antigen-antibody reactions.This novel ELISA, based on multiple denatured HPV16 antigens, is able to detect antibodies in women infected by HPV16 and it is not genotype-specific, as it detects antibodies also in women infected by other genital HPVs. The assay is easy to perform and has low cost, making it suitable for monitoring the natural history of HPV infections as well as for detecting pre-existing HPV antibodies in women who receive VLP-based HPV vaccination. Human Papillomaviruses (HPVs) infect cutaneous, genital, and respiratory epithelia in tissue-specific manner. Papillomaviruses are non-enveloped viruses with a double-strand DNA genome of 8 Kb. The genome is encapsidated in an icosahedral structure of 55 nm-diameter, composed of the L1 and L2 proteins, which are the major and minor capsid protein, respectively. The HPV16 genome codes for several non-structural proteins: E1, E2, E4, E5, E6 and E7. E1 and E2 are essential in viral transcription and replication, E4 binds cytokeratins and is involved in modification of the cytoskeleton network; E5 affects cellular receptors of growth factors, whereas E6 and E7 are the major transforming proteins [HPV infections are widespread in the general population, and viral infection is closely associated to both benign and malignant lesions ,3. StudiThe main conclusion from these sero-epidemiological studies is that the antibody response to HPV16 proteins does not invariably occur during a natural HPV infection. For example a humoral immune response to VLPs is induced in about half of the women with normal cytology and HPV DNA presence in their cervical epithelium. The VLP-specific antibodies are neutralizing and genotype-specific and have been crucial for the development of preventive HPV vaccines ,9. An anEscherichia coli (E. coli). The assay uses the recombinant viral L1 and L2 capsid proteins, E6 and E7 oncoproteins, and non-structural E4 protein, all in denatured form. The assay has been tested to monitor the antibody response in women enrolled in the HPV-PathogenISS cohort to study the HPV-related pathogenetic mechanisms of CIN progression in HIV-negative and HIV-positive women [This paper reports the development of an in-house ELISA system, based on five HPV16 proteins expressed in ve women .Before testing the 99 sera for the presence of HPV antibodies, DNA samples from cervical smears of the same patients were analysed for the presence of HPV DNA, followed by virus genotyping. HPV sequences were found in all the samples, but the identification of a specific HPV genotype was possible only in 66 samples. In the present study these 66 cases were considered Table . Among tE. coli and purified. The protein pattern shows bands of expected molecular mass; the level of protein purity is over 95%. The proteins were inoculated into mice to generate specific hyper-immune sera. All the viral proteins showed high immunogenicity, inducing specific antibodies reacting with the corresponding antigen in ELISA, Immunoprecipitation and in Western blotting experiments (data not shown).To study the HPV-specific antibody response, several ELISAs were set up in parallel using the recombinant HPV16 L1, L2, E4, E6 and E7 proteins as coating antigens. Figure In the attempt to identify HPV specific and pan-reactive antibodies, we set up an ELISA using the HPV16 recombinant proteins in denatured form, with carefully determined cut-off values (see below). Among the 66 sera examined, 22 derived from women infected by HPV16, while 44 cases from women infected by other HPV genotypes. The results of a representative multiple HPV-ELISA are shown in Figure Among the 44 sera of women infected by other HPV genotypes, 39 (89%) reacted at least to one of the five HPV16 proteins, and only 5 (11%) did not react at all. Of all the 66 sera analysed, 57 (86%) were reactive at least to one HPV16 protein, and 9 sera (14%) were entirely negative. The reproducibility of the assay was high as indicated by the inter-assay Coefficient of Variation (CV) of about 5%. The response of the different sera to the same antigen is variable and the differences might reflect the different stage of viral infections.Figure The percentage of negative sera, shown in Figure The Avidity Index (AI) of specific immunoglobulins is a useful parameter for diagnosis of primary or past infection in a large number of viral diseases . It is wIn this study, an in-house ELISA based on the recombinant HPV16 L1, L2, E4, E6 and E7 proteins in denatured form has been developed and used for the parallel detection of specific antibodies in sera collected from women with abnormal PAP smears. The results show that this assay has an increased sensitivity compared to the tests based only on one viral protein, resulting in detection of a higher number of positive sera. The assay is not genotype-specific but it can be used to detect antibodies raised against genital HPVs other than type 16. This suggests that the 5 proteins belonging to different HPV genotypes share linear epitopes, as could be deduced from the high level of amino acid identity of these proteins. By comparing the amino acid sequences of the HPV16 L1, L2, E4, E6 and E7 to those of the other genotypes found in our study, the highest identity (>65%) is found among the L1 proteins. The homology among the E7 proteins is over 50%, similar to that of the E6 proteins, in contrast to L2, where homology is less than 50%. The sequence identity among the E4 proteins of related genotypes is less than 40%, and even less among distantly related genotypes (data not shown).The specificity of the antigen-antibody interactions in the ELISA was indirectly measured by the avidity test, where immunoglobulins non-specifically bound to denatured proteins were removed by the urea treatment. As discussed above, most of the HPV-specific antibodies bound to the 5 antigens had AI higher than 40%, implicating specific antigen-antibody reactions, even in the sera of women infected with HPV other than type 16. These results suggest that the ELISA combined with the Avidity Index might improve the diagnostic value of serology survey in HPV infections.Due to the limited number of sera tested in the present study, it is evident that this ELISA system needs validation in large-scale population studies, especially for the detection of HPV pan-reactive antibodies in people infected by genotypes different from HPV16. Nevertheless, in view of the known cross-reactivity between the most common HPV genotypes circulating in Europe ,25, thisE. coli are cost-effective and easy-to-standardize serological reagents [In conclusion, the results of the ELISA introduced in this study are encouraging even though further investigations are needed to evaluate its usefulness in population screening for HPV infections. Moreover the assay is easy to set up since the recombinant proteins expressed and purified from reagents -30. The reagents  and the reagents ,33.NC_001526) [6 tag proteins (Table E. coli JM109 strain. The authenticity of the constructs was confirmed by DNA sequencing. The deduced amino acid sequences of the inserts were compared to those of the HPV16 (NC_001526), showing 100% identity.The HPV16 genes were generated by PCR on pMHPV16d plasmid containing the complete virus genome (Accession number NCBI _001526) , kindly 2HPO4 300 mM NaCl, 10 mM Tris, 1% Triton X-100, pH 8), containing 8 M urea, while bacterial cells transformed by E6 plasmids were lysed in Phosphate buffer, containing 6 M Guanidine-HCl. Proteins were purified by affinity chromatography on Ni-NTA resin and eluted by a pH gradient. The fractions containing the proteins were pooled, adjusted to a neutral pH by adding Tris pH 8.8 and stored in urea buffer at -30°C, until use. Protein concentration was determined by standard method . To evaluate the purity, each protein samples of L1, L2, E4, E6 and E7 were denatured in SDS-loading buffer , separated in pre-cast gel Nu-PAGE MES-SDS (INVITROGEN), stained by Gel Code Blue Stain Reagent (PIERCE). The proteins were identified by Western blot using the monoclonal anti poly-Histidine antibody Clone HIS1 (Sigma-Aldrich). A peroxidase-conjugate goat anti-mouse IgG (H+L) (SBA-INC USA) was used as secondary antibody. The immune-complexes were revealed by chemiluminescence (Amersham Bioscience).The recombinant proteins were purified using a denaturing protocol: bacterial cells transformed by L1, L2, E4, and E7 plasmids were lysed in a Phosphate buffer . To assess DNA integrity, the beta-globin gene was PCR amplified in each samples by the primers PCO3 and PCO4 . To deteThe sera analysed in this study were collected from women enrolled in the HPV-PathogenISS study. The women had an abnormal PAP smear as requested for the study design . Only 99The denatured HPV16 recombinant proteins (0.25 μg/well) were adsorbed in carbonate buffer (pH 9.4) into Polysorp microtiter plates (NUNC) at 4° O/N. After a blocking step of 2 h at 37°C in phosphate buffer saline (PBS) containing 3% Non Fat Dry Milk (NFDM), the plates were incubated with 100 μl of human sera diluted 1:50 in 1% NFDM-PBS, at 37°C for 1 hr. The specific antigen-antibody complexes were detected by a peroxidase-conjugated goat anti-human IgG (H+L) (SBA INC. USA), using the TMB (VECTOR) as a substrate. After 30 min, the enzymatic reaction was stopped by adding 50 μl of 1 M sulphuric acid/well. Optical density was read at 450 nm. Washing steps were done with 200 μl/well of PBS containing 0.05 % Tween 20. The Monoclonal anti-poly-Histidine antibody was used as control of the coating step.The avidity test was performed as described by Roque-Afonso et al. . Serum sTo determine the cut-off values for each antigen, 40 control human sera were tested in ELISA against each of the five HPV recombinant proteins. The assay included the endpoint titre of the corresponding hyper-immune HPV sera as a positive control. The cut-off value was calculated as the arithmetic mean of the absorbance values of the negative sera, plus two standard deviation (SD). After the first cut-off calculation, the values of outlier serum samples were excluded, and the cut-off was re-calculated ,44. To cThe author(s) declare that they have no competing interests.PDB conceived the study, participated in acquisition, analysis and interpretation of data and in drafting the manuscript. FG, SM, MGD participated in acquisition, analysis and interpretation of data, LA participated in acquisition, analysis and interpretation of data and critically revised the manuscript, MB, SC; LM, AA, MC, participated in acquisition of the data; KS participated in design of the study and critically revised the manuscript. CG conceived the study, participated in its design, coordination, in acquisition of funding and helped to draft of the manuscript.All authors have read and approved the final version of the manuscript.


HPV infection in women from developing countries is an important public health problem. Therefore, we sought to determine the prevalences of HPV infection and HPV genotypes in a female population of Durango City, Mexico. Also to determine whether any socio-demographic characteristic from the women associated with HPV infection exists.Four hundred and ninety eight women seeking cervical Papanicolaou examination in three public Health Centers were examined for HPV infection. All women were tested for HPV DNA PCR by using HPV universal primers. In addition, all positive HPV DNA PCR samples were further analyzed for genotyping of HPV genotype 16, 18 and 33. Socio-demographic characteristics from each participant were also obtained.Twenty-four out of four hundred and ninety-eight (4.8%) women were found infected by HPV. HPV genotype 16 was found in 18 out of the 24 (75%) infected women. Two of them were also coinfected by HPV genotype 18 (8.3%). In the rest 6 PCR positive women, genotyping for HPV genotypes 16, 18 and 33 were negative.The prevalence of HPV in women of Durango City is low; however, most infected women have high risk HPV genotype. The women who were studied showed low frequency of risk factors for HPV infection and this may explain the low prevalence of HPV infection. The high frequency of high risk HPV genotypes observed might explain the high rate of mortality for cervical cancer in our region. Human papillomavirus virus (HPV) is the main causal factor of cervical cancer . This viWe have studied 498 women seeking for cervical Papanicolaou examination and attending three public health centers of Durango City, Mexico. One hundred and sixty six women from each health center were included in the study. Health centers were a University outpatient clinic (Institute for Scientific Research), a hospital of the Instituto Mexicano del Seguro Social (IMSS), and a hospital of the State Health Office . All 498 participants were enrolled consecutively from July to December 2003.Socio-demographic data including age, birth place, residence place, marital status, occupation, age at start of active sexual activity, number of sexual partners, use of condom, and history of smoking from all 498 women studied were obtained.A cervical smear was obtained from each participant by using Ayre spatula and cytology brush. Papanicolaou smears were evaluated according to the Bethesda diagnosis criteria .A second cervical specimen was obtained with the aid of a cervical brush for HPV DNA PCR and HPV genotyping. DNA extraction was performed by using DNAzol . With this method, good quality DNA was obtained inferred by the MW>20 kb for the genomic human DNA analyzed by means of 1% agarose electrophoresis, stained with ethidium bromide. In addition, the latter was confirmed by obtaining positive results for beta globin in all samples. HPV DNA PCR was carried out by using MY09/11 primers as described elsewhere . The MY-This study was approved by the institutional ethical committee. The purpose and procedures of the study were explained to all participants, and a written informed consent was obtained from all of them.2 test. A level of P < 0.05 was considered significant.Results were analyzed with the aid of the software Epi Info 6. To assess the association between the characteristics of the subjects and the infection, the crude odds ratio with a 95% exact confidence interval was used. In addition, comparison of the frequencies between groups was performed by the χMost (87%) women were born in Durango State, and 37% were born in Durango City. All 498 women had a residence in Durango City. The mean age was 39 years (range: 20 to 72 years). Their occupations were: 60% housewives, 16.8% professionals, 16.4% employees, 4.2% factory workers, 1% students, and 0.7% other activities. Their marital status included 70% married, 3.8% widowed, 12% never married, 8% living together, and 6% divorced. History of smoking was present in 18% of the women. The mean age at start of active sexual activity was 20 years (range: 13 to 37). The mean number of sexual partners was 1.8 (range: 1 to 20), and 18% used condom during intercourse.Eleven (2.2%) out of the 498 samples showed abnormalities in the Papanicolaou examination. Table Women found infected by HPV were 24 out of 498 (4.8%). HPV genotype 16 was found in 18 out of the 24 (75%) infected women. Two of them 8.3%) were also infected by HPV genotype 18. While six out of the 24 HPV DNA PCR positive women showed negative results in PCR for genotyping of HPV genotypes 16, 18 and 33. A representative gel obtained in HPV genotyping is shown in Figure % were alIn this study, we found a 4.8% prevalence of HPV infection in women seeking cervical Papanicolaou examination of Durango City, Mexico. This prevalence is lower than those reported in other regions of Mexico and the majority of those reported abroad. In two previous Mexican studies, prevalences of 14% and 15% were found ,17. WhilWe concluded that prevalence of HPV infection in women seeking cervical Papanicolaou examination in Durango City, Mexico was low. However, the high risk HPV genotype 16 is responsible for the majority of infections. Women studied showed low frequency of risk factors for HPV infection and this may explain the low prevalence of HPV infection. The high frequency of high risk HPV genotypes observed might explain the high rate of mortality for cervical cancer in our region.The author(s) declare that they have no competing interests.LFSA conceived and designed the study protocol, applied the questionnaires, performed the Papanicolaou examination of the women, wrote the manuscript and performed the data analysis. CAE conceived and designed the study protocol, participated in the coordination and management of the study, performed the data analysis and wrote the manuscript. MRR standardized the PCR methodology and analyzed the samples for HPV DNA detection and genotyping. MCR performed the protocol and monitored the study.The pre-publication history for this paper can be accessed here:


Epidermodysplasia verruciformis patients. One factor by which this anatomical tropism could be determined is the regulation of HPV gene expression in the host cell. The HPV long control region (LCR) contains cis-responsive elements that regulate HPV transcription and the epithelial tropism of HPV is determined by epithelial specific constitutive enhancers in the LCR. Since HPV-16 and other types infecting the mucosa differ in host cell from HPV types infecting skin, it has been hypothesized that it is the combination of ubiquitous transcription factors working in concert in the host cell that determines the cell-type-specific expression. To study if HPV tropism could be determined by differences in transcriptional regulation we have cloned the transcriptional regulating region, LCR, from HPV-16 and HPV-5 and studied the activation of a reporter gene in cell lines with different origin. To analyse promoter activity we transfected the plasmids into four different cell lines; HaCaT, C33A, NIKS and W12E and the efficiency of HPV-5 and HPV-16 LCR in the different cell lines was compared. In HaCaT cells, with a skin origin, the HPV-5 LCR was two-fold more efficient in transcriptional activation compared to the HPV-16 LCR. In cervical W12E cells the HPV-16 LCR was almost 2-fold more effective in activating transcription compared to the HPV-5 LCR. The ability to initiate transcription in the other cell lines was independent on cell origin and HPV-type.Human papillomavirus type-16 (HPV-16) infects mucosal epithelium and is the most common type found in cervical cancer. HPV-5 infects cornified epithelium and is the most common type found on normal skin and belongs to the types frequently associated with skin cancers of  Papillomaviridae family and consist of a large family of non-enveloped double stranded DNA viruses that infect the cutaneous or mucosal epithelia of a dozen vertebrate species with a strict species tropism. Over 100 human papillomavirus (HPV) types have been completely described and identified in human tissues and they together with animal papillomaviruses are divided into 18 genera based on their nucleotide sequence identity of the major capsid protein L1 open reading frame (ORF) [Epidermodysplasia verruciformis. All HPVs cause lesions of epithelial origin, but nothing is known regarding why certain HPVs are only detected in skin while some are only found in mucosal cells. It has been suggested that the different biology of transcriptional regulation of alpha and beta HPVs could be important for this tropism [Papillomaviruses belong to the me (ORF) . HPV-5 ime (ORF) . It belo tropism .97 is the major promoter which directs the expression of E6 and E7 as well as several other early gene products. It is analogous to the P97 of HPV-31 and P105 of HPV-18. For HPV-5, two promoters are suggested to exist within the LCR, an early E6 promoter and a late promoter [175 and P7535 [Once HPV has entered the cell, the viral DNA is transported to the nucleus for transcription and replication. The long control region (LCR) of HPV contains enhancer elements responsive to cellular factors and virally encoded transcriptional regulatory factors and is positioned between the L1 and E6 gene. The transcription is tightly regulated by the differentiation state of the infected epithelial cell -9 and thpromoter . Their pnd P7535 ,14. In and P7535 . Dependind P7535 -19.Since mucosal and cutaneous epithelium has been shown to express different proteins  it is co175 (E6) and P7535 (late) promotors. The HPV-16 LCR fragment (nt 7155-103) contained the P97 major promoter in the beginning of the E6 gene. The correct cloning of the LCR-fragments was verified through sequence analysis. To analyse promoter activity we transfected the plasmids into HaCaT cells from adult trunk skin [The LCR fragment was cloned from the end of the viral L1 gene to the beginning of E6. The HPV-5 LCR fragment (nt 7468-199) ended with the first ATG of the E6 open reading frame (ORF), including the suggested HPV-5 Punk skin , C33A ceunk skin , a spontunk skin  and W12Eunk skin . Both thunk skin ,26. Whenin silico and shown to have different sets of transcription factor binding sites [97 promoter [97 seems to be the major early promoter in HPV-16 although other transcription initiator sites have been observed. Two promoters are described within the HPV-5 LCR, one just in front of the E6 gene and the other in the middle of the LCR [A factor determining HPV tropism could be viral entry into the host cell and we have found some differences in virus entry dependent on HPV type and cell origin , but the anatomical tropism could not be completely explained by these results and other functional differences, like transcriptional activation, should be important for this tropism. The LCR of HPV-16 is much longer (832 bp) than HPV-5 478 bp) and has several additional binding sites for transcription factors when compared . The LCR78 bp andng sites . A numbeng sites . The HPVng sites . HaCat cpromoter . P97 see the LCR . The las the LCR . None of the LCR . The HPV the LCR . Our conThe HPV E2 protein can regulate the transcription from adjacent promoters by interacting with transcription factors like TATA-box binding protein and Sp1 . E2 recoThe author(s) declare that they have no competing interests.NM participated in the design of the study, carried out molecular cloning and transfection experiments and drafted the manuscript. MS carried out molecular cloning, transfection and transcriptional activation experiments. ME conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


The outcome of treatment by age and socio-economic status was examined for 1,588 women with invasive cancer of cervix resident in the West of Scotland and diagnosed between 1980 and 1987. There was no difference in prognosis according to either variable once analysis was controlled for stage at presentation, treatment type and tumour grade. Tumour histology, date of treatment and health board of residence had no significant effect on survival independent of other variables. A strong correlation was found between socio-economic status and the incidence of cervical cancer in the West of Scotland. Women aged 45 and over and living in deprived areas were more likely to present with later stage tumours and to survive less well than younger patients from the more affluent parts of the region. Any additional resources which may be made available for cervical cancer screening should be directed more effectively towards those most at risk.


Eighty-three women, mean age 45 years, successfully treated by surgery (S) or radiotherapy (RT) for stage 1b cervical cancer were assessed a mean of 97 weeks post treatment. Forty to 50% reported persistent tiredness, lack of energy and weight gain. Sixty per cent had not resumed their full premorbid functional status. Mean scores for anxiety and depression were higher than general population means and this sample scored higher for psychological distress than published data quoted for disease free cancer patients. These women reported many concerns about cervical cancer, most commonly fear of recurrent disease (91%). More than one-third blamed themselves for the disease. There were no significant differences in functional outcome or psychological status between treatment groups or by age or time since treatment. Psychological distress scores were significantly correlated with physical complaints (P < 0.001) and functional outcomes (P < 0.02). For the 61 women who were sexually active, sexual function post-treatment was rated as significantly poorer than subjectively recalled premorbid sexual function (P < 0.005). RT treated patients were more likely to report pain on intercourse and loss of enjoyment. Psychological as well as physical problems were highly correlated with sexual outcome (P < 0.01) 44% were unable to talk adequately with their partners about their experience. The majority felt they needed more information about cervical cancer, its treatment and how to help themselves rehabilitate. Forty-nine per cent would have liked to have had counselling. Even with the same physical morbidity the functional, emotional and sexual status of these women could be improved by giving more attention to their psychological and sexual concerns.


The prevalence of human papillomavirus (HPV) infection and other risk factors were studied in a high risk area for cervical cancer (Greenland) and in a low risk area (Denmark). From Nuuk (Greenland) and Nykøbing Falster (Denmark), random samples of 150 women aged 20-39 years were drawn. A total of 129 and 126 women were included in Greenland and Denmark, respectively. The proportion of HPV infected women assessed by ViraPap was similar in Denmark and Greenland (4.8 vs 3.9%). When type specific polymerase chain reaction (PCR) was used, the total HPV detection rate was 38.9% in the Danish population and 43.4% in the Greenlandic. A similar interrelationship between Greenland and Denmark applied to the HPV types 11, 16, 18 and 33. No relationship was observed between HPV detection and number of partners for any of the diagnostic methods. Significantly more Greenlandic than Danish women had antibodies to HSV 2, 76.0% and 26.2%, respectively. The prevalence of self-reported histories of selected venereal diseases was also highest among Greenlanders, except for genital warts where the prevalence was similar in the two areas. Greenlandic women had significantly more sexual partners, earlier age at first intercourse, more current smokers and less use of barrier contraceptives compared to the Danish women. This study confirms the results of our previous population-based cross-sectional comparison study in these areas, corroborating the conclusion that the prevalence of detectable HPV infection does not seem to be a determinant of cervical cancer incidence. However, by using DNA hybridisation techniques, temporal virus shedding is only measured at one point in time. Detectable virus shedding may not correlate with the risk of cervical cancer. In fact, HPV DNA detection may have different implications in different populations. In Denmark, HPV DNA detection may reflect transient, recently acquired infection, whereas in Greenland, it is more indicative of chronic persistent infection.


Human papillomavirus type 16 (HPV-16) infection is an important cause of cervical cancer, other anogenital cancers and, possibly, some oral and pharyngeal cancers. The association of HPV-16 with oesophageal and with prostate cancers has not been firmly established.We analysed sera from 3,757 HIV seronegative black South Africans using an anti-HPV IgG enzyme-linked immunosorbent assay (ELISA). The subjects were recruited from 1995 to 2000 as part of an ongoing cancer case control study. Cases were patients with newly diagnosed cancers of the cervix (n = 946), other anogenital organs (n = 80), the oral cavity and pharynx (n = 102), the oesophagus (n = 369) or the prostate (n = 205). The comparison group consisted of 2,055 age and sex-matched patients randomly selected from the same data base, diagnosed at the same hospitals, but with a vascular disease or with a cancer unrelated to HPV infection. Subjects' sera were randomly and blindly allocated onto ELISA plates. Optical density (OD) levels of anti-HPV-16 IgG of > 0.45 and ≥ 0.767 were taken to be cut-offs for negative, medium and high antibody levels.After adjustment for potential confounders, cancer types that showed a statistically significant association with increased anti-HPV-16 IgG antibody (Ab) levels were cancer of the cervix , cancers of other anogenital organs , and cancer of the oesophagus . Cancers of the oral cavity and pharynx showed a borderline significant association in the unadjusted model (p = 0.05) but after adjustment for confounding the trend in relation to Ab levels was positive but not statistically significant . Prostate cancer was not associated with HPV-16 seropositivity .If there is indeed an association between HPV-16 and oesophageal and possibly also some oral cavity and pharyngeal cancers, then emerging HPV vaccines may also reduce, at least in part, the incidence of these leading cancer types. Cancers of the cervix, oesophagus, oral cavity and prostate gland are among the leading cancer types in South Africa. InfectiThe aim of this study was to assess, in a standardised manner, the known association between anti-HPV-16 IgG antibodies and cancers of the cervix and anogenital region and to explore the possible associations between anti-HPV-16 IgG serological responses and cancers of the oral cavity and pharynx, oesophagus and prostate. Measuring the presence and viral loads of various HPV DNA genotypes in the target organs would have been the method of choice, but collecting tissue from cases and controls is not practical in large-scale epidemiological studies. Thus serological markers of HPV infection were used.An ongoing case-control study on lifestyle factors, infection and cancer began in 1995 in the main public referral hospitals for cancer treatment in greater Johannesburg, South Africa. Infectious agents already tested include HIV , human hSera from subjects were batch tested for HIV-1 by the South African National Health Laboratory Service Serology Laboratory using standard serological methods described previously . The curSerum aliquots from all participants were air freighted in dry ice to the University of Cape Town, Division of Medical Virology for anti-HPV-16 IgG testing by ELISA. The ELISA was a modification of that described by Studentsov and colleagues ,18, usinTo minimise laboratory variation and case-control bias, serum aliquots from the cases and the comparison group were randomly allocated on each ELISA plate by five age groups  and by case-comparison status. Specimens from males and females were run on separate plates. The laboratory staff were 'blinded' with regard to the distribution of the aliquots on the plates. In addition, for quality control purposes, ten adult consenting volunteer donors provided anonymous blood samples to use as plate controls. These donors were tested for anti-HPV-16 IgG antibody responses prior to the commencement of this study, and the sera of three donors who were found to have low, medium and high anti- HPV-16 IgG optical density values were added on wells on each of the 111 ELISA plates. As a secondary precaution, pooled sera of children collected for other purposes were added on wells of each ELISA plate to represent an additional reference group. Children's anti-HPV IgG titres are usually low, and therefore could represent a true negative (uninfected) group [The ELISA optical density (OD) cut-off for positivity was 0.45, determined by calculating the mean plus 4 standard deviations (SD) of the absorbance values obtained from children's serum samples tested by VLP-16 IgG ELISA . OD valuIn the comparison group, bivariate, chi-squared and t-statistics were calculated to measure the association between anti-HPV-16 IgG optical density and a number of demographic and lifestyle factors, in males and females, such as age , years of education , lifetime number of sexual partners , residence , birthplace , alcohol consumption (ever/never), tobacco smoking , and parity , all of which were found in some studies to be associated with HPV acquisition and with the disease conditions of interest [Odds ratios to compare anti-HPV-16 IgG levels in patients with each cancer type of interest  and the comparison group were calculated using unconditional logistic regression and shown in table The anti-HPV-16 IgG levels of pooled children's sera were indeed low  compared to the mean antibody level of the (adult) study subjects . To measure the reproducibility of the ELISA assay between plates, the intra-class correlation coefficient was calculated for the high, medium and low anti-HPV IgG levels of the volunteer sera and was found to be excellent (ρ = 0.9724).2 = 5.34, p = 0.02). In females, there was a significant inverse trend in the HPV-16 seropositivity in relation to age, being higher in the youngest age group 18–34 years (67%) and lower in the older age group  or high anti-HPV-16 IgG levels .This study is part of an ongoing cancer case control study run in the two major tertiary hospitals in Johannesburg, where the degree of diagnostic accuracy is high. Controls (comparison group) were selected from the pool of incident cancer patients and patients admitted for the fist time with vascular disease. In this study the controls were selected by excluding subjects whose cancer type was thought associated with the exposure of interest, e.g. those with squamous cell skin cancers and laryngeal cancers. We also excluded subjects with endometrial and ovarian cancer to avoid misclassification of the genital cancers of interest. Whilst a number of co-factors are thought to be important in the aetiology of the cancer types of interest (e.g. smoking), these were not part of the main hypothesis and so smoking related cancers were retained in the comparison group.Virus-like particles (VLPs) engineered from various HPV types and used in enzyme-linked immunoassay (ELISA) have been validated as type specific antigens to detect serological markers of past and present HPV infection  StudentsViral persistency and high viral loads are associated with an increase in the risk of cervical and anogenital cancers-31. AntiIn this study, a number of measures were introduced to minimise laboratory variation and bias. These included: i) using individual sera from a group of local volunteers to assess the reliability of the assay at low, moderate and high anti-HPV 16 IgG optical density levels; ii) adding a group of children's sera as a cross check for negative control values; iii) randomly allocating cases and the comparison groups on each plate to minimise case control bias; iv) 'blinding' the laboratory so that the assays were performed without knowledge of the disease or demographic status of the subjects; and v) testing cancers that are known to be associated with HPV  and those with an uncertain association  using the same assay, under the same laboratory conditions.Whilst there is no gold standard to evaluate whether the anti-HPV-16 IgG optical density cut-off levels truly reflect infection by HPV 16, a cut-off of 4 SD above that found among healthy children in a previous study (>0.45) seemed appropriate for the purposes of this study . This isIn this study, the anti-HPV 16 IgG seropositivity patterns observed in the comparison group generally concur with other studies, namely that females have a higher seropositivity or prevalence of anti-HPV 16 IgG (and higher mean OD levels) than males, despite the latter reporting a higher number of sexual partners ,34. StonAfter adjustment for a number of potential confounding factors, in keeping with other studies, women with medium and high anti-HPV-16 IgG OD levels showed increasing risks of developing cancer of the cervix. Similarly, women and men with cancers of other anogenital organs showed at least a doubling of risk in relation to anti-HPV-16 IgG positivity. Consistent with a recent multi-centre study , we obseBy contrast, no association in relation to anti-HPV-16 IgG antibodies was observed for prostate cancer. The literature regarding the association with HPV and the prostate (or a co-related infection) has been inconclusive. For example Dillner et al found anIf there is indeed an association between HPV-16 and oesophageal and possibly also some oral cavity and pharyngeal cancers, then emerging HPV vaccines may also reduce, at least in part, the incidence of these leading cancer types.


Objective: To compare two protocols for intrapartum antibiotic prophylaxis (IAP) against neonatal group Bstreptococcal (GBS) sepsis, with respect to staff compliance, in a prospective cohort study in the obstetric unitsof a community hospital (A) and a university teaching hospital (B).Methods: Cohorts comprised about 500 women attending antenatal clinics at each hospital . Womenidentified as GBS carriers at 26–32 weeks'gestation and those who had intrapartum clinical risk factors (CRF) wereeligible for IAP. Compliance was defined as the proportion of women eligible for IAP who received it accordingto protocol–as determined by audit of case records–and compared between hospitals and according to indication.Results: Overall, 39% of women were eligible for IAP. Indications were GBS carriage alone (21%), CRF alone(13% ) and both (5% ). Compliance was similar for GBS carriers at both hospitals: 78% at Hospital A and 76% atHospital B. However, because of the poor predictive value of screening before 32 weeks, only 65%of intrapartumGBS carriers actually received IAP. For women with CRF only, compliance was significantly lower at Hospital Bthan Hospital A .Conclusions: According to currently recommended protocols, about one-third of healthy women are eligible forintrapartum antibiotics to prevent neonatal GBS sepsis. In practice, antibiotics are often used inefficiently becauseof poor compliance with protocols and poor predictive values of selection criteria. Better implementationstrategies should improve compliance, but GBS vaccines are needed to replace prophylactic antibiotic use, withits associated disadvantages.


Despite the lack of randomised controlled trials and paucity of the published data, the current evidence suggests that the post-mastectomy radiation therapy (PMRT) does not represent a contraindication to skin-sparing mastectomy (SSM) and immediate breast reconstruction (IBR) in the multidisciplinary setting. Although PMRT is associated with a higher incidence of complications, a satisfactory cosmetic outcome can be achieved in most patients. Radiation has a deleterious effect on autologous flap reconstruction that relies on fat for volume replacement such as the deep inferior epi-gastric perforator (DIEP) flap reconstruction and this method of reconstruction should be delayed until RT is completed. Until better methods of RT delivery are developed to minimise complications, women at high risk of requiring PMRT, can be safely offered SSM and IBR with a sub-pectoral saline-filled tissue expander and this can be replaced with a permanent prosthesis or converted into an autologous flap reconstruction after the completion of RT. Any capsule formation can be surgically treated at this stage. This new concept, known as immediate-delayed reconstruction, can avoid the cosmetic and RT delivery problems that can occur after IBR.Furthermore, prior RT does not represent a contra-indication to SSM and IBR, however it increases the incidence of complications. Both immediate and delayed breast reconstruction following mastectomy provide substantial psychosocial benefits for women undergoing mastectomy for breast cancer . DespiteMost women undergoing mastectomy for breast cancer do not require post-operative RT. However, patients with several positive regional lymph nodes and/or large tumours are offered RT in view of the proven reduction in loco-regional recurrence and improved survival . SimilarAlthough results from individual series vary considerably, it appears that the complications of RT following IBR occur in a high proportion of patients .A study of immediate TRAM reconstructions showed the commonest complications were fat necrosis (16%) and radiation fibrosis (11%) , althougThe other concern regarding RT in the reconstructed breast is related to the use of implants, either alone or in conjunction with a flap reconstruction. The fibrosis in particular often causes subsequent shrinkage of the reconstructed breast around the implant – termed 'capsular contraction'. One study compared 39 irradiated implant reconstructed breasts with 338 non-irradiated reconstructions and found a significant negative effect on the reconstructive outcome with implants , the maiThe adverse effects of RT on SSM and IBR can be minimised by optimising RT delivery and by placing a sub-pectoral saline-filled tissue expander in order to facilitate delayed revision of reconstruction using an implant, an autologous flap with or without implant after removing the tissue expander ,14. ThisHultman and Daiza investigated the effects of previous RT on subsequent SSM and IBR in 37 breasts, although not all patients had received previous RT . TRAM anMore recently, Bacilious et al reported that tissue expansion was reliable with low morbidity in Hodgkin's patients with prior mantle irradiation . Second-However, these studies are small and larger studies with longer follow-up are required to verify these findings.We conclude that, as long as a higher complication rate is accepted and patients are fully informed, it would appear safe for women to undergo SSM and IBR after breast RT or before PMRT. Until better methods of RT delivery are developed to minimise complications, women at a high risk of requiring PMRT, can be safely offered SSM and IBR with a sub-pectoral saline-filled tissue expander and this can be replaced with a permanent prosthesis or converted into an autologous flap reconstruction after the completion of RT.


The authors discuss a new study of Bangladeshi migrants to the UK, which found that adult women who had migrated before the age of 8 years had greater luteal phase progesterone secretion than those who had migrated after that age. Progress in the biomedical and clinical sciences has relied heavily on experimental animal research. However, the impact of other comparative disciplines such as evolutionary biology, and in particular its subdisciplines of life history biology and ecological developmental biology, on human medicine has been limited. Despite its heritage and contributions, evolutionary biology has rarely been applied to understanding human development, particularly from a medical rather than an anthropological perspective.PLoS Medicine:This Perspective discusses the following new study published in 10.1371/journal.pmed.0040167Núñez de la Mora A, Chatterton RT, Choudhury OA, Napolitano DA, Bentley GR (2007) Childhood conditions influence adult progesterone levels. PLoS Med 4(5): e167. doi:This migrant study involved first- and second-generation Bangladeshi women living in the UK, women still resident in Bangladesh, and women of European descent in the UK. The study found that women who spent their childhood in conditions of low energy expenditure, stable energy intake, good sanitation, low immune challenges, and good health care had higher levels of progesterone and an earlier maturation than women who developed in less optimal conditions.Recently this gap in applying evolutionary biology to medicine has started to close, with a growing recognition that this discipline is critical to understanding the human condition and the risks of health and disease. Randolph Nesse and colleagues have proposed that evolutionary biology and “evolutionary medicine” should be a core discipline of medical training . For exaLife history theory is a framework developed to explain the very different strategies of growth, maturation, and reproduction used by different species across different environmental niches. The framework also attempts to explain the variation in reproductive and other strategies that individuals in a species may adopt, particularly when in different energetic states. Simply put, it posits that the organism must trade off its limited energetic capacity against different key components of its life course strategy so as to maximise the probability of gene flow to the next generation.Selection acts on the mechanisms underlying this trade-off capacity. Important trade-offs affect patterns of reproductive and mating strategy as well as patterns of growth and relative investment in repair, and thus lifespan. There is an extensive theoretical basis for this approach  supportIn human biology, life history theory is applied to explain biodemographical observations such as patterns of reproduction and ageing, often obtained from studies in modern hunter-gatherer communities. In human females, trade-offs affecting reproductive strategy could emerge as differences in ages of menarche or menopause, in fecundity, or in inter-birth interval. For example, a recent study showed a trade-off between the age of menarche and mortality risk across a broad sample of hunter-gatherer societies . ClearlyEvidence for developmental effects of environment on mammalian reproductive strategy is increasing. For example, in sheep it is well documented that seasonality of birth  determines the timing of puberty . In humaThis override or a similar mechanism also operates beyond puberty, as shown by the well-established relationship between energy balance and ovarian function in adult women . The setPLoS Medicine, Alejandra Núñez-de la Mora et al. [In a new paper published in a et al.  take theThis finding adds considerable weight to the utility of applying life history concepts to the study of human reproduction. Such perspectives may be important in understanding the conflict between the sociological drive to later first pregnancy and the declining fertility of older women, particularly those attempting their first pregnancy, who then must face the increased use of assisted reproductive technologies. Whether the conditions of early life are intertwined with the pattern of later reproductive decline merits further research.In the final part of their paper the authors speculate that the increased adult levels of progesterone experienced by younger migrants may play a role in the changing incidence of breast cancer. This is a rather broad extrapolation from their data, and there are many other possible explanations for the epidemiological observations. Yet there is other evidence associating early life events with the changing risk of breast cancer—larger babies have a greater risk of premenopausal breast cancer, for which several alternative mechanisms, including increased levels of intrauterine growth factors, have been proposed . Thus, aThis paper reminds us of the broader importance of considering early life effects in human medicine. The “developmental origins” model originally focused on the effect of prenatal nutrition on later risks of disease  and was


Primary esophageal adenocarcinomas are thought to arise from Barrett esophagus, an acquired condition in which the normal esophageal squamous epithelium is replaced by a specialized metaplastic columnar-cell-lined epithelium.Since the early 1970s, a dramatic change has occurred in the epidemiology of esophageal malignancy in both North America and Europe: the incidence of adenocarcinomas of the lower esophagus and esophagogastric junction is increasing. Several lifestyle factors are implicated in this change, including gastroesophageal reflux disease , severity of gerd, endoscopic and histologic findings, and a panel of biomarkers will be developed to better identify patients with Barrett esophagus at increased risk for malignant progression, leading to more rational endoscopic surveillance and screening programs.Today,  Although diet Since the early 1970s, a dramatic change has occurred in the epidemiology of esophageal malignancy in both North America and Europe: the incidence of adenocarcinomas of the lower esophagus and esophagogastric junction , thereby promoting cellular proliferation and inducing specialized columnar metaplasia of the normal squamous epithelium lining the esophagus. Dysplasia is widely regarded as the precursor of invasive cancer, and high-grade dysplasia in Barrett epithelium is frequently associated with esophageal adenocarcinoma. That association underlies the rationale for endoscopic surveillance programs: to detect malignancy at an earlier and potentially curable stage. However, the surveillance strategy remains controversial.An established risk factor for Barrett esophagus is The recent identification of molecular markers associated with the Barrett metaplasia–dysplasia–carcinoma progression may have potential clinical application to identify individuals with Barrett esophagus who are at increased risk for progression to invasive adenocarcinoma The present review focuses on the pathogenesis of Barrett esophagus and esophageal adenocarcinoma, with emphasis on selected molecular biomarkers that may have future clinical application to the management of this disease.The diagnosis of intestinal metaplasia is made histologically and is characterized by the presence of goblet cells egj egj The definition of intestinal metaplasia (Barrett esophagus) may be further categorized by endoscopic measurement of the length of the columnar epithelium. Classic “long-segment” Barrett esophagus refers to specialized intestinal metaplasia extending more than 3 cm above the anatomic From a practical standpoint, intestinal metaplasia of the esophagus and cardia cannot be accurately differentiated on the basis of routine histology alone. Recent immunohistochemical studies have reported a potential role for cytokeratins 7 (CK7) and 20 (CK20) in differentiating the two The reported prevalence of Barrett esophagus has varied between populations, and most studies have reported incidence and prevalence rates for the classic long-segment disease dna abnormalities. Such dysplasia is generally classified as indefinite, low-grade, or high-grade Dysplasia within a segment of Barrett esophagus can be identified histologically based on phenotypic nuclear alterations resulting from hgd) is regarded as the most reliable predictor for progression to invasive cancer; hgd is frequently associated with esophageal adenocarcinoma hgd to invasive adenocarcinoma is quite variable; reports have indicated that most patients with hgd actually die from non-malignant causes Although the natural history of dysplasia is not known, it was recently reported that the presence of low-grade dysplasia in Barrett epithelia conferred an increased risk of progression to malignancy Relatively few studies have investigated the prevalence of dysplasia within Barrett epithelia. Based on careful pathologic examination of resected esophageal adenocarcinomas, dysplastic change is reported in a relatively high percentage of associated Barrett epithelia egj or cardia. The Siewert system is increasingly being used to classify adenocarcinomas of the esophagus and esophagogastric junction. This system is based on an estimate of the tumour centre in relation to the egji  arise 1 cm to 5 cm above the egj, and type ii (cardia adenocarcinomas) arise from the region 1 cm above to 2 cm below the egj. Type iii (subcardia gastric adenocarcinomas) arise 2 cm to 5 cm below the egj. One disadvantage of the Siewert classification is that precise measurements may be difficult for large, advanced-stage tumours.The definition of primary esophageal adenocarcinoma is controversial, especially when the tumour involves the i) from adenocarcinomas arising at the egj (type ii) Since the mid-1990s, our group has used strict clinicopathologic criteria to stratify primary esophageal adenocarcinomas The most important criterion for establishing a diagnosis of a primary esophageal adenocarcinoma is the presence of Barrett epithelium. However, because Barrett epithelium may not be identified in up to 50% of surgically resected esophageal adenocarcinomas (likely as a consequence of tumour progression), criteria 2 to 5 should be met to establish a primary esophageal origin .egj range from 0.74 to 1.34 per 100,000 population The prevalence of esophageal adenocarcinoma has recently been well studied and, as mentioned earlier, has increased steadily since the mid-1970s. Current estimates of the annual incidence of adenocarcinoma of the esophagus and hgd or cancer at an early and potentially curable stage.The goal of any cancer surveillance program is the detection of premalignant or early invasive disease which, when treated, will ultimately result in improved survival. For patients with Barrett esophagus, endoscopic surveillance refers to esophagogastroscopy and biopsy performed at regular intervals to detect The efficacy and utility of Barrett surveillance is controversial. Among the proposed disadvantages are the difficulty of identifying early neoplastic lesions with current endoscopic techniques and the frequency of sampling error It has been suggested that new strategies are required to improve the efficacy of surveillance for Barrett esophagus. Proposed methods of reducing endoscopic variability and sampling error include the use of chromoendoscopy, magnification endoscopy, fluorescent endoscopy, or optical coherence tomography gerd. This theory is supported by a number of physiologic abnormalities that have been identified in patients with Barrett esophagus—among them, increased acid exposure, a defective lower esophageal sphincter, and impaired esophageal motility and clearance Although the precise causes and natural history of Barrett esophagus remain unknown, the condition is generally accepted to be acquired as a result of gerd leads to acute mucosal injury, promotes cellular proliferation, and induces specialized intestinal metaplasia of the esophagus. Studies have shown Barrett esophagus to be hyperproliferative, reflected by an increased S-phase fraction on flow cytometry pcna) pcna immunostaining is limited to the basal layer of metaplastic Barrett epithelia; in hgd, it extends to the more superficial layers. Similar patterns of staining for Ki67 suggest a functional instability of Barrett mucosa.The hypothesis is that gerd or Barrett esophagus (or both) has been reported egj, predisposing to gerd or to stasis.To date, no genetic locus for familial gerd (and hence be associated with Barrett esophagus); others have found stronger associations with chronic gastritis, Helicobacter pylori infection, gastric intestinal metaplasia, and gastric malignancy H. pylori, because gastric infection with S100A8-positive strains  has been found to have an inverse association with the development of esophageal adenocarcinoma Similarly, the causes and pathogenesis of cardia intestinal metaplasia are unknown. A number of reports suggest that this entity may represent an early manifestation of A number of molecular alterations have been reported in Barrett esophagus, and these molecular alterations are implicated in the molecular pathogenesis of esophageal adenocarcinoma hgd or invasive adenocarcinomaMolecular diagnosis for early detection of Prediction of risk for disease progression in endoscopic surveillance programsStaging and prognosisPrediction of chemosensitivityIntermediate biomarkers in chemoprevention studiesNovel targets for anticancer therapiesnci) Early Detection Research Network (edrn) has developed five phases to validate novel biomarkers used in the early detection of cancer  in Barrett epithelia is associated with risk of progression to malignancy Several studies have reported that aneuploidy  appeared to be at low risk for progression to adenocarcinoma Flow cytometry has also been used to study cell-cycle kinetics, including S-phase fraction, in esophageal pre-malignancy in a number of phase 1 and 2 studies TP53 is located on chromosome 17p13 and encodes a 53-kDa polypeptide (Tp53) that regulates cell cycle progression, dna repair, apoptosis, and neovascularization in both normal and malignant cells via highly complex dna and protein interactions CDKN1A , which sequesters a number of cyclin-dependent kinases (cdks), Tp53 mediates both G1 and G2/M arrest. Point mutations leading to loss of function of TP53 is a common mechanism of inactivation, and more than 90% of TP53 mutations have been located in the conserved dna binding domain (exons 5–8). Through the 1990s, TP53 was extensively characterized; it appears to have a central role in human malignancy The tumour suppressor gene TP53 gene were initially reported in primary esophageal adenocarcinomas and associated Barrett epithelium in 1991 TP53 alterations in Barrett esophagus has been extensively characterized TP53 mutations in non-dysplastic Barrett epithelia suggests that TP53 may be altered early in the metaplasia–dysplasia–carcinoma sequence, and it may therefore be a useful biomarker in endoscopic surveillance programs.Mutations in the TP53 mutations were associated with poor tumour differentiation and with reduced disease-free and overall survival following surgical resection TP53 mutation in esophageal adenocarcinomas were predominantly G:C to A:T transitions at CpG dinucleotides, suggesting that TP53 mutations result from endogenous mechanisms that likely involve spontaneous deamination into thymine of the 5′-methylated cytosine that frequently occurs at CpG di-nucleotides. Because this mechanism is enhanced by exposure to oxy-radicals and nitro-radicals, we hypothesized that local overproduction of nitric oxide, a consequence of chronic gerd, may enhance the rate of formation of spontaneous TP53 mutations in Barrett esophagus.In a 10-year prospective study of surgically resected esophageal adenocarcinomas, TP53 mutations or protein overexpression in Barrett epithelia as predictors of malignant progression, loss of heterozygosity (loh) of 17p (inclusive of TP53) was evaluated in one ongoing phase 4 study in conjunction with flow cytometry loh ranged from 6% in non-dysplastic Barrett epithelia to 57% in hgd and was a significant independent predictor of progression to esophageal adenocarcinoma, with a relative risk of 16. In this study of 325 patients with Barrett esophagus, only 6 of 26 patients who progressed to malignancy lacked 17p loh. And 17p loh was also associated with increased risk for aneuploidy, tetraploidy, and hgd, with relative risks of 7.5, 6.1, and 3.6 respectively.Although no phase 4 studies have evaluated CCND1 gene located on chromosome 11q13 Cyclin D1 is a key regulator of cell-cycle progression, particularly at the transition from G1 to the S phase; it is encoded by the CCND1, alternative gene splicing is thought to give rise to two functional transcripts cdks 4 and 6 (CDK4/6); the resulting complex phosphorylates the RB1 tumour suppressor gene, thereby resulting in cell-cycle progression to S phase. The variant transcript (cyclin D1b), a consequence of the polymorphic A-allele, encodes a truncated protein isoform with an altered C-terminal domain that has been implicated in neoplastic transformation CCND1 A/A genotype were at increased risk for gerd, Barrett esophagus, and esophageal adenocarcinoma, supporting the hypothesis that this polymorphism is an individual susceptibility factor in the molecular progression of esophageal adenocarcinoma As a result of a single base polymorphism (G870A) of CDKN2A gene , which is localized to chromosome 9p21, encodes a protein, P16, that belongs to a family of cdk inhibitors. The P16 protein binds to and inhibits CDK4/6, resulting in reduced phosphorylation of RB1 and inhibition of cell-cycle progression through G1. An alternative transcript  functions to sequester MDM2, thereby stabilizing the TP53 tumour suppressor gene The CDKN2A are reported frequently in various human malignancies, but mechanisms of CDKN2A inactivation appear to vary between tumour types. Point mutations in Barrett esophagus and esophageal adenocarcinoma are relatively uncommon, but 9p loh and promoter hypermethylation appear to be frequent mechanisms of CDKN2A inactivation CDKN2A alterations have been the subject of phase 1 and 2 studies only, they are increasingly recognized as critical early molecular lesions associated with clonal proliferation within Barrett epithelia Alterations of hgd remains the most reliable predictor of progression to invasive esophageal adenocarcinoma, but potentially promising biomarkers include aneuploidy  loh and TP53 mutations CDKN2A alterations gerd, endoscopic and histologic findings, and a panel of biomarkers will be developed to better identify patients with Barrett esophagus at increased risk for malignant progression, leading to more rational endoscopic surveillance and screening programs.Despite advances in multimodality therapy, esophageal (Barrett) adenocarcinoma remains a highly lethal malignancy. To substantially improve outcomes with this disease, future management strategies will need to focus on prevention and early detection based on an improved understanding of esophageal tumour biology. Although several molecular alterations in the progression of Barrett esophagus to invasive esophageal adenocarcinoma have been identified, relatively few will ultimately prove to have clinical application. Currently,


Chlamydia (C.) trachomatis upper genital tract infection can cause distal tubal damage and occlusion, increasing the risk of tubal factor subfertility and ectopic pregnancy. Variations, like single nucleotide polymorphisms (SNPs), in immunologically important host genes are assumed to play a role in the course and outcome of a C. trachomatis infection. We studied whether genetic traits (carrying multiple SNPs in different genes) in the bacterial sensing system are associated with an aberrant immune response and subsequently with tubal pathology following a C. trachomatis infection. The genes studied all encode for pattern recognition receptors (PRRs) involved in sensing bacterial components.In women, C. trachomatis IgG antibody testing and genotyping  of the PRR genes TLR9, TLR4, CD14 and CARD15/NOD2. In all women, a laparoscopy was performed to assess the grade of tubal pathology. Tubal pathology was defined as extensive peri-adnexal adhesions and/or distal occlusion of at least one tube.Of 227 subfertile women, serum was available for C. trachomatis infection (i.e. C. trachomatis IgG positive), subfertile women carrying two or more SNPs in C. trachomatis PRR genes were at increased risk of tubal pathology compared to women carrying less than two SNPs (73% vs 33% risk). The differences were not statistically significant (P = 0.15), but a trend was observed.Following a C. trachomatis PRR genes tends to result in an aberrant immune response and a higher risk of tubal pathology following a C. trachomatis infection. Larger studies are needed to confirm our preliminary findings.Carrying multiple SNPs in  Chlamydia (C.) trachomatis infection. Some women clear a C. trachomatis infection adequately without developing tissue damage, whereas others get a persistent infection which may ascend to the upper genital tract, increasing the risk of tubal damage and subfertility. The susceptibility, course and outcome of infectious diseases are determined by environmental factors, virulence factors of the pathogen and host factors.A large variation exists in the individual response to a C. trachomatis infection).Immunogenetic studies evaluate the role of genetic variations in immunologically important host genes as determinants of the susceptibility, course and outcome of infectious diseases. Among these variations are single nucleotide polymorphisms (SNPs), in which one nucleotide has been substituted, inserted or deleted. This may lead to synthesis of a potentially aberrant protein, or to up- or downregulation of the normal protein, and subsequently to an aberrant immune response, increasing the risk of late sequelae of infectious diseases . PRRs are present on or in circulating cells of the innate immune system (e.g. macrophages) and local cells . PRRs are involved in the bacterial sensing pathways of the innate immune system by recognizing the so-called pathogen-associated molecular patterns (PAMPs), which are pathogen-specific cell wall components or intracellular components. Since different PRRs recognize different PAMPs, pathogen recognition and initiation of the immune response is a complex and flexible system.C. trachomatis-associated tubal pathology, as shown for the PRR toll-like receptor (TLR) 4  have. (2003) . Further. (2003) ,7.C. trachomatis-associated tubal pathology in subfertile women. According to their biological function  may increase the risk of The study was performed in women who visited the Academic Hospital Maastricht between December 1990 and November 2000 because of subfertility. In all patients blood was drawn at their initial visit for a Chlamydia IgG antibody test (CAT). All spare sera were cryopreserved. Only patients who had undergone a laparoscopy and tubal testing as part of their fertility work-up were included in the present study. Since the prevalence of SNPs may depend on ethnical background, only Dutch Caucasian women were included. Patients who had undergone previous pelvic surgery (except for an uneventful appendectomy or Caesarean section) were excluded. In the Netherlands, for retrospective analysis of anonymized patient data and stored sera no ethical committee approval is required. In the fertility clinic of the Academic Hospital Maastricht, all couples are informed at intake about possible use of their anonymized data and stored sera for research purposes, and a "no objection procedure" is followed. Only patients having not objected participated in the present study.Two independent investigators, who were unaware of the CAT results, scored 259 successive laparoscopy reports to assess the grade of tubal pathology. Tubal pathology was defined as extensive peri-adnexal adhesions and/or distal occlusion of at least one tube . In caseC. trachomatis-related abnormalities  and were excluded.Of the 259 women who underwent a laparoscopy, 43 (17%) had tubal pathology (according to the above-mentioned definition) and 184 (71%) had no tubal pathology , and these 227 women participated in the present study. Thirty-two women (12%) had minor or non-C. trachomatis were detected using the species-specific Chlamydia pneumoniae IgG micro-immunofluorescence (MIF) test , as described previously [C. pneumoniae and C. trachomatis . We have previously studied the test performances of five commercially available C. trachomatis IgG tests, including the C. trachomatis IgG spot in the C. pneumoniae MIF (AniLabsystems) [C. trachomatis IgG titre obtained by the C. pneumoniae MIF (AniLabsystems) had the best predictive value for tubal factor subfertility [IgG antibodies to eviously . This spsystems) . In our ertility . TherefoFor the immunogenetic analyses, genomic DNA was extracted from the cryopreserved serum samples using either the MagNaPure LC isolator according to the manufacturers' instructions  or the High Pure PCR Template Preparation (HPPTP) Kit according to the manufacturers' instructions . Both techniques provide enough DNA for reproducible genetic analyses. Genotyping was performed using a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP)-assay or TaqMan-assay as described previously ,11-13. T2 tests were used to compare the single genotypes between C. trachomatis IgG-positive and IgG-negative subfertile women with and without tubal pathology and the healthy control group. Subsequently, the single genotypes were used to define carrier traits. The carrier traits were tested in χ2 and trend analyses. P < 0.05 was considered statistically significant.The genotype distribution was tested for Hardy-Weinberg equilibrium to assess Mendelian inheritance. Fisher's exact or χC. trachomatis IgG antibodies were present in 39 women, of whom 26 (67%) had tubal pathology and 13 (33%) did not have tubal pathology. C. trachomatis IgG antibodies were absent in 188 women, of whom 17 (9%) had tubal pathology and 171 (91%) did not have tubal pathology.Of all 227 subfertile women participating in the present study, 43 (19%) had tubal pathology, whereas 184 (81%) did not have tubal pathology. For all genes studied, the genotype distribution was in Hardy-Weinberg equilibrium in the subfertile women and the ethnically-matched control group.C. trachomatis IgG-positive subgroup of subfertile women and the healthy control group  but a clear trend was observed.The SNPs in the single genes were combined in carrier traits. The risk of tubal pathology was assessed in C. trachomatis-related tubal pathology and we confirmed our hypothesis that a carrier trait based on genes in bacterial sensing pathways had a stronger association with the risk of tubal pathology than a single gene analysis.Over the last decade, immunogenetic studies have provided insight in the pathogenesis of and susceptibility to infectious diseases. So far, the role of SNPs in immunologically relevant genes has been established in numerous diseases, e.g. sexually transmitted infections -16 and iC. trachomatis infection as compared to carrying a single SNP.Recent studies have shown the value of genetic traits in complex diseases. Carrying multiple SNPs in the same gene, or multiple SNPs in multiple genes, has been associated with an increased risk of infectious diseases and malignancies ,4,6,7. AC. trachomatis . The differences did not reach statistical significance (P = 0.15) due to the small sample size . If the association found in this pilot study would be confirmed in a larger cohort, a 50% increase in sample size (to 341 subfertile women) would result in a significant difference (P = 0.047) between C. trachomatis IgG-positive subfertile women carrying two or more SNPs versus C. trachomatis IgG-positive subfertile women carrying less than two SNPs regarding the risk of tubal pathology. A 100% increase in sample size (to 454 subfertile women) would result in a strong association  between carrying two or more SNPs and an increased risk of tubal pathology. Increasing the sample size twofold would not be possible however in our setting in a reasonable time frame. Although the present study was performed in a large fertility clinic and patients were included during a ten-year period, the number of affected women remained small, due to the low prevalence of IgG-positivity in combination with both carrying multiple SNPs and tubal pathology.Our data show a doubling of the risk of tubal pathology in C. trachomatis PRR genes tends to increase the risk of an aberrant immune response and tubal pathology. To draw significant conclusions, our hypothesis should be retested in further studies using a larger cohort.From our results it can be concluded that an adequate recognition of the pathogen at the site of infection seems to be a relevant step in the immune response, and that carrying multiple SNPs in multiple C. trachomatis IgG-positive women and C. trachomatis IgG-negative women was observed. In previous studies, the presence of C. trachomatis IgG antibodies, an indicator of a previous C. trachomatis infection, has been associated with tubal pathology [C. trachomatis infection synergistically enhance the risk of tubal pathology, while carrying these SNPs does not influence the risk of tubal pathology in the absence of a previous C. trachomatis infection  that likely result in an aberrant immune response are associated with tubal pathology following a The author(s) declare that they have no competing interests.JEdH: Data acquisition, statistical analysis, drafting the manuscript.SO: Data acquisition, statistical analysis, critically revising for immunogenetic content.JAL: Sample collection, critically revising for medical content.JML: Partial gene selection based on knock out work, critically reading the manuscript.JII: Partial gene selection based on knock out work, critically reading the manuscript. ASP: Critically revising for immunogenetic content.SAM: Study design, conception and coordination, statistical analysis, critically revising for immunogenetic content.All authors contributed to writing of the final manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


P=0.019), among which HPV 16, 18, and 31 were the most important. The negative predictive value of HR-HPV detection for histologically confirmed high-grade lesions was 100%. An age limit for HPV reflex testing may be motivated in cases of low-grade squamous intraepithelial neoplasia (LSIL), because of high HR-HPV prevalence among younger women. By using HPV reflex genotyping, additional extensive workup can safely be avoided in about 50% of all cases of atypical squamous cells of undetermined significance (ASCUS) and LSIL among women ⩾30 years. This screening strategy could potentially reduce the total abnormal cytology-reporting rate in the Swedish screening programme by about 1% and provide more accurately directed follow-up, guided by cytological appearance and HPV test results.The aim was to evaluate human papillomavirus (HPV) ‘reflex genotyping’ in cases of minor cytological abnormalities detected in the gynaecological screening programme in Stockholm, Sweden. Liquid-based cytology samples showing minor cytological abnormalities were analysed using HPV genotyping . Colposcopically directed cervical biopsies were obtained and the HPV test results were correlated with the histological results. In all, 63% (70/112) of the samples were high-risk (HR) HPV (HR-HPV) positive. A statistically significant correlation was found between high-grade cervical lesions and HR-HPV ( Globally, cervical cancer is the second most common cancer, after breast cancer, among women . It is uIn Sweden, significant decreases in the incidence and mortality of cervical cancer have been observed since the introduction of a population-based gynaecological screening programme in the 1960 s (Liquid-based cytology (LBC) has shown higher sensitivity for high-grade cervical lesions than conventional cytology , but has not been generally recommended in cases of low-grade squamous intraepithelial lesions (LSIL) because of a high prevalence of oncogenic HPV in this group , 4204 liquid-based consecutively obtained samples from routine screening were prepared, using the ThinPrep 2000 Processor  .Minor cytological abnormalities were found in 149 (3.5%) of these samples, comprising the cytological classifications of ASCUS, atypical glandular cells of undetermined significance (AGUS), and LSIL.The cytological diagnoses were defined using the Bethesda nomenclature (P=0.028), although the samples were consecutively derived from the same screening cohort. The age range was similar for both groups: 22–58 years for ASCUS and 22–59 years for LSIL.Mean age of patients was 33 years, median age 30 years, and age range 22–59 years. Women with ASCUS were older than women with LSIL; mean ages of 35 years and 31 years, respectively . HPV-DNA detection and genotyping were performed using the Linear Array HPV Genotyping Test (LA) according to the manual provided by the manufacturer (Roche Diagnostics). Thus, HPV-DNA was amplified by PCR using a pool of biotin-labelled primers that hybridise in the L1 region. Then, HPV-DNA amplicons were chemically denatured to single-stranded DNA, hybridised with matching type-specific DNA probes, immobilized on nylon strips, and detected by colorimetric determination.55, 61, 62, 64, 67, 69, 70, 71, 72, 81, 83, 84, IS39, and CP6108. Those of undetermined risk  will be considered together with the LR-HPV types -HPV types 26, 53, 66, 68, 73, and 82; and those considered to be low-risk (LR)-HPV or of undetermined risk including types 6, 11, 40, 42, 54, The ability of the HPV test to identify or exclude a high-grade cervical lesion was evaluated with the histopathology as gold standard.χ2 statistics (exact tests), and Student's t-test were used. The null hypothesis of no difference was rejected at a significance level of P⩽0.05.To determine the statistical significance of differences between groups, The primary end point was histopathologically verified CIN2+. The sensitivity, specificity, negative predictive value (NPV), and PPV of HR-HPV detection using LA to detect such high-grade lesions (CIN2+) were calculated, with 95% confidence intervals CI; .Of the 149 cases of mild cytological abnormalities studied, histopathological follow-up was obtained in 112 cases. Histology results were available for 39/56 (70%) of the ASCUS and 73/93 (78%) of the LSIL samples. These are the cases that will be discussed here later.The cytological findings in relation to the histological diagnosis in 112 women are summarised in In all, 81% (91/112) of all samples were HPV positive. There was a significant proportion of multiple infections and overlap of infections with HPV types from one, two, or all three risk categories .P=0.026).The prevalence of HPV-infected samples increased with the grade of dysplasia: 74% (43/58) of the WNL, 85% (33/39) of the CIN1, and 100% (15/15) of the CIN2+ cases were HPV positive (P=0.019). In all, 63% (70/112) of the samples were HR-HPV positive (P=0.112).positive . In thisP=0.018).In P=0.045), 18 (P=0.039), and 31 (P=0.006) with high-grade cervical lesions. Presence of coinfection with other HPV types has not been taken into consideration when performing the calculations ; however, never as a single HR-HPV infection. In this limited material, we were able to demonstrate a significant correlation between HPV types 16  of all cases, and there were no HPV 11 infections.vs 36, and 42%, respectively). However, none of these differences were statistically significant, but of course, a multiple HPV infection increases the probability of harbouring a highly oncogenic HPV type.In all, 54% (61/112) of the cases had multiple HPV infection, and of all 91 HPV-infected individuals, 67% (61/91) were infected by more than one HPV type. Multiple HPV infection was slightly more common in cases of histological CIN2+ than in CIN1 and WNL cases . Among the HR-HPV-positive samples, 41% (29/70) were infected by more than one HR-HPV type. Also in the case of HR-HPV, multiple infections were slightly more common in cases of CIN2+  than in In the Swedish population-based gynaecological screening programme, abnormal samples are associated with follow-up investigations, and clinical recommendations for the management of minor cytological abnormalities vary. In Stockholm County, all women with any degree of atypia are referred for a colposcopic examination and targeting biopsy. The question of whether HPV testing may improve the Swedish screening programme is under discussion, especially for use as a follow-up test to identify possible precancerous lesions among mild cytological abnormalities and distinguish these from reactive and degenerative lesions. This is an approach, which is widely accepted internationally (see introduction), but has only been randomly adopted in Sweden.vs 10–15%), discounting subtle changes in deference to achieving cytological certainty  NPV for high-grade cervical dysplasia during a maximum follow-up time of 1 year . This isIn the LSIL group, HR-HPV prevalence is high, about 80–85%, among women younger than 30 years, and drops to about 50% among women aged 30 years, or older. For this reason, specificity of HPV detection test can be improved by setting an age limit of 30 years for HPV reflex testing in cases of LSIL . An age In cases of ASCUS, where HR-HPV prevalence is considerably lower (about 40%), and the HR-HPV prevalence was not significantly higher among women under the age of 30 years than among older women, for which reason the diagnostic value of HPV testing in this group is independent of age.Thus, in HPV triage using the LA-HPV test for all cases of ASCUS and for cases of LSIL with age ⩾30 years, the need for extensive follow-up investigations would be reduced by about 50%, with good safety. In such cases, cytological evaluation and virological analysis could be performed using the same material (reflex testing). Because there is only one sampling event, sampling error would be reduced. Screening programme costs would also be reduced, because both analyses can be performed without recalling the patient. The decision of whether to set an age limit for HPV reflex testing in LSIL cases should be based on health–economic calculations.According to our results, only the HR-HPV group had a statistically significant correlation with CIN2+, for which the HR-HPV types 31, 18, and 16 were the most important . In thisIn cases where histology findings were WNL or CIN1, more than 50% were HR-HPV positive. These women need to be followed up, given their elevated risk for developing a high-grade cervical lesion. Repeated HPV genotyping allows diagnosing persistent HPV infection and risk stratification of women with cytological abnormalities, and might be useful for clinical management (Liquid-based cytology (LBC) combined with reflex detection of HR-HPV types in cases of minor cytological abnormalities probably provides increased sensitivity and increased specificity for the detection of high-grade cervical lesions, when used in population-based screening.Based on the fact that minor cytological abnormalities constitute 75–80% of all abnormal cytological results in Sweden, our preliminary calculations show that combining LBC with HPV reflex testing in gynaecological screening reduces the total abnormal cytology-reporting rate by approximately 1%. However, LBC screening generally yields a somewhat higher abnormal cytology-reporting rate than conventional cytology screening. Conventional cytology is the predominating screening method in Sweden today, which makes the potential 1% reduction of the abnormal cytology-reporting rate uncertain. The main value of introducing LBC combined with HPV reflex testing is probably an improved ability to identify women with an increased risk of developing premalignant and malignant cervical lesions, without increasing the abnormal cytology-reporting rate. A cost–efficiency analysis is ongoing. Unnecessary investigations and unnecessary psychological stress could be avoided, and resources could be used for more accurately directed follow-up of women with abnormal cytological findings.