The distribution of human papillomaviruses (HPVs) varies greatly across populations and HPV surveys have been performed in different geographical regions in order to apply appropriate vaccine strategies. Little information, however, exists regarding HPV genotypes distribution in immigrant women from countries at high incidence for cervical cancer. The aim of this study was to determine the spectrum of HPVs and their variants among HIV-positive and HIV-negative women immigrants in South Italy mainly from West Africa and with a history of prostitution.Cervical cytological samples have been collected from 14 HIV-positive and 31 HIV-negative immigrants (38 out of 45 were born in Nigeria), attending a gynecological outpatient clinic in the Campania region. Human papillomaviruses were detected by broad spectrum consensus-primer-pairs MY09/MY11 and GP5+/GP6+-based polymerase chain reaction and characterized by nucleotide sequence analysis. Altogether, 42.2% (19/45) of samples were HPV positive with detection rates of 57.1% (8/14) in HIV-positive and 35.5% (11/31) in HIV-negative women. Among the twelve different viral genotypes identified, HPV33, 58, 70 and 81 were the prevalent genotypes with a frequency of 6.7% each, followed by HPV16, 35, 42, 54, 31, 52, 56 and 67, in descending order of prevalence. Sequence homology studies performed on the L1 amplified fragments of HPV16, 52 and 58 isolates allowed the identification of nucleotide changes distinctive of non-European variants.The overall HPV prevalence (42.2%) was high in this immigrant women group with the most common viral types other than HPV16 and 18, against which current vaccine strategies have been developed. The distribution of HPV genotypes and their variants in high-risk immigrants reflects that of their original countries. The surveillance of risk groups that may act as viral reservoirs of uncommon genotypes within different countries are necessary to determine the severity of HPV infection with the different viral types and to monitor a possible shift of prevalent strains following vaccination. To daoperties . The preoperties -12. The operties ,13,14. Toperties . Other moperties ,15,16. Foperties . The mosoperties -27, whicoperties ,25,26,28operties -31. Genooperties .The recent success of HPV16 and 18-based prophylactic vaccines in preventing persistent viral infections and HPV-associated cervical lesions is encouraging ,34. The Several epidemiological studies have been performed among Italian women reporting the HPV type prevalence in cytological normal women, in low grade and high grade squamous intraepithelial lesions, in cervical cancer patients and in HIV-positive women -38. All No studies have been performed in Italy on the prevalence of HPV types in immigrant women with diverse risk profiles. The goal of the current study was to analyze HIV-positive and HIV-negative immigrant women, mainly from sub-Saharan Africa and with a history of prostitution, and to describe the prevalence of HPV genotypes and their variants in order to identify viral types that are spreading trough population mobility.This study enrolled 45 women attending the outpatient Unit of Obstetric and Gynecology Clinic of Pineta Grande Hospital from January through October 2004. This Hospital serves the highest number of long and short term immigrants in Campania region . All women with sufficient Italian or English language skills, who voluntarily gave their consent to participate in the study, were interviewed using a structured questionnaire on reason for the visit, demographic characteristics and sexual behaviors. Fifteen women attended the clinic for prenatal care early in pregnancy, five for post-partum follow-up and 25 for routine gynecological care or various gynecological disorders including sexual transmitted diseases (STDs).The cervical cell scrapings were collected with a cytobrush from the ecto- and endocervix of each woman. After spreading of cells on slides (Pap smears), the remaining cells were suspended in 1 ml of lysis buffer  and stored at -20°C until analysis. Cytological evaluation of Pap smears, according to the 2001 Bethesda System, revealed two low-grade squamous intraepithelial lesions (LSIL) and 43 normal samples.The study protocol was approved by the local ethical review board of authors' Institutions.Genomic DNA was extracted from cervical-scraping cell lysates according to published procedures. In particular, samples were digested with Proteinase K (150 μg per ml at 60°C for 30 min) in 100 μl of lysis buffer , followed by DNA purification by phenol and phenol-chloroform-isoamyl alcohol (25:24:1) extraction and Ethanol precipitation in 0.3 M Sodium Acetate (pH 4.6).DNA quality test, performed by amplification with specific oligoprimers targeting a fragment of the exon 7 within the TP53 gene ,39, and HPV detection was performed by 1) single round PCR using the degenerated oligoprimers MY09/MY11 as previously described ; and 2) HPV genotypes were identified by direct sequence analyses of single round MY09/MY11 amplified DNA and/or GP5+/GP6+ nested PCR amplified products obtained from each HPV-positive sample as previously described . PCR proSmaI pBS-minus vector (Stratagene) and subjected to sequence analysis following procedures previously described [Two MY09/MY11 and three GP5+/GP6+ amplified DNA samples showing sequence patterns compatible with multiple infections have been subcloned in escribed .HPV variants were searched by direct sequence analyses of MY09/MY11 amplified products (>400 bp long) obtained from each sample with a single HPV infection. The common sequence analysis limitation of low DNA amount obtained in some samples(< 50 ng) with single round MY09/MY11 PCR was circumvented by the use of AmpliCycle sequencing kit following the manufacturer's protocol of AmpliTaq DNA polymerase , specific for 0.5 ng to 30 ng of PCR products. Two viral variants, amplified from samples containing multiple infections, have been characterized following subcloning into pBS-minus vector.Phylogenetic analysis was performed by multiple sequence alignments of the 5' region of L1 genes from different HPV isolates with MegAlign program of the Lasergene software .2 test and, where appropriate, Fisher's exact test to calculate all P values related to the differences between groups with Epi Info 6 Statistical Analysis System Software . Differences were considered to be statistically significant when P values were less than 0.05.The data were analyzed using χIn this study 45 randomly selected immigrant women (38 out of 45 were born in Nigeria) attending the gynecological outpatient unit of Pineta Grande Hospital in the Campania region have been enrolled from January through October 2004. Cervical cytological samples have been collected from all women consenting to the study after the administration of a standardized questionnaire regarding their life style and sexual activity. The selected demographic characteristics of the 14 HIV-positive and the 31 HIV-negative women have been reported in Table P = 0.1732). Fifteen subjects (60%) among the 25 reporting to have had sexual risk behavior tested positive for HPV sequences (P = 0.0165). Only 4 (20.0%) of the 20 women, without sexual risks behavior, were positive for HPV  of the samples with detection rates of 57.1% (8/14) in HIV-positive and 35.5% (11/31) in HIV-negative women , 6822 (G to A), 6827 (C to A), 6828 (A to G), common to all three analyzed viral isolates and previously described in HPV58 samples from Mali and Brazil -50; the P < 0.001) for detection of all mucosal HPV genotypes and allows the identification of some HPV types, such as HPV30 and 70, not detected by either MY09/MY11 nor GP5+/GP6+ alone [The prevalence of type specific HPVs and their variants has been analyzed in randomly selected immigrant women, mostly from Nigeria, living in Campania region. Viral infections have been detected by PCR using the MY09/MY11 oligoprimers alone or followed by nested PCR with GP5+/GP6+ primer pairs. Genotyping and variant characterization has been performed by direct sequencing analysis of PCR products. Samples which appeared to contain multiple infections for a mixed-sequence pattern were subcloned and nucleotide sequence has obtained from individual clones. The nested PCR method, performed with MY09/MY11, as outer primer pairs, and GP5+/GP6+, as inner primer pairs, has been previously shown to be significantly more sensitive , as reported by the UNAIDS HIV Surveillance .in vitro studies that HPV70 together with 53 and 82, which are phylogenetically related to high risk viruses, are indistinguishable with respect to their ability to degrade p53 and to immortalize keratinocytes, suggesting the need to monitor this HPV types in more extended epidemiological studies [Several reports pointed out the possibility of certain HPV types being more common in sub-Saharan African women than elsewhere. HPV35, for instance, was slightly more common than HPV16 in Mozambique both in women with normal cytology and in those with high-grade squamous intraepithelial lesions or worse . HPV52 wPhylogenetic studies of HPV16, 18, and the less prevalent HPV types 31, 33, 35, 52, 58, and 67, obtained from different geographical regions, showed that specific variants can be identified in different ethnic groups from different geographical regions ,48,50.In this study only one HPV isolate out of six, analyzed within the MY09/MY11 region, presented homology with the reference sequence of the corresponding genotype. A second HPV16 isolate obtained from a Nigerian woman showed nucleotide signatures distinctive of HPV16 African 2 lineage, which was never detected in the Italian population. The single HPV52 isolate obtained from an Uruguayan woman, showed nucleotide changes specific of viral variants previously identified in samples from Panama and Brazil ,29,48,48Although this study has the limitation to include a relatively low number of women, due to the low participation rate, it clearly shows that migrant women carries the viral types and their variants present in their original countries, with possible implications in the spreading of uncommon HPV genotypes in the resident populations.In conclusion, the immigrant women analyzed in this study, mainly of African origin and with sexual risk behavior, have a high HPV prevalence with the most common viral types other than HPV16 and 18, against which current vaccine strategies have been developed. Wide population based epidemiological studies, including the surveillance of risk groups that may act as niches of uncommon viral types and their variants, are necessary, particularly in countries like Italy with high immigration rates, to monitor eventual spreading of uncommon genotypes for vaccine considerations.The author(s) declare that they have no competing interests.FMB, MLT and SP were responsible for the overall planning and coordination of the immigrant women community based study.  LB was involved in phylogenetic analyses. GV and RP were involved in the patients enrollment and cervical sample collection. MLD was responsible for specimen processing and HPV-DNA analysis. MLT and FMB compiled and finalized the manuscript. All authors read and approved the final manuscript.


Objective: Human papillomavirus (HPV) is the major cause of cervical carcinoma and cervical intraepithelial neoplasia worldwide. Certain HPV types have a strong association with and probably a causative role in thepathogenesis of premalignant cervical lesions. Epidemiologic studies in women infected by the human immunodeficiencyvirus (HIV) have shown an increased incidence of squamous intraepithelial lesions (SILs), whichwere predominantlyhigh-grade. Six to 30 per cent of women diagnosed with atypical squamous cells of undeterminedsignificance (ASCUS) on a Papanicolaou (Pap) smear harbor SIL in normal screening populations. This study wasundertaken to determine the presence of low- and high-risk HPV types in women infected by HIV and to correlatethe results to those of the Pap smear.Study design: HPV DNA typing (low- and high-risk) by Digene™  hybrid capture methodology was performed on cervical swabs from 209 HIV-positive women. The results of HPV typingwere correlated with those of the Pap smear in a retrospective analysis.Results: One hundred and one women (48%) tested positive for HPV subtypes by DNA typing by the hybrid capture method. Of these, 64 patients (63%) had Pap smears whichwere read as being normal, having benign cellularchanges, or having ASCUS (favor reactive process). Of these, 19 patients tested positive for both high-risk andlow-risk subtypes, 32 patients tested positive only for high-risk subtypes, and 13 patients tested positive only forlow-risk subtypes.Conclusion: HPV subtyping identifies a significant group of HIV-positive women who are at risk for developing cervical intraepithelial neoplasia, although they may not show significant abnormalities on their Pap smears.


The increasing incidence of human papillomavirus (HPV) infection and HPV-associated conditions such as genitalwarts in women is a global concern. Genital warts are a clinical manifestation of HPV types 6 and 11, and areestimated to affect 1% of sexually active adults aged between 15 and 49. HPV infection is also strongly associatedwith cervical cancer, and is prevalent in as many as 99% of cases. The psychological stress of having genital warts isoften greater than the morbidity of the disease, and therefore successful treatment is crucial. Current treatmentsare patient-applied and provider-administered therapies. Imiquimod 5% cream, a patient-applied therapy, is anefficacious treatment with tolerable side-effects and a low recurrence rate, and has the potential to be an effectivestrategy for the management of genital warts.


Objective: To evaluate the functional capacity of granulocytes and monocytes from pregnant and nonpregnant                                   women in relation to group B streptococcus (GBS) colonization status.                                   Methods: Engulfment of fluorescent GBS by peripheral blood phagocytes from GBS-colonized and noncolonizedwomen was measured by flow cytometry. Intracellular superoxiode generated in response to GBS challenge tomonocytes and granulocytes enriched from peripheral blood of these women was also measured by flowcytometry, and extracellular superoxide was determined by colorimetric assay.Results: Monocytes and granulocytes from pregnant, GBS-colonized women engulfed significantly greaternumbers of GBS than phagocytes from pregnant, noncolonized women. No difference in intracellular superoxideproduction was detected between any of the groups of women; however, monocytes from pregnant, colonizedwomen released significantly more superoxide into the extracellular milieu than did granulocytes from the samewomen. No differences in extracellular release of superoxide were observed among noncolonized womenwhether they were pregnant or not.Conclusions: Monocytes from pregnant, colonized women engulf more GBS and release more of the superoxideinto the extracellular environment, where it is unlikely to be an effective defense mechanism against intracellularbacteria. This suggests that components of the innate immune system that should serve in a protective role mayfunction suboptimally, thereby contributing to the colonization process by GBS.


Objective To study the microbial etiology of tubo-ovarian abscess (TOA).                Methods We recruited 11 women in Nairobi, Kenya who failed antibiotic therapy alone and requiredsurgical drainage of a presumptive TOA. Pus from the nine abscesses and two pyosalpinges were collected andcultured for aerobic, facultative and anaerobic microorganisms.Results Eleven women suspected of having a TOA were hospitalized and treated for a median of 6 days (range3–14 days) prior to surgical drainage of the abscess. Nine (82%) specimens were culture positive. Aerobeswere present in all nine specimens. Seven of the nine positive cultures (78%) were polymicrobial and five ofthe polymicrobial cultures contained both anaerobes and aerobes. Anaerobic Gram-negative bacilli  and Streptococcus sp.  were themost common microorganisms isolated. Neisseria gonorrhoeae and Chlamydia trachomatis were not isolated byculture or detected by polymerase chain reaction.Conclusions In Kenya, persistent TOAs are associated with endogenous flora similar to that normally foundin the gastrointestinal tract.


Background: Human papillomavirus (HPV) is the most significant cause of cervical cancer. In view of the numberof drawbacks associated with endocervical sampling, the gold standard for HPV detection, this study examined theutility and specificity of vaginal sampling as an alternative for endocervical sampling for the routine detection of  HPV.Case study: The study comprised 51 women who tested positive and 54 women who tested negative forendocervical HPV by polymerase chain reaction (PCR), confirmed by histopathology. At the time of specimencollection, both (speculum-assisted) endocervical and vaginal (no speculum) scrapings were isolated from HPV positiveand negativewomen, and  HPV DNA was assessed by PCR using the MY09/MY11 primer system;HPV typewas identified by hybridization of PCR products with type-specific biotinylated DNA probes. Each participantserved as her own control. HPV was detected in vaginal and cervical scrapes from all HPV-positive but notHPV-negative women. In HPV-positive  women the same HPV type was found in vaginal and endocervical scrapings.Conclusion: Correlation between vaginal and endocervical sampling methods was excellent in detecting thepresence of HPV DNA and for identifying distinct HPV genotypes. Utilization of vaginal testing for routine HPVdetection, and for the long-term follow-up of persistent HPV infection, is therefore recommended.


BMC Public Health 2006, 6:251) we found an error in the equation of our measurement of trait anxiety.In review of our article  differed between the two sub-samples.That is to say that the response alternative "almost never" was coded as "1" in the first sub-sample whereas in the second sub-sample it was coded as "4". Therefore we can also deduce that the responses of the participants from the different sub-samples were coded into the different direction.This error is corrected by recoding the variable so that in both sub-samples the higher score indicates higher trait anxiety. We re-conducted the analyses and found only minor differences compared to the original figures given and we also found that the error did not alter the main results or conclusions drawn.The following corrections should be incorporated into any future analysis of our original article .Trait anxiety), the Cronbach's Alpha for Trait Anxiety should be 0.85 instead of 0.88.Within the Methods section, paragraph 10 , the last sentence should now read as "The associations with trait anxiety and magnitude of changes in work were weaker." rather than "In contrast, the associations with trait anxiety and magnitude of changes in work were much weaker."Within the Results, paragraph 6 , the 3rd sentence should read "Further adjustment for personality trait anxiety attenuated the association in both sexes, but the results remained statistically significant." rather than "Further adjustment for personality factor trait anxiety had no effect on the associations."Within the Results, paragraph 7  can indicate that the construct of social capital might be more than an aspect of an individual's personality." rather than "The significant association in multilevel models together with the substantially weak association with one personality measure (trait anxiety) can indicate that the construct of social capital might be more than an aspect of an individual's personality."And finally, within the discussion, paragraph 5 (paragraph beginning "We assessed criterion") The 3The pre-publication history for this paper can be accessed here:


S. aureus in the causation of mastitis in breastfeeding women.Mastitis is a common problem for breastfeeding women. Researchers have called for an investigation into the possible role of maternal nasal carriage of S. aureus nasal carriage in mastitis. Other factors such as infant nasal S. aureus carriage, nipple damage, maternal fatigue and oversupply of milk were also investigated. A case-control design was used. Women with mastitis  were recruited from two maternity hospitals in Melbourne, Australia . Breastfeeding women without mastitis  were recruited from maternal and child health (community) centres and the rooms of a private obstetrician. Women completed a questionnaire and nasal specimens were collected from mother and baby and placed in charcoal transport medium. Women also collected a small sample of milk in a sterile jar.The aim of the study was to investigate the role of maternal S. aureus in breastfeeding women with mastitis  and control women . However, significantly more infants of mothers with mastitis were nasal carriers of S. aureus  than controls . The association was strong  after adjustment for the following confounding factors: income, private health insurance, difficulty with breastfeeding, nipple damage and tight bra. There was also a strong association between nipple damage and mastitis .There was no difference between nasal carriage of S. aureus and mastitis, but nasal carriage in the infant was associated with breast infections. As in other studies of mastitis, we found a strong association between nipple damage and mastitis. Prevention of nipple damage is likely to reduce the incidence of infectious mastitis. Mothers need good advice about optimal attachment of the baby to the breast and access to skilled help in the early postpartum days and weeks.We found no association between maternal nasal carriage of  Mastitis is a common problem for breastfeeding women -3 yet itStaphylococcus aureus (S. aureus): present in 32% to 44% of breast milk samples . WS. aureus, mother anaemic, baby prefers one breast. Variables were eliminated one at a time using logistic regression. Only those with a p-value of the Wald statistic ≤ 0.05 were retained in the model. The process was repeated until only significant variables remained. Then all independent variables eliminated in the original univariate analysis were added back into the model one at a time to check that none was now significant given the reduced model. The lroc test identified that the area under ROC curve was 0.8778, that is a high sensitivity, and the lstat test showed 80.73% correctly classified. The final model (Model 1) is presented in Table The initial model included the following variables: mother's age, income (2 levels), private health insurance, past history of staphylococcal infection, baby having difficulty with breastfeeding, nipple cracked, engorged breast/s, missed feed/s, tight bra, too much milk, using lanolin on nipple/s, baby positive for nasal The adjusted Odds Ratio for infants of mothers with mastitis to be nasal carriers was 3.23  after adjusting for possible confounding factors (Model 1). In order to explore the effects of breastfeeding factors and baby nasal carriage without including demographic factors, a second model was developed (Model 2). Without including the demographic variables , the second model is very similar to Model 1. In a third model (not shown), the demographic variables were included while limiting the analysis to private patients (n = 126), and the results were also similar. A fourth model , excluding all babies over 7 weeks also found that infant nasal carriage was significant .S. aureus was 44.4% . This is consistent with the mean of 37.2% in general populations calculated by Kluytmans and colleagues from eighteen studies in 13,873 people [The study showed that there was no difference in the proportion of women with mastitis and without mastitis who were nasal carriers (43% and 46% respectively). The overall proportion of women with a positive nasal culture for 3 people , but see3 people , 24% [23S. aureus in the first eight weeks, falling to 21% by six months [We found that a very high proportion of infants of mothers with mastitis were nasal carriers: 82% and this was statistically significantly higher than infants of other women (56%). Infant nasal carriage remained significant after adjusting for other variables. Younger infants were most likely to be nasal carriers than older infants in this study. In a similar manner, Peacock and colleagues found that 40–50% of infants were colonised with x months .An association was not found between parity or a history of mastitis and being a case in this study, in contrast to previous studies ,16. ThisThe presence of a cracked nipple was associated with a high odds for mastitis, 9.34 , after adjusting for other factors. Foxman and colleagues also found an association with "nipple cracks or sores" with an OR of 3.4  on logistic regression . PreventThis study found that using lanolin on nipples was significantly associated with mastitis on univariate analysis; however this was no longer significant on multivariate analysis. We expect that the association between creams on nipples and mastitis  is more S. aureus was isolated in the milk of 46% of women with mastitis, a similar proportion to studies over the last thirty years [S. aureus was also isolated in the milk of 17% of women without mastitis; mostly reported as "sparse" (11/15), probably reflecting contamination of the milk by bacteria on the skin of the nipple or the hands. In other studies between 0 and 20% of milk specimens from healthy women are positive for S. aureus [ty years . S. aure. aureus ,28.S. aureus was transmitted from the infant to the mother. In 1957, Wysham and colleagues demonstrated that 7 of 9 infants with positive throat cultures for S. aureus transmitted the organism to their bottle of formula milk [S. aureus [Although it is not possible to conclude whether transmission occurred from the infant to the mother's breast or visa versa, it is likely that ula milk . Babies . aureus ,30. StapS. aureus strains present in mothers with mastitis and in their infants. However, funding for this study was not sufficient to conduct PFGE.The diagnosis of mastitis relied on clinical signs and symptoms as there are no definitive tests for mastitis in women. The women in this study experienced either fever or systemic symptoms for at least 12 hours. Future studies could assess the usefulness of testing milk for the presence of leukocytes . MoleculIt was originally planned to recruit women for the control group through MCH centres in the community (n = 25). However, we relied on women being referred to the study and recruitment was slow, so we started recruiting women attending a private obstetrician for their six-week postnatal check up. It would have been inappropriate to recruit women attending the hospital (Emergency Department or Breastfeeding clinic) as we were seeking women without problems for the control group. Therefore the controls were more likely to be private patients than cases, which resulted in more women from the higher income group as controls than cases. In order to assess if this had an effect on the study results, a logistic regression model was developed using only women with private insurance and similar results were obtained as when the model included all women.S. aureus carriage in infants and mastitis in their mothers  appears to be robust as it was significant in each logistic regression model. However, it does not tell us if this association proves a link between nasal carriage and mastitis nor in which direction the transmission is occurring.A limitation of the case-control design is that any associations identified cannot be concluded to be causal. For example, private health insurance appears to be protective against mastitis , but this association is due to the selection bias that occurred during recruitment. The association between nasal Mastitis is an acute painful illness, not limited to the breast, and often associated with a negative emotional response . In ordeS. aureus is present in mother and child, and the direction of transmission of organisms between mother and child.Future studies need to aim to collect clinical specimens prospectively in order to determine the transmission dynamics between mother and infant. Molecular microbiology (e.g. PFGE) can be used to confirm that the same strain of S. aureus may be carried transiently on the hands [It is not standard practice for mothers to wash hands before breastfeeding . Future studies could focus on hand washing as he hands  and can S. aureus in mothers and infants where mothers have already experienced an episode of mastitis?Another topic for future research is recurrent mastitis. Would it be possible to reduce recurrences of mastitis by reducing nasal carriage of S. aureus and mastitis, but nasal carriage in the infant was associated with breast infections. As in other studies of mastitis, we found a strong association between nipple damage and mastitis.In conclusion, we found no association between maternal nasal carriage of The author(s) declare that they have no competing interests.The study was conducted by LHA as part of her PhD. SMG and JL supervised the project and contributed to the study design, analysis and writing.The pre-publication history for this paper can be accessed here:


One of the major mechanisms of metabolism of TAM and one of its major active metabolites, 4-hydroxytamoxifen (4-OH-TAM), is via glucuronidation. In the present study, the glucuronidating activities of three common variant isoforms encoded by the human UDP-glucuronosyltransferase (UGT) 1A4 gene were examined against TAM, 24Thr/48Leu and UGT1A424Pro/48Val variants were generated by site-directed mutagenesis of the pcDNA3.1/V5-His-TOPO plasmid expressing wild-type UGT1A424Pro/48Leu. Levels of UGT1A4 expression in UGT-overexpressing cell lines were measured by western blot analysis.HPLC was used to detect glucuronide conjugates in microsomes from UGT1A4-overexpressing HK293 cells. The UGT1A4 wild-type cDNA was synthesized by RT-PCR using normal human liver total RNA. The UGT1A424Pro/48Leu-overexpressing HK293 cells exhibited significant levels of activity against TAM, trans-4-OH-TAM and cis-4-OH-TAM, forming exclusively the tamoxifen quaternary ammonium glucuronide (TAM-N+-glucuronide) and the 4-hydroxytamoxifen quaternary ammonium glucuronides (trans-4-OH-TAM-N+-glucuronide and cis-4-OH-TAM-N+-glucuronide) with apparent Km values of 2.0 μM, 2.2 μM, and 2.1 μM, respectively. Higher glucuronidation activities were found by kinetic analysis for microsomes from the variant UGT1A424Pro/48Val-overexpressing cell line as compared with microsomes from wild-type UGT1A424Pro/48Leu-overexpressing cells against TAM and against both the trans and cis isomers of 4-OH-TAM. A significantly (P < 0.02) lower Km value (~1.6-fold to 1.8-fold) was observed for both 4-OH-TAM isomers, while a near-significant (P = 0.053) decrease in Km was observed for TAM for the UGT1A424Pro/48Val variant as compared with wild-type UGT1A4. The Vmax/Km ratio for the UGT1A424Pro/48Val variant was significantly (P ≤ 0.005) higher than that observed for the wild-type UGT1A4 isoform for both the trans and cis isomers of 4-OH-TAM after normalization for UGT1A4 expression by western blotting. No significant effect on enzyme kinetics was observed for the UGT1A424Thr/48Leu variant against either isomer of 4-OH-TAM or with TAM.Microsomes from wild-type UGT1A4These data suggest that the UGT1A4 codon 48 Leu>Val polymorphism significantly alters glucuronidation rates against TAM and its active hydroxylated metabolites, and that this polymorphism may play an important role in individual pharmacological response to TAM therapy. Tamoxifen (TAM) (1- [4-(2-dimethylaminoethoxy)-phenyl]-1,2-diphenylbut-1(Z)-ene) is a nonsteroidal antiestrogen commonly used for the treatment and prevention of estrogen-dependent breast cancer . In addiThe mechanisms underlying variability in response to TAM and to TAM-related toxicities remain obscure. Since there is compelling evidence that TAM is converted to antiestrogenic metabolites more potent than TAM itself, one hypothesis is that altered patterns of metabolism of TAM and/or its primary metabolites might contribute to this interindividual variability.N-desmethyl-TAM (endoxifen). Since both trans-4-OH-TAM and endoxifen exhibit up to 100-fold the levels of antiestrogenic activity compared with TAM and other TAM metabolites  ion at m/z 563.70 (data not shown). These data suggest that the glucuronides derived from both isomers were in fact monoglucuronides.This pattern is similar to that observed for ompounds  – suggesn Figure . The mas24Pro/48Leu-overexpressing cells, significant glucuronidating activities were observed against both trans-4-OH-TAM and cis-4-OH-TAM . No TAM-4-O-glucuronide formation was observed in assays with either the trans or cis isomer of 4-OH-TAM with UGT1A4-overexpressing cell microsomes, and no glucuronidation activity was observed for untransfected HK293 cell homogenates for any substrate examined in this study (results not shown).In assays with microsomes prepared from wild-type UGT1A4M Figure . Single M Figure , with thM Figure  and weretrans-4-OH-TAM and cis-4-OH-TAM in our assay conditions. Using a concentration range of 4-OH-TAM (1–10 μM for trans-4-OH-TAM and 1–15 μM for cis-4-OH-TAM) and a specific incubation time (30 min), the apparent Km values and the Vmax/Km ratios for UGT1A4-induced glucuronidation of trans-4-OH-TAM and of cis-4-OH-TAM were 2.2 ± 0.4 μM and 29.3 ± 2.7 μl/min/μg, and 2.1 ± 0.4 μM and 2.0 ± 0.3 μl/min/μg, respectively (performed in three independent experiments for both isomers).As shown in a concentration curve for both isoforms of 4-OH-TAM  lower Km value (~1.6-fold to 1.8-fold) was observed for both 4-OH-TAM isomers, while a near-significant (P = 0.053) decrease in Km was observed for TAM – for the UGT1A424Pro/48Val variant compared with wild-type UGT1A4. The Vmax/Km ratio for the UGT1A424Pro/48Val variant was significantly (P ≤ 0.005) higher than that observed for the wild-type UGT1A4 isoform after normalization for UGT1A4 expression by western blotting for both the trans and cis isomers of 4-OH-TAM. No significant effect on enzyme kinetics was observed for the UGT1A424Thr/48Leu variant against either isomer of 4-OH-TAM or against TAM itself.As shown in Table cis-4-OH-TAM, while a second hepatic UGT (UGT1A4) was suggested to possess limited activity against both the cis and trans isomers of 4-OH-TAM [N, N-dimethylaminoalkyl side chain. The fact that UGT1A4 was shown to be active against TAM is not surprising given that this activity is consistent with UGT1A4's activity spectrum to produce N-glucuronidated metabolites with other compounds [The present study is the first to examine the potential role of UGT genetic polymorphisms on the metabolism of TAM and its active metabolites. Limited studies have previously been reported identifying the UGT enzymes involved in TAM metabolism. The hepatic UGT2B15 was shown to be the major UGT active against 4-OH-TAM . In more4-OH-TAM , UGT1A4 ompounds -34,39-41Km values against both trans-4-OH-TAM and cis-4-OH-TAM. The single glucuronide peaks observed for incubations of UGT1A4-overexpressing microsomes with either trans-4-OH-TAM or cis-4-OH-TAM were sensitive to alkali as well as to β-glucuronidase, suggesting that these glucuronides corresponded to 4-OH-TAM-N+-glucuronide. This pattern of glucuronide formation was similar to that observed in human liver microsomes, where both the TAM-4-O-glucuronide and the 4-OH-TAM-N+-glucuronide were detected in incubations with either the trans or cis isomers of 4-OH-TAM, with the peaks corresponding to 4-OH-TAM-N+-glucuronide sensitive to treatment with alkali and β-glucuronidase. Both trans-4-OH-TAM-N+-glucuronide and cis-4-OH-TAM-N+-glucuronide peaks were confirmed by LC–MS to be 4-OH-TAM monoglucuronides.To better characterize the glucuronide specificity of UGT1A4 against 4-OH-TAM in the present study, HPLC assays were conducted using UGT1A4-overexpressing cells. In this study, UGT1A4 exhibited significant activity with very low apparent N, N-dimethylaminoalkyl side chain of 4-OH-TAM. This activity is consistent with UGT1A4's activity spectrum to produce N-glucuronidated metabolites with other compounds [These data suggest that – identical to previous observations for TAM , and as ompounds -34,39-41Km values reported in the present study for wild-type UGT1A4-induced glucuronidation of trans-4-OH-TAM and cis-4-OH-TAM were 2.2 μM and 2.1 μM, respectively. These values are 35-fold lower than that observed previously for UGT1A4 against the trans and cis isoforms of 4-OH-TAM [The 4-OH-TAM . The diftrans-4-OH-TAM exhibits approximately 100 times the level of antiestrogenic activity compared with nonmetabolized tamoxifen and is probably a significant contributor to overall TAM-associated antiestrogenic activity [N position on endoxifen is demethylated, suggesting that endoxifen may perhaps be a less effective substrate for N-glucuronidation by enzymes such as UGT1A4. Studies aimed to identify the UGTs that are active against endoxifen are currently ongoing.While activity -21, its activity ,43,44. Sin vitro assays using individual human liver microsomes, exhibited no association with altered glucuronidation capacity in vitro for 4-OH-TAM or for TAM in the present study. This pattern is also different from the apparent decrease in activity observed for this variant against β-naphthylamine, benzidine, trans-androsterone and dihydrotestosterone, although statistical analysis for glucuronidation rates between variants against each of these compounds were not provided in these studies [in vitro functional analysis of the codon 24 polymorphism using a cell-line-overexpression system may not be optimal for studies of its effect on glucuronidation kinetics. A significant association was, however, observed for the UGT1A4 codon 48 polymorphism and 4-OH-TAM glucuronidation. The increased activity observed for the UGT1A424Pro/48Val variant as compared with the wild-type UGT1A424Pro/48Leu isoform against trans-4-OH-TAM, cis-4-OH-TAM and TAM suggests that the codon 48 polymorphism may significantly alter UGT1A4 enzymatic function against TAM and its active metabolites. As it is not yet clear whether UGT1A4 is active against endoxifen, the role of the UGT1A424Pro/48Val variant on endoxifen glucuronidation is presently not known.The two previously identified missense UGT1A4 polymorphisms examined in this study are located at codons 24 and 48, resulting in Pro>Thr and Leu>Val amino acid changes, respectively ,36. The  studies . This sutrans-4-OH-TAM formation in human liver microsomes from individual subjects [Previous studies have demonstrated that variant genotypes for enzymes involved in the TAM metabolism may significantly alter overall patient response to TAM. The low-activity SULT1A1*2 allele was linked to increased rates of mortality in breast cancer patients treated with TAM . In addisubjects .24Pro/48Val variant could potentially be similarly impacted with respect to individual patient response to TAM treatment and TAM-related toxicities. The prevalence of the UGT1A424Pro/48Val variant is relatively high  so this24Pro/48Val variant could potentially be similarly impacted with respect to individual patient response to TAM treatment and TAM-related toxicities.Results from this study indicate that UGT1A4 exhibits very high activity against TAM and against both isomers of 4-OH-TAM, and that the UGT1A4 codon 48 polymorphic variant is associated with altered TAM and 4-OH-TAM glucuronidation activities. These studies suggest that subjects with the UGT1A4Km = Michaelis-Menten equilibrium constant; LC–MS = liquid chromatography–mass spectrometry; Leu = leucine; 4-OH-TAM = 4-hydroxytamoxifen; 4-OH-TAM-N+-glucuronide = 4-hydroxytamoxifen quaternary ammonium glucuronide; TAM = tamoxifen; TAM-N+-glucuronide = tamoxifen quaternary ammonium glucuronide; PCR = polymerase chain reaction; Pro = proline; RT = reverse transcriptase; Thr = threonine; UDPGA = UDP-glucuronic acid; UGT = UDP-glucuronosyltransferase; Val = valine.DMEM = Dulbecco's modified Eagle's medium; HPLC = high-performance liquid chromatography; The authors declare that they have no competing interests.DXS designed the experimental plan, performed all kinetic analysis and contributed to tissue culture experiments for the studies described in this paper. GC helped to develop HPLC separation protocols and contributed to all data analysis and discussions. RWD guided all molecular aspects of this paper and contributed to mammalian tissue culture experiments and cell microsome preparations. KD performed the western blot analysis and helped to establish UGT1A4 cell lines. J-LF created the UGT1A4 cell lines. PL was instrumental in the planning, supervision and data analysis, and cowrote the manuscript with DXS. All authors participated in the revision of the manuscript and gave final approval of the version to be published.


Studies in rats and monkeys suggest that TCDD may affect ovarian function. We examined the relationship of TCDD and age at menopause in a population of women residing near Seveso, Italy, in 1976, at the time of a chemical plant explosion. We included 616 of the women who participated 20 years later in the Seveso Women’s Health Study. All women were premenopausal at the time of the explosion, had TCDD levels measured in serum collected soon after the explosion, and were ≥ 35 years of age at interview. Using proportional hazards modeling, we found a 6% nonsignificant increase in risk of early menopause with a 10-fold increase in serum TCDD. When TCDD levels were categorized, compared with women in the lowest quintile (< 20.4 ppt), women in quintile 2 (20.4–34.2 ppt) had a hazard ratio (HR) of 1.1 (p = 0.77), quintile 3 (34.3–54.1 ppt) had an HR of 1.4 (p = 0.14), quintile 4 (54.2–118 ppt) had an HR of 1.6 (p = 0.10), and quintile 5 (> 118 ppt) had an HR of 1.1 (p = 0.82) for risk of earlier menopause. The trend toward earlier menopause across the first four quintiles is statistically significant (p = 0.04). These results suggest a nonmonotonic dose-related association with increasing risk of earlier menopause up to about 100 ppt TCDD, but not above.2,3,7,8-Tetrachlorobenzo- The ageThere is limited epidemiologic evidence that endocrine-disrupting chemicals affect the natural timing of menopause. Data from a case–control study of breast cancer in North Carolina (USA) show that women with serum dichlorodiphenyldichloroethene (DDE) levels in the upper 10th percentile had an earlier onset of natural menopause than did women with levels below the median [hazard ratio (HR) = 1.4], but poly-chlorinated biphenyl (PCB) levels were not related to age at menopause . Women fIn this study, we examine the relationship of TCDD and age of onset of natural menopause in a population of women residing near Seveso, Italy, in 1976, at the time of a chemical plant explosion. These women were exposed to the highest levels of TCDD known in residential populations .The Seveso Women’s Health Study (SWHS) is the first comprehensive epidemiologic study of the reproductive health of a female population exposed to TCDD. Women eligible for SWHS were 1 month to 40 years of age in 1976, had resided in one of the most highly contaminated areas (zone A or B), and had adequate stored sera collected soon after the explosion . RecruitDetails of the study are presented elsewhere . BrieflyDetails of serum sample selection are presented elsewhere . The TCDn = 71), a serum TCDD level equal to one-half the detection limit was assigned (n = 40) or the Filser model (n = 1). For the 7 women with post-1977 measures whose TCDD levels were < 10 ppt, measured values were used. The study median serum sample weight was 0.65 g, and the median limit of detection was 18.8 ppt, lipid-adjusted.We measured TCDD in sera collected between 1976 and 1977 for 564 women, between 1978 and 1982 for 28 women, and between 1996 and 1997 for 24 women whose earlier samples had insufficient volume. For nondetectable values  and a categorical variable based on quintiles of serum levels near the time of the explosion . To evaluate the relationship between serum TCDD and age at natural menopause, we performed Cox proportional hazards analyses with the robust method of calculating the variance-covariance matrix (10 TCDD). Scaled Schoenfeld residuals were generated for the final multivariate model and used to test the proportional hazard assumption   . Analysep < 0.10) or if they changed the regression coefficient for TCDD exposure by > 10%. We considered the following as potential covariates: current body mass index (BMI), premenopausal smoking history, education, marital status, current physical activity, age at menarche, parity, OC use, and history of heart disease. We also created a variable for premenopausal history of medical conditions that could potentially be related to age at menopause, including type 1 diabetes (n = 1), rheumatoid arthritis (n = 4), radiation for cancer (n = 4), epilepsy (n = 2), hyperthyroid (n = 10), and untreated hypothyroid (n = 2) (n = 23). The results were similar; therefore, we present only the results including the women with other medical conditions. One covariate (education) was found to violate the proportional hazards assumption (p = 0.06); therefore, estimates stratified by education were obtained.Covariates were considered for the multivariate Cox proportional hazards analysis if they had been reported in previous literature to be related to age at menopause. Covariates were kept in the multivariate model if they were statistically significant ( (n = 2) . In addin = 7), OC use (n = 19), or other hormone use (n = 1).For women who met the definition of natural menopause, age (in years) at last menstrual period was assigned as their age at menopause. Surgically menopausal women were censored at the age at which they had surgery. Premenopausal and perimenopausal women were censored at their age at interview. Women using OCs or other hormones and those with a history of chemotherapy were censored at the age at which they began use or treatment. Each woman was entered into the analysis at 35 years of age, before which natural menopause was unlikely to occur. Twenty-seven women (4%) were censored before 35 years of age because of surgical menopause . To assess the possibility that TCDD exposure is associated with conditions that would lead to surgical menopause or that it is associated with a longer menopausal transition, we also reran the final models with a redefined outcome including surgical menopause, perimenopause, impending menopause, and natural menopause.The final models were also rerun considering alternative TCDD exposure scenarios including cumulative exposure dose (area under the curve measured in parts per trillion-years) and estimated exposure dose at time of failure or censorship (parts per trillion). These doses were estimated for each year of exposure (time dependent) following the first-order kinetic model assuming a half-life of 9 years for TCDD . Ages atn = 39), other hormone use (n = 17), chemotherapy (n = 2)]. The mean (± SD) age at menopause for those in natural menopause (n = 169) was 49.2 ± 3.7 years , which was older than those in surgical menopause .Characteristics of the 616 women in the analysis are presented in 2), two-thirds had never smoked, about half had ever used OCs, and nearly all had been married and were parous .For each menopausal category, the median lipid-adjusted serum TCDD level and inter-quartile range (IQR) are presented in 10 TCDD) was 1.02  . However, when a square term in log10 TCDD was added to the continuous variable model, it was statistically significant, suggesting a curvature in the dose–response curve (results not shown).In Cox proportional hazards modeling, the unadjusted HR associated with a 10-fold increase in TCDD (log = 0.89] . That isp = 0.77), women with TCDD levels in quintile 3 (34.3–54.1 ppt) had a 40% increase in hazard of natural menopause , and women with TCDD levels in quintile 4 (54.2–118 ppt) had a 60% increase in hazard of natural menopause . Women in the highest quintile , however, had only a 10% increase in hazard of earlier natural menopause . Although no increasing trend of earlier natural menopause was observed across the five quintiles (p = 0.44), a significant trend to earlier natural menopause across the first four quintiles was found (p = 0.04). Furthermore, when we excluded the 24 women who had back-extrapolated TCDD levels from 1996, the association is strengthened. Compared with women in the lowest quintile (< 20.4 ppt), women in quintile 2 (20.4–34.2 ppt) had an HR of 1.2 (p = 0.5); quintile 3 (34.3–54.1 ppt) had an HR of 1.6 (p = 0.08); quintile 4 (54.2–118 ppt) had an HR of 1.7 (p = 0.05); and quintile 5 (> 118 ppt) had an HR of 1.2 (p = 0.5) for risk of earlier menopause. The trend toward earlier menopause across the first four quintiles is statistically significant (p = 0.02).When serum TCDD levels were categorized into quintiles, risk of earlier menopause trended upward in the first four quintiles but not in the highest quintile in the unadjusted  and adjuThe results did not change when women in the impending menopause category were classified as menopausal in the analysis (data not shown). Similar results were found when women in surgical menopause and peri-menopause were also combined with natural and impending menopause as one outcome (data not shown).When TCDD exposure was extrapolated to the time of failure or censorship, the results were no different. Cumulative TCDD exposure (parts per trillion-years), however, was not related to age at onset of menopause .In the final models described above, nulliparity was associated with earlier natural menopause , and history of OCs for at least 5 years was associated with later natural menopause . We observed a nonsignificant earlier natural menopause for women who were current smokers . BMI, however, was not associated with age at natural menopause.The results of this study of women residing in Seveso, Italy, in 1976, at the time of a chemical plant explosion that resulted in very high levels of TCDD exposure, suggest a nonmonotonic dose-related association of TCDD levels in sera collected near the time of exposure with earlier onset of natural menopause; the trend for increasing risk is observed with TCDD levels up to about 100 ppt, but not above. Our finding is supported by the earlier mean age of menopause observed in our study (49.2 ± 3.7 years) relative to that (mean = 50.9 years) reported in an Italian clinic-based study of > 4,300 menopausal women during the same time period (1995–1997) . It is aTo our knowledge, no previous epidemiologic studies have examined the relation of TCDD exposure and age at menopause. However, amenorrhea was observed in a case report of an Austrian woman with extremely high levels of serum TCDD  . Our finThe potential impact of TCDD exposure on age at menopause is biologically plausible, as animal studies indicate . In a raBax gene expression in oocytes, which is necessary for the oocyte loss related to premature ovarian failure .If TCDD exposure induces earlier menopause, it is unlikely to occur via oocyte apoptosis. Recent data in mice suggest that TCDD does not induce  failure . Althougin utero exposure on adult weight of offspring). The present results as well as those in animals suggest a reevaluation of the presumed monotonic dose–response relationships with exposure to endocrine-disrupting chemicals that are typically tested in statistical modeling of epidemiologic data.We observed an inverted U-shaped relationship between TCDD serum levels and earlier menopause. An inverted U-shaped dose response has been hypothesized by This study has some limitations. One limitation is the retrospective recall of age of natural menopause. However, previous studies have reported moderately high reliability and accuracy based on interview . FurtherAlthough smoking has been associated with earlier menopause in a number of studies , we did p-dioxins and PCBs during this time period . TherefAn advantage of this study is that we were able to measure TCDD levels in individual serum samples collected near the time of exposure, and there was a wide range of exposure. For the few women whose samples were of inadequate volume, we used serum collected between 1996 and 1997. If we exclude these women, the relation is strengthened. We have examined multiple exposure scenarios including exposure soon after the explosion as well as exposure extrapolated to each age at risk.in utero and lactational TCDD exposure may have significant effects on ovarian follicles (In summary, we observed a nonmonotonic dose–response relationship between serum TCDD levels and age of onset of natural menopause. The women in this study experienced substantial TCDD exposure during the postpubertal–adult developmental period. Animal evidence suggests that ollicles ; therefo


Current and recent users of hormone replacement therapy (HRT) have an increased risk of being recalled to assessment at mammography without breast cancer being diagnosed , but there is limited information on the effects of different patterns of HRT use on this. The aim of this study is to investigate in detail the relationship between patterns of use of HRT and false positive recall.A total of 87,967 postmenopausal women aged 50 to 64 years attending routine breast cancer screening at 10 UK National Health Service Breast Screening Units from 1996 to 1998 joined the Million Women Study by completing a questionnaire before screening and were followed for their screening outcome.p (heterogeneity) < 0.0001); 1.65 (1.43–1.91), 1.49 (1.22–1.81) and 2.11 (1.45–3.07) for current HRT used orally, transdermally or via an implant, respectively (p (heterogeneity) = 0.2); and 1.84 (1.67–2.04) and 1.75 (1.49–2.06) for sequential and continuous oestrogen-progestagen HRT, respectively (p (heterogeneity) = 0.6). The relative risk of false positive recall among current users appeared to increase with increasing time since menopause, but did not vary significantly according to any other factors examined, including duration of use, hormonal constituents, dose, whether single- or two-view screening was used, or the woman's personal characteristics.Overall, 399 (0.5%) participants were diagnosed with breast cancer and 2,629 (3.0%) had false positive recall. Compared to never users of HRT, the adjusted relative risk (95% CI) of false positive recall was: 1.62 (1.43–1.83), 1.80 (1.62–2.01) and 0.76 (0.52–1.10) in current users of oestrogen-only HRT, oestrogen-progestagen HRT and tibolone, respectively (Current use of oestrogen-only and oestrogen-progestagen HRT, but not tibolone, increases the risk of false positive recall at screening. Around one-third of women attending the UK National Health Service Breast Screening Programme (NHSBSP) from 1996 to 2000 were using hormone replacement therapy (HRT) at the time and about 16% were former users . Among pThis study investigates in detail the effects of different patterns of use of HRT on the risk of false positive recall, as well as the consistency of HRT effects. It also presents a meta-analysis of results from studies examining the effect of current and past use of HRT on false positive recall.At the time of the study, all 50 to 64 year old women in the UK who were registered with a general practitioner were invited to attend the NHSBSP approximately once every three years for routine mammographic screening. The NHSBSP is aimed at screening asymptomatic women, whereas women with symptoms are encouraged to consult their general practitioner.Women recruited into the Million Women Study (described in detail elsewhere ) who attThe variables for these analyses, including use of HRT, were defined according to what was reported on the recruitment questionnaire, that is, at baseline. Women were asked which specific proprietary preparation of HRT they had used most recently, and for these analyses the preparations are grouped according to the hormonal constituents of each preparation listed in the British National Formulary . Women win situ), according to the pathology information recorded by the screening centre. 'False positive recall' was defined, according to screening centre records, as being recalled to assessment following initial screening mammography, without a subsequent diagnosis of screen-detected breast cancer during that screening episode. Women with technically inadequate films that needed to be repeated were classified according to the results of their repeat mammogram.An outcome of screen-detected breast cancer was defined as histologically confirmed screen-detected breast cancer  at recruitment.  who were aged 49 or 65 years when screened, but were close to their 50th or 65th birthday, since women are invited to screening according to their year of birth, rather than their exact age.)2, ≥25 kg/m2) and history of previous breast surgery for a condition other than breast cancer . All adjustment factors include an 'unknown' category. Adjustment for other potential confounding factors, such as deprivation index, educational attainment, family history of breast cancer, age at first birth and parity, had no material effect on the findings and are, therefore, not included in the analyses. All tests for trend by duration and time since last use of HRT were calculated using the appropriate median duration or time since last use in each category. Where results are presented graphically, relative risks are shown as black squares with areas inversely proportional to the variance of the log relative risk, indicating the amount of statistical information available for that particular estimate. The corresponding confidence intervals are shown as horizontal lines.Relative risks (RR) are estimated as odds ratios and 95% confidence intervals (CI) calculated by conditional logistic regression, stratifying by screening centre, age at screening , time since menopause , reported previous breast screening , body mass index  were recalled to assessment and 3,028 attended this assessment; 15 women (0.02%) were lost to follow-up due to having defaulted attendance at assessment and/or having chosen to be investigated outside the National Health Service. Overall, 399 (0.5%) women were diagnosed with screen-detected breast cancer, leaving 2,629 (3.0%) women with false positive recall.21(heterogeneity) = 13.7, p < 0.001). There was a significant trend in recall according to time since stopping use of HRT (χ21(trend) = 14.0, p < 0.001), suggesting the wearing off of the effect of HRT over time in past users [Table Among current users of HRT, the relative risk was significantly elevated in all duration groups, including those women who had been using HRT for less than one year, and there was no significant trend in the relative risk according to duration of use Table . In cont22(heterogeneity) = 22.2, p < 0.0001)  = 21.0, p < 0.0001). There was no significant difference in the relative risk of false positive recall between women using oestrogen-only and oestrogen-progestagen HRT (χ21(heterogeneity) = 2.3, p = 0.1) [The relative risk of false positive recall differed significantly according to whether a woman was currently using oestrogen-only, oestrogen-progestagen or other types of HRT: RR (95% CI) 1.62 1.43–1.83), 1.80 (1.62–2.01) and 0.95 (0.73–1.23), respectively  = 0.5, p = 0.5) or type of progestogen  = 4.3, p = 0.1) in use, according to the dose of oestrogens currently being used  = 3.0, p = 0.08; for oestradiol ≤1 mg versus >1 mg, χ21(heterogeneity) = 0.5, p = 0.5; for oestradiol ≤50 μg versus >50 μg, χ21(heterogeneity) = 3.4, p = 0.07) or according to whether the oestrogen was taken orally, via a patch or an implant (χ22(heterogeneity) = 2.8, p = 0.2)  = 0.3, p = 0.6).Among women currently using oestrogen-only or oestrogen-progestagen HRT, there was no significant difference observed according to the type of oestrogen  = 38.2, p = 0.01), which was chiefly the result of variation according to the closely related factors of age within screening round and time since menopause. Although this heterogeneity appeared to be greater for time since menopause than for age, there were insufficient data to investigate their dual effects. There was no significant heterogeneity in the effect of HRT according to whether or not women had had a previous screen, after accounting for age. No significant heterogeneity was present for current use of HRT for any other factors examined, nor was significant heterogeneity found for the effect of past use of HRT  = 18.1, p = 0.6). These included comparisons between the different screening centres  = 1.1, p = 0.3; χ21(heterogeneity for past use) = 0.2, p = 0.7).To examine how consistent the effects of current and past use of HRT are across other possibly relevant factors, the adjusted relative risks of false positive recall and 95% confidence intervals in current and past users of HRT compared to never users are presented Fig. . SignifiFigure Women who have ever used HRT are at an increased risk of false positive recall compared to never users. The relative risk is greatest in current users and, among past users, declines significantly with increasing time since ceasing use . The relThe study described here is large and population-based, meaning that major effects of HRT on false positive recall can be quantified and generalised to the screened population in the UK. Detailed prospective data were gathered on the use of HRT immediately prior to screening, including information on current and past use, along with the type  and dose of the preparation currently being used. These data have shown excellent agreement with general practice prescription records . It shouCurrent use of HRT was associated with increased false positive recall soon after use commenced. This is consistent with three previous comparable publications, which all show significant increases in the risk of false positive recall with current use of HRT Fig. . There iThe effect of current use of HRT was not significantly related to duration of use and, among women using oestrogen alone or combined oestrogen/progestogen, did not differ significantly according to the type of oestrogen and/or progestogen in current use, according to the dose of oestrogen currently in use or according to the mode of delivery of the oestrogen. Two previous smaller studies with data on false positive recall and use of oestrogen-only and oestrogen-progestagen HRT also report no substantive differences in the risk between users of these types of HRT ,9; one oOverall, former users of HRT had a significantly lower risk of false positive recall than current users but a significantly higher risk than never-users. Although results from previous studies were apparently inconsistent, the totality of the evidence to date shows an increased risk of false positive recall in past users of HRT without significant variation between studies Fig. . The relThere is evidence to suggest that women who are currently using oestrogen-only and oestrogen-progestogen HRT have a higher proportion of their mammograms occupied by relatively radiodense tissue, compared to women who have never used HRT -15 and tBeing recalled to assessment is a stressful experience for most women ,30, and Current use of oestrogen-only and oestrogen-progestagen HRT, but not tibolone, increases the risk of false positive recall at screening. The elevated risk of false positive recall in users of oestrogen-only and oestrogen-progestagen HRT was observed consistently among the different HRT preparations in use.CI = confidence interval; HRT = hormone replacement therapy; NHSBSP = National Health Service Breast Screening Programme; RR = relative risk.The authors declare that they have no competing interests.Emily Banks, Gillian Reeves, Valerie Beral and Julietta Patnick had the original idea for the study, with important input on practical aspects of study design from Diana Bull, Barbara Crossley, Elizabeth Hilton and Moya Simmonds. Emily Banks and Diana Bull analysed the data and Emily Banks, Gillian Reeves, Valerie Beral and Julietta Patnick interpreted the data. Stephen Bailey, Nigel Barrett, Peter Briers, Ruth English, Alan Jackson, Elizabeth Kutt, Janet Lavelle, Linda Rockall, Matthew Wallis and Mary Wilson contributed to local study design and conduct. All the authors participated in drafting the paper and gave final approval of the version to be published. Emily Banks, Gillian Reeves, Valerie Beral and Diana Bull are guarantors for the study.


The mixture analysis of sex-adjusted values of 1 of these traits, the event-related potential obtained at the parietal midline channel (ttth4), found the appearance of a 3-component normal mixture in cases and controls. The mixtures differed in that the cases had significantly lower mean values than controls and significantly different mixing proportions in 2 of the 3 components. Implications of this study are: 1) Sex needs to be taken into account when studying risk factors for alcohol dependency to prevent finding a spurious association between alcohol dependency and the risk factor. 2) Mixture analysis indicates that for the event-related potential "ttth4", the difference observed reflects strong evidence of heterogeneity of response in both the cases and controls.We consider 12 event-related potentials and one electroencephalogram measure as disease-related traits to compare alcohol-dependent individuals (cases) to unaffected individuals (controls). We use two approaches: 1) two-way analysis of variance , and 2) likelihood ratio tests comparing sex adjusted values of cases to controls assuming that within each group the trait has a 2 (or 3) component normal mixture distribution. In the second approach, we test the null hypothesis that the parameters of the mixtures are equal for the cases and controls. Based on the two-way analysis of variance, we find 1) males have significantly ( Disease-related traits (DRTs) may provide more powerful phenotypes than the disease itself for identifying alcohol dependency genes. For example, an alcohol DRT phenotype might be due to a single major gene with high penetrance, while alcohol dependency may result from the action of several genes and environmental factors. In characterizing a DRT, we first compare affected to unaffected individuals. If the DRT is due to 1 of many disease predisposing genes, then the responses in both affected and unaffected individuals may be a mixture with 2 or 3 components depending on the effect of genotype on the DRT in affected individuals and unaffected individuals. Lo et al. [We considered Collaborative Study on the Genetics of Alcoholism (COGA) family data provided by Genetic Analysis Workshop 14 (GAW14) Problem 1 . One affThe 12 event-related potentials (ERPs), phenotypes ttth1, ttth2, ttth3, ttth4, ttdt1, ttdt2, ttdt3, ttdt4, ntth1, ntth2, ntth3, and ntth4, and one electroencephalogram (EEG) phenotype, ecb21, were considered, as well as the sex of the individual.The 2-way analysis of variance used sex, disease status (affected vs. unaffected), and the sex-disease status interaction as factors.Adj = Y - df, where  for females and df = 0 for males.A mixture model analysis incorporated the findings on sex obtained in the 2-way analysis of variance, and was done on sex-adjusted values for those traits in which we found a difference between males and females. The adjustment was YX = 1 when an individual is a control and X = 2 when an individual is a case, then the density of the trait, Y, isThis analysis assumed that conditional on whether an individual is a case or control, the distribution of the trait has a 2-component normal mixture distribution. If we let fx(y) = πx 1φ + πx 2φ for x = 1, 2,     (1)y; μ, σ) denotes the normal density with mean, μ, and standard deviation, σ and πx 1+ πx 2= 1. Without loss of generality, μx 1< μx 2and 0 < πix < 1 for x = 1, 2.where φHcan be tested against the alternative of equal component means and unequal mixing proportions01: μi2 = μi1 and πi2 ≠ πi1 for i = 1, 2     (3)Husing a likelihood ratio test (LRT) statistic. Under the null hypothesis, the LRT statistic has an asymptotic chi-square distribution with 1 df. We can also consider an alternative of unequal means and equal mixing proportion, i.e.,10: μi2 ≠ μi1 and πi2 = πi1 for i = 1, 2.     (4)HFinally we also consider an alternative of unequal means and unequal mixing proportions11: μi2 ≠ μi1 and πi2 ≠ πi1 for i = 1, 2.     (5)H00 we might want to consider the alternative H11 given in (5) versus H01 using a 2 df chi-square test or H10 using a 1 df chi square test.If we reject HFollowing the same logic, we considered a set of 3-component normal mixture models for cases and controls. Similar to model (1), we considered 3 component mixtures with equal within component variances. Thus the general equation for the mixture density isfx(y) = πx 1φ + πx 2φ + πx 3φ for x = 1, 2,     (6)x 1+ πx 2+ πx 3= 1 and 0 < πix < 1 for x = 1, 2, i = 1, 2, 3. We again set μx 1< μx 2< μx3, and refer to the component having mean μix as the ith component. As in comparing cases to controls assuming a 2-component normal mixtures, we estimate the parameters and test hypotheses under various 3-component normal mixture models. These include 1) equal parameter values for cases and controls (6 parameters); 2) unequal mixing proportions, but equal component means (8 parameters); 3) unequal component means but equal mixing proportions (9 parameters); 4) equal first component means and equal first component mixing proportions (9 parameters); 5) unequal mixing proportions and unequal within component means (11 parameters).where πix, μix and σ for i = 1, 2 (or 3) and x = 1, 2.The expectation-maximization algorithm (EM) , a generA method of Maller and Zhou  allows up > 0.17). For 7 traits, sex was a significant factor; disease was a significant factor for only 3 traits. This was unexpected, because based on the data description, we expected to observe differences between cases and controls on all measures. In Table Table The means observed for males were slightly lower than those observed for females wherever there was a significant sex difference. Thus the adjusted values for females were slightly smaller than the original values. The values of the adjustment used in the females for the electrophysiological measures, when there was a significant sex difference, range from 0.32 to 2.00, with the sex adjustment value for the ERP obtained at the parietal midline channel, ttth4, equal to 0.45.00 for the alternative H01  for every DRT considered. We rejected H00 at the 0.05 level for alternative H10  in the case of trait ttth2 . In the case of ttth4, the DRT obtained at the parietal midline channel, we reject H00 , H01 , and H10  for H11, indicating that we may have both unequal mixing proportions and unequal means for cases and controls. Thus, while the analysis of variance shows that the controls have a higher mean value of ttth4, the mixture analysis indicates more complex distribution differences. Both component means are higher in the controls than the cases (3.83 vs. 3.33 and 6.00 vs. 4.64), whereas the estimated proportion of controls in component with the higher mean is lower than that for the cases (0.04 vs. 0.31).We failed to reject H2 = 12.6, df = 2, p-value = 0.002) and of equal component means for cases and controls . Upon exploring further, we could not reject a hypothesis that cases and controls had equal means and proportions in the first component, i.e., the component with the lowest mean .Applying similar methods we used LRTs to find the most parsimonious 3-component normal mixture distribution for ttth4. Upon doing this we rejected hypotheses of equal mixing proportions for cases and controls , to a 2-component normal mixture model , and to a 3-component normal mixture model . The AIC values of the single normal density, 2-component mixture model and 3-component mixture model are 381.2, 380.8, and 371.5, respectively. The BIC values for the above three models are 390.3, 402.1, and 398.8, respectively. AIC leads to a 3-component mixture model, while a single density model is indicated by BIC. When there are inconsistencies in model selection based on AIC and BIC, Leroux [f1(y) = 0.19 φ + 0.77 φ + 0.04 φ for controlsandf2(y) = 0.19 φ + 0.52 φ + 0.29 φ for cases.Figure In the case of ttth4, the first component mean and corresponding mixing proportion are the same for cases and controls, and there is a general shift, in the direction that the mean ttth4 is lower for alcohol-dependent individuals than their unaffected relatives in the other 2 component means. From the final model, we can see that the explanation for a lower mean in the cases is the lower mean and a lower estimated proportion in the second component compared to the control group., with a sample size n = 50 in each group, power is equal to or larger than 0.50. Thus we have reasonable power to detect differences between cases and controls. Whenever our alcohol-dependent sample has a larger percentage of males than our control sample, any differences observed between cases and controls may reflect these sex differences rather than differences in the disease groups. Regardless of the sample makeup, taking sex into account should always be done when studying factors related to alcoholism. Another reason we do not see large differences between our controls and the cases may be that these controls all have a family history of alcoholism.An interesting result is that, with sex controlled, there are few significant differences between cases and controls, namely ttth4, ttdt3, and ttdt4. For moderate estimated effect size few significant findings are observed on comparing cases to controls.In this study we report significant findings observed on investigating 13 correlated measures. As in any study in which a large number of tests have been done, we would expect some significant findings due to chance. Thus the results here must be considered as preliminary. On the other hand, given that these measures were included in the COGA dataset  as beingTwo-way analysis of variance (sex and disease) indicates that controlling for sex there is a significant difference between alcohol-dependent cases and controls for only 3 ERPs, namely ttth4, ttdt3, and ttdt4. Comparison of both the 2-component and 3-component normal mixture parameters for ttth4, the ERP obtained at the parietal midline channel, indicate these differences may reflect the same mixing proportion and mean in the component having the lowest mean, but unequal mixing proportions and unequal component means in the other 2 components.AIC: Akaike information criterionBIC: Bayesian information criterionCOGA: Collaborative Study on the Genetics of AlcoholismDRT: Disease-related traitEEG: ElectroencephalogramEM: Expectation-maximization algorithmERPs: Event-related potentialsGAW14: Genetic Analysis Workshop 14LRT: Likelihood ratio testMLE: Maximum likelihood estimationNRM, SJF, KQY, and GAC conceived of the study, participated in its design and coordination, and helped to draft the manuscript. NRM presented this work. TD carried out all of the analyses including the genetic analyses, data reduction, and statistical analyses.


A nested case-control study was undertaken in the Maribo County cohort of 27,811 women with negative Pap smears. Sixty women who later developed invasive cervical cancer constituted the cases, and five matched controls were selected from the cohort for each case. A total of 633 previous, negative smears for the cases and controls were reviewed independently by two pathologists. The review showed misclassification to be frequent in these smears collected in the period 1966-82. Thirty-five smears were considered positive at the review. The misclassification was differential in respect to the women's later disease status. The odds ratio for patients compared with controls for having at least one positive smear was 22.12 (95% CI 7.54-64.94). We were unable to identify specific characteristics of misclassified smears coming from later cases. Koilocytosis/dyskeratosis, herpes virus changed cells and hyperkeratosis were equally rare in smears from patients and controls. The Maribo County data indicate that the fraction of preventable cases of invasive cervical cancer in women aged 30-64 within the first 5 years after a negative smear could be increased from 62-72% to 83-86%, if misclassification of true positive smears could be eliminated. As a rough estimate, this would be at the cost of a 2% increase in the work load. It should be remembered that there is a large element of extrapolation in applying these results based on relatively poor quality specimens from 1966-82 as compared to a modern screening service.


The origin of microbial ORFans, ORFs having no detectable homology to other ORFs in the databases, is one of the unexplained puzzles of the post-genomic era. Several hypothesis on the origin of ORFans have been suggested in the last few years, most of which based on selected, relatively small, subsets of ORFans. One of the hypotheses for the origin of ORFans is that they have been acquired thru lateral transfer from viruses. Here we carry out a comprehensive, genome-wide study on the origins of ORFans to quantify the strength of current evidence supporting this hypothesis.We performed similarity searches by querying all current ORFans against the public virus protein database. Surprisingly, we found that only 2.8% of all microbial ORFans have detectable homologs in viruses, while the percentage of non-ORFans with detectable homologs in viruses is 7.9%, a significantly higher figure. This suggests that the current evidence for the origin of ORFans from lateral transfer from viruses is at best weak. However, an analysis of individual genomes revealed a number of organisms with much higher percentages, many of them belonging to the Firmicutes and Gamma-proteobacteria. We provide evidence suggesting that the current virus database may be biased towards those viruses attacking Firmicutes and Gamma-proteobacteria.We conclude that as more viral genomes are sequenced, more microbial ORFans will find homologs in viruses, but this trend may vary much for individual genomes. Thus, lateral transfer from viruses alone is unlikely to explain the origin of the majority of ORFans in the majority of prokaryotes and consequently, other, not necessarily exclusive, mechanisms are likely to better explain the origin of the increasing number of ORFans. ORFans are defined as ORFs (Open Reading Frames) having no sequence homologs in other genomes . ORFans Since we coined the term "ORFan" in 1999 ,7-10. A Escherichia coli MG1655 genome against sequenced bacteriophage genomes and found that only ~9% of the ORFans have homologs in phages ).For each ORF, g, we calculated the value Hg as the number of genomes having at least one homolog of g, and Ug, a measure of the "uniqueness" of g as:i corresponds to a genome having a homolog of g.where each BORFs with H = 1 and one BLASTP hit were classified as singleton ORFans; ORFs with H = 1 and more than 1 BLASTP hits were classified as paralogous ORFans; ORFs with H > 1 and U <= 0.1 were classified as orthologous ORFans.We downloaded the Refseq release 13 (Sept. 2005) virus database from NCBI , containAll two sample tests used are Wilcoxon nonparametric tests and are conducted by using R language .YY conducted the computation, analyzed data, and drafted the manuscript. DF designed the research, supervised this project and finalized the manuscript. All authors read and approved the final manuscript.the numbers of ORFans and nonORFans, the numbers of ORFs having homologs in viruses in 277 prokaryotic genomes, using different BLASTP cutoff to define ORFans.Click here for file