cervical cancer represents the third most common cancer in women worldwide , with 86% of all deaths occurring in developing countries , where there is lack of access to prevention , early diagnosis and treatment ( jemal et al . , 2011 ) .
in brazil , cervical cancer ranks as the second most frequent cancer among women between 15 and 44 years of age ( who / ico , 2010 ) .
most cervical cancers can be detected early through a routine exfoliative cytology test ( papanicolaou test ) , a screening which has been used for more than 50 years in an effort to decrease the number of deaths from cervical cancer ( stack , 1997 ) .
dna testing has also been reported as an important tool for detecting hpv ( mayrand et al . , 2007 ) and is being introduced in some countries as an adjunct to the papanicolaou test or as the primary screening test ( castle et al . , 2012 ) . in developing countries ,
this introduction has been controversial , mainly because cytology - based screening programs suffer from lack of basic healthcare infrastructure ( bradford and goodman , 2013 ) . in a meta - analysis study ,
hpv types 16 , 18 , 31 , 33 and 45 were shown to be the most prevalent types associated with cervical carcinomas in latin america ( ciapponi et al . , 2011 ) .
chromosomes or chromosome segments that fail to be incorporated into nuclei during cell division configure micronuclei ( mn ) .
the mn test , readily performed via microscopy , has been successfully used to screen population groups at risk for cancer , representing a sensitive indicator of genetic damage ( samanta and dey , 2012 ) .
the frequency of mn appears to increase in carcinogen - exposed tissue before there are any clinical symptoms ( gayathri et al . , 2012 ) .
considering the fact that oncogenic hpv viral proteins may induce cytogenetic changes in exfoliated cells of the uterine cervix , a positive correlation between micro - nucleated cells and dysplasia in papanicolaou smears has been reported ( gandhi and kaur , 2003 ; guzmn et al .
, 2003 ; corts - gutirrez et al . , 2010 ; aires et al . , 2011 ) .
taking this into account , the aim of the present study was to investigate the association between hpv - dna and mn in women from southern brazil with normal cervical cytology in order to contribute additional information to cervical cytology analysis . additionally , some of the most prevalent high - risk hpv types were investigated .
a total of 158 normal cervical smears were analysed cytologically for hpv - dna and mn comparisons .
the smears were collected through the use of a cytobrush in a gynecology clinic at a healthcare medical centre ( posto de sade modelo ) in porto alegre , rio grande do sul , southern brazil from march to december 2009 . all women who agreed to participate signed an informed consent form .
this study was approved by the ethics committee of universidade luterana do brasil ( number 2008 - 601h ) .
two slides were made , one for the conventional papanicolaou exam and another for mn analysis .
after the slides were prepared , the cytobrush was placed in a tube containing 2 ml te 1x and stored at 20c for later dna extraction . for the papanicolaou test
, the analyses were based on the bethesda system ( solomon et al . , 2002 ) .
the slides for detecting and counting mn were stained with giemsa , and evaluated four times , twice by each of two observers independently .
a total of 1000 cells were counted under a light microscope at a 1000 magnification following the criteria described by tolbert et al .
a non - organic method developed in our laboratory was used to extract the dna from the clinical specimens ( michelon et al . , 2011 ) .
the resulting sediment was resuspended in 50 l of 1x te and incubated at 99 c for 10 min .
the dna was then purified using 5 l of a glass matrix ( concert extraction systems ; life technologies , rockville , md , usa ) .
the amplification of a 150 bp fragment of the l1 region of the hpv genome was performed using the gp5+/biogp6 + consensus primers ( de roda husman et al . , 1995 ) .
the pcr was carried out in a final volume of 50 l containing 50 mm kcl , 10 mm tris - hcl ph 8.5 , 2.5 mm mgcl2 , 0.1 mm of each deoxynucleoside triphosphate , 50 ng of each primer , 2.5 u of taq dna polymerase ( invitrogen ) and 10 l of extracted dna .
a negative control ( no dna ) was included in each pcr run to ensure that there was no cross contamination .
samples were amplified under the following conditions : initial denaturation step at 95 c for 5 min , 40 cycles at 95 c for 1 min , 52 c for 1 min and 72 c for 1 min , and a final elongation step at 72 c for 10 min .
dna of caski ( hpv-16 positive ) cells were used as a positive control in all pcr reactions .
the final pcr product ( 10 l ) was visualized in electrophoresis agarose gel ( 1.5% ) under uv light .
hpv dna genotyping of high - risk types 16 , 18 , 31 , 33 , 39 , 45 and 59 was carried out using a microplate colorimetric hybridization assay ( mcha ) developed in our laboratory ( barcellos et al . , 2011 ) .
seven dna plasmids containing the l1 gene of a high - risk hpv type ( hpv16 , 18 , 31 , 33 , 39 , 45 and 59 ) were used as positive controls .
the absolute and relative frequencies of the categorical variables and the distribution of the continuous variables were evaluated to define the profile of the population under study .
chicago , il , usa ) . the  test was used to study differences in categorical parameters
the mean age of the 158 women was 42.7 13.9 years , the mean age of menarche was 11.6 1.7 years and the mean number of children per woman was 1.4 1.2 children . in terms of other baseline characteristics ,
107 ( 65.5% ) had a stable marital status , 119 ( 73% ) were caucasian , 25 ( 25.2% ) smoked , 58 ( 35.6% ) used oral contraceptive , and 78 ( 47.9% ) were post - menopausal .
the mean age of hpv - dna positive women ( 34.2  12.6 ) was significantly lower than the mean age of hpvdna negative women ( 43.9 13.7 ) ( p = 0.003 ) .
the positive samples were tested for the presence of hpv 16 , 18 , 31 , 33 , 39 , 45 and 59 .
fourteen of the 20 positive samples ( 70.0% ) were positive for hpv 16 , with 7 out of 20 ( 35.0% ) being positive for hpv 31 ; 4 out of 20 ( 20.0% ) for hpv 33 ; 2 out of 20 ( 10.0% ) for hpv 39 ; 3 out of 20 ( 15.0% ) for hpv 45 ; and 2 out of 20 ( 10.0% ) for hpv 59 .
infection by one and multiple hpv types was found in 11 out of 20 ( 55.0% ) and 9 out of 20 ( 45.0% ) samples , respectively .
the cellular changes consistent with the presence of mn were observed in 47 ( 29.7% ) of 158 slides , of which 11 were hpv - positive and 36 were hpv negative .
the presence of mn was significantly associated with hpv - dna positivity ( p = 0.016 ) ( table 1 ) . on the other hand
, the absence of mn observed in a considerable number of hpvdna negative samples ( 102 ) may be of great value in predicting the absence of hpv .
in developed countries , the variability of performance in cytology - based screening has led to the development of more effective alternatives , such as reliable molecular testing .
several large randomized controlled trials have concluded that hpv testing is more sensitive than cytology for cervical cancer screening ( mayrand et al . , 2007 ; castle et al . , 2012 ) . despite the importance of hpv testing
, its use requires advanced technology and still has a high cost , limiting the possibility of its introduction in developing countries .
considering the importance of cervical cancer control programs and the limited number of studies on mn scoring in normal cervical cytology ( aires et al .
2012 ) , the present study was intended to evaluate whether mn is a suitable test to complement the results of the papanicolaou smear in a low - resource setting , analyzing the association between hpv - dna and mn in normal cervical cytology of women from southern brazil .
mn scoring on the cytology smear is easy , cheap , reproducible and can be performed on routinely stained papanicolaou smears as a biomarker of chromosomal damage ( gayathri et al . , 2012 ; samanta and dey , 2012 ) .
in the present study , the prevalence of hpv - dna was 12.7% and was similar to that found in a meta - analysis study , in which the estimated global hpv prevalence in women with normal cytology was 11.7% ( bruni et al .
, brazil has the highest incidence of cervical cancer , with a prevalence of hpv infection among women with normal cervical cytology of 21.1% , being the northern and northeastern regions with higher mortality than eastern and southern states ( dutra et al . , 2012 ) .
in another study developed in rio grande do sul state , the prevalence of hpv - dna found in women with normal cytology was of 32.7% ( coser et al . , 2013 ) .
it is reasonable to assume that these differences in hpv prevalence can be due to risks factors associated to the characteristics of populations in different countries , sensibility of the study method used to detect dna , and/or the number of women included in each study ( sample size ) .
this finding supports previous research , which concludes that hpv prevalence is higher in women younger than 35 years of age , decreasing in women of more advanced age ( sanjos et al . , 2007 ) .
this result corroborates earlier findings that hpv 16 is the most prevalent type of hpv woldwide ( guan et al . , 2012 ) .
in the present study , the occurrence of mn was associated with the presence of hpv - dna in normal cervical cytology , thus corroborating data obtained by other authors ( aires et al . , 2011 ;
this finding suggests that the presence of mn , even with no cytological signs of hpv , can be used by the physician as a criterion for repeating the test within a shorter interval of time . on the other hand
, the high number of samples with negative results in both tests ( mn and hpv - dna ) suggests that these women are at low risk of hpv infection and can be called in for a new round of screening at the normal interval .
other authors have used the micronucleus test as a biomarker for chromosome instability and malignancy , observing higher frequencies of micronucleated cells in samples with cervical cancer than in controls ( majer et al . , 2001 ;
, 2008 ; corts - gutirrez et al . , 2010 ; gayathri et al . ,
finally , there is a consensus that the most clinically effective and cost - effective methods for reducing the incidence of cervical cancer are those that limit the number of required visits ( hoppenot et al . , 2012 ) . since
cervical screening is usually based on cytology alone , the study of mn in papanicolaou smears can improve the quality of diagnosis and screening .
 the aim of the present study was to investigate the association between hpv - dna and micronucleus ( mn ) frequency in women with normal cervical cytology . 
 a total of 158 normal cervical smears were analyzed cytologically . 
 the hpv genome was amplified using the gp5+/biogp6 + consensus primers . 
 hpv - dna of high - risk types 16 , 18 , 31 , 33 , 39 , 45 and 59 were also investigated . 
 of the 158 samples , 20 ( 12.7% ) and 47 ( 29.7% ) were positive for hpv - dna and mn , respectively . 
 evidence for mn was found in 11 out of 20 ( 55% ) hpv - dna positive samples and in 36 out of 138 ( 26.1% ) hpv - dna negative ones . 
 mn presence was significantly higher in hpv - dna positive samples ( p = 0.016 ) 
 . on the other hand , the absence of mn observed in a considerable number of hpv - dna negative samples ( 102 ) may be of great value in predicting the absence of hpv . the mean age of hpv - dna positive women ( 34.2  12.6 ) was significantly lower than the mean age of hpv - dna negative women ( 43.9  13.7 ) ( p = 0.003 ) . 
 infection by one or multiple hpv types was found in 11 out of 20 ( 55.0% ) and 9 out of 20 ( 45.0% ) samples , respectively . 
 the evaluation of mn using cervical smears collected for cytology tests could , thus , be used as additional information to monitor a population s exposure to hpv . 
Introduction
Material and Methods
Results
Discussion