it is estimated that , globally , 466,000 new cases of cervical cancer are diagnosed 
 each year . as per estimate 231,000 women in a year 
 die of cervical cancer and 80% of them are from the developing world . use of cytological screening technique , papanicolaou ( pap ) test , has significantly increased the detection of cervical 
 cancer . cancer of cervix emerges from a defined series of preneoplastic 
 lesions with increasing cellular dysplasia , referred to as cervical 
 intraepithelial neoplasia ( cin ) grade i , ii , and iii . however , cin lesions 
 frequently regress , and not all cin iii lesions , progress to invasive cancer 
 . persistent infection with high - risk - type human papilloma viruses ( hpvs ) is the strongest independent 
 risk factor for cervical cancer . cin lesions induced by hpv infection and 
 genomes of these viruses were detected in the vast majority of cervical cancer 
 specimens [ 57 ] . during the course of tumor initiation in an hpv - infected cervical epithelial cell , an aneuploid dna pattern develops , 
 which along with its descendants shows genetic instability . aneuploidy reflects 
 a situation of uncontrolled increase of dna and loss of essential information 
  and plays an important role in neoplastic transformation . increased 
 aneuploid dna value with the increase in grades of cervical dysplasia has long 
 been considered to be a specific prognostic marker of malignancy . however , 
 at present , no well - established technique , which , qualitatively , predicts the 
 clinical outcome of cervical dysplasia except pap smear screening , is 
 available . approximately 1530% of all women who developed low - grade squamous intraepithelial lesion ( lsil ) have a 
 probability of developing moderate - to - severe cervical intraepithelial neoplasia 
 ( cin ii and iii ) identified on a subsequent cervical biopsy . to prevent 
 cervical malignancy and its precursors , several adjuvant diagnostic methods 
 have been proposed to strengthen the accuracy of cytological and histological 
 diagnosis . as chromosomal aneuploidy has been used as an early key event 
 in tumorigenesis caused by genetic instability [ 13 , 14 ] , the cytometric 
 equivalence of chromosomal aneuploidy detected by dna flow - cytometry may serve 
 as a marker of neoplasia and provide valuable information for the diagnosis and 
 understanding of pathogenesis of cervical cancer . since aneuploid pattern of 
 dna is a useful parameter for the prediction of progression and persistence of  atypical squamous cells of 
 unknown significance ( ascus )  , low - grade squamous intraepithelial 
 lesion ( lsil / mild dysplasia / cin i ) , and high - grade squamous intraepithelial 
 lesion ( hsil / moderate and severe 
 dysplasia / cin ii and iii ) in clinical 
 practice , the present study was designed to investigate whether 
 alteration in cellular / nuclear content predicts the occurrence of cancer in 
 cervical smears . we have thus estimated their dna content using flow cytometry 
 and compared it with the cytological findings . the cases were derived from the ongoing outpatient cytology screening at department 
 obstetrics and gynecology , king george 's medical university ( lucknow , india ) for 
 the present study . cytologically diagnosed 127 cases of dysplasia ( 79 mild , 36 
 moderate , and 12 severe ) , 57 cases of atypical 
 squamous cells of unknown significance ( ascus )  and 69 controls , 
 which enrolled during the period of 20042006 , were recruited from a 
 prospective followup study to understand the biological behavior and the 
 natural history of cervical precancerous lesions . none of the patients had any previous history of cin or had been treated 
 for cin . the blunt end of the second spatula was 
 transferred into a cryo tube immediately , containing 3  ml chilled pbs ( 7.2  ph ) 
 and quick frozen at 20c until further use for flow - cytometric 
 analysis . precancerous lesions of the uterine cervix were classified as normal , 
 ascus , lsil ( mild dysplasia / cin 
 grade i ) , and hsil ( moderate dysplasia / cin grade 
 ii , severe dysplasia / cin grade iii and carcinoma in situ / cis ) . to understand the biological behavior 
 of cervical precancerous lesions , the following criteria were followed.the cases initially diagnosed as lsil ( cin i ) and hsil 
 ( cin ii and iii ) were made to undergo a second pap smear test after 30 days.cytological confirmed dysplasia 
 cases were enrolled for the followup with the consent of the patients . cases initially diagnosed as cin 
 iii were subjected to colposcopy directed biopsy on day 30 and were directed to 
 gynecologist for further management . a cytological control examination was done 
 after every 6 and 3 months for lsil and hsil subjects , respectively , and were 
 categorized as  returned to normal  ( after the morphology normalized ) ; 
 progressed ( when presented with higher grade of dysplasia ) ; and with no change 
 ( when remained at the same grade of dysplasia ) . the samples were also tested 
 for the dna ploidy pattern.ascus and all aneuploid cases from control group were also followed for a 
 period of one year . the cases initially diagnosed as lsil ( cin i ) and hsil 
 ( cin ii and iii ) were made to undergo a second pap smear test after 30 days . cytological confirmed dysplasia 
 cases were enrolled for the followup with the consent of the patients . cases initially diagnosed as cin 
 iii were subjected to colposcopy directed biopsy on day 30 and were directed to 
 gynecologist for further management . a cytological control examination was done 
 after every 6 and 3 months for lsil and hsil subjects , respectively , and were 
 categorized as  returned to normal  ( after the morphology normalized ) ; 
 progressed ( when presented with higher grade of dysplasia ) ; and with no change 
 ( when remained at the same grade of dysplasia ) . ascus and all aneuploid cases from control group were also followed for a 
 period of one year . to determine the cellular dna content , the protocol of nicoletti 
 et al . was followed . in brief , cells were fixed with chilled ethanol for 
 30 minutes at 4c temperature . after adding propidium 
 iodide ( sigma - aldrich , miss , usa ) , the mixture was stored in dark for 1 hour at 
 room temperature . dna histogram cell - cycle analysis was performed as 
 described by rabinovitch , using modfit lt for mac v2.0 
 software 
 after excluding debris by using electronic gate . the events in g0-g1 , s , and g2-m 
 phases of the cell cycle were counted . cases were regarded as acceptable for 
 analysis if the cv of the g0-g1 peak was 7.0 . if two distinct g0-g1 peaks were present 
 with a dna index of 1.15 ( each containing 10% of 
 total cell population ) , the histogram was considered as aneuploid ( dna 
 content ) . by convention , a histogram was 
 classified as tetraploid only if a large peak observed in g2-m ( dna index of 
 1.802.2 ) , containing 20% of 
 the total curve and was also associated with a corresponding 8  n peak . epi - info software was used for 
 calculating the odd ratio and  ( chi - square ) values . a p - value 
 of .05 or less was 
 considered statistically significant ( 95% ci ) . the third and fourth groups comprised of lsil 
 ( 79 cases ) and hsil ( 48 cases ; 36 cases in moderate dysplasia and 12 cases in 
 severe dysplasia ) . a comparison of dna content analysis 
 with behavior of lsil or hsil in 
 patients having cytologically diagnosed various grades of dysplasia along with 
 the control and ascus groups were made ( table 1 ) . 127 cases of different grades 
 of dysplasia were employed for analysis of dna ploidy out of which 78 ( 62.99% ) 
 cases were aneuploid ( 39 mild , 28 moderate , and 11 cases of severe dysplasia ) 
 and remaining 49 ( 37.01% ) cases ( 40 mild , 8 moderate , and 1 severe ) were nonaneuploid , 
 that is , diploid ( see figure 2 ) . in the control and ascus groups , 6 ( 8.70% ) and 
 8 ( 14.04% ) cases were found to be aneuploid , respectively ( see figure 2 ) . the 
 association of aneuploidy with increasing grades of dysplasia was found to be 
 significant ( p  .001 ; 
 table 1 , see figure 3 ) . all these cases with aneuploid dna 
 content and cytologically diagnosed ascus and various grades of dysplasia were 
 followed up for 1 year at aforementioned intervals , in order to study the 
 behavior of aneuploid dna in progression / regression / persistence of disease . 
 among 57 patients with ascus , 49 ( 85.96% ) diploid cases had smears that returned 
 to normal cytology and remaining 8 ( 14.04% ) aneuploid cases showed signs of 
 progression / regression . one patient presented with hsil and three with lsil 
 after one year period of followup . remaining four aneuploid cases of ascus 
 group regressed to normal , as evident from diploid dna . out of six aneuploid 
 cases with normal cytological diagnosis , 4 cases were followed , where only one 
 case represented progression of disease from normal to mild dysplasia after a 
 year . a total of 79 patients who had diagnosis 
 of lsil , 75 cases were followed up . out of 39 aneuploid cases , 7 developed 
 moderate dysplasia ( cin ii ) and 30 cases presented with no change in cytology 
 ( cin i ) , after a period of six months from the date of registration . 35 cases who had lsil with nonaneuploid 
 dna returned to normal and one patient presented with no change in cytology , that 
 is , remained at cin i. the sample for this particular subject showed aneuploid 
 dna content of cell in followup study while 35 normal cases were diploid ( table 2 ) . out of 36 cases of moderate dysplasia , 20 patients out of 25 who were 
 diagnosed with moderate dysplasia ( hsil / cin ii ) and with aneuploid dna 
 remained cin ii ( 71.79% ) with an aneuploid dna in their followup study after 
 three months of registration . in addition , 2 other patients were diagnosed 
 later with cis , who were positive for aneuploid dna in their followup . 1 patient 
 with diploid dna remained at cin ii in cytological followup and other 4 diploid 
 showed a regressive pattern . out of 11 only 3 aneuploid cases of severe 
 dysplasia ( cin iii ) were followed , rest underwent prescribed treatment . of the 3 
 cases , which were aneuploid , 1 case progressed to cis and 2 remained at cin 
 iii . the association of aneuploidy with progression of disease was found to be 
 significant ( table 2 ) . test value for lsil 
 ( p  .001 ) and hsil ( p  .001 ) was significant . there is evidence that cervical 
 carcinoma evolves on the basis of persistence of aneuploidy . studies have 
 shown that aneuploid conditions indicate either invasive carcinoma or 
 prospective neoplastic development in cervical dysplasia [ 18 , 19 ] and play a 
 causal role in neoplastic development [ 20 , 21 ] . genetic abnormalities , such as numerical chromosomal aberrations ( aneuploidy and 
 tetraploidy ) , have been reported in women diagnosed with precancerous and 
 cancerous cervical lesions and are observed frequently in the presence of hpv 
 infections [ 10 , 22 , 23 ] . according to melsheimer et al . , deregulated viral 
 oncogene expression appears to result first in chromosomal instability and 
 aneuploidization and is subsequently followed by integration of hpv genomes in 
 the affected cell clones . in cin and 
 invasive cervical cancer , dna ploidy assessment by flow cytometric technique has 
 been established as a prognostic factor that allows the estimation of the 
 relative progressive risk into more advanced lesions [ 8 , 2427 ] . dysplastic 
 epithelium represents potentially precancerous conditions in which the risk of 
 progression to cancer is usually unknown . bocking and motherby   suggested 
 that the identification of dna aneuploidy in dysplastic squamous epithelia 
 could increase the predictive value for malignant transformation to over 90% . similarly , the results of our study have also confirmed the predictive value of 
 aneuploidy with 92% aneuploid cases in all grades of dysplasia showing 
 progressive pattern of disease ( table 2 ) . evidences indicate aberrant dna content 
 in cells as a cause rather than as a consequence of malignant transformation [ 29 , 30 ] . duesberg et al . pointed out that aneuploid cancers 
 are generally considered to be more dangerous and aggressive than the diploid 
 . . carried out a prospective study to determine the 
 progression to malignancy of cervical smears with ascus . researchers concluded that the 
  aneuploid lesions should immediately be controlled histologically or be removed  . in 
 accordance with the above findings , the results of the present study also 
 demonstrate the progressive pattern of disease among aneuploid cases from ascus 
 group . suggested that ascus cases , those with high - risk hpv and 
 with abnormally high dna content , represent similar biologic features as hsil 
 with elevated risk to develop cancer . thus , dna content may reflect 
 genomic instability of the cells very early in the malignant process , at a 
 point where morphological changes in malignancies or premalignancies are not 
 yet evident . conceivably , lesions that are defined as nondysplastic , and 
 therefore considered to represent minimal or no risk for developing cancer 
 cervix , may contain readily detectable large - scale genomic alteration viz dna 
 aneuploidy which could signal a malignant transformation [ 34 , 35 ] . the 
 identification of a molecular biomarker of persistent hpv infection in women 
 who had been diagnosed as normal / ascus in pap smear could be a valuable tool in 
 the proper management of this disease . we have also found 6 aneuploid cases 
 from control group and among them , after one - year duration , one case presented 
 the progression of disease . in this study , we have shown that 
 aneuploid dna content could be used as a biomarker of progressive cervical 
 changes towards malignancy . progressive 
 increase in aneuploidy increases the severity of the lesions ( 49.36% for mild 
 dysplasia , 77.77% for moderate dysplasia versus 91.66% for severe dysplasia ) . our followup results clearly demonstrate that more and more progressive 
 pattern of disease was noted in aneuploid cases and that dna ploidy could be 
 used as a prognostic marker for cervical malignancies . comparison of results obtained with pap screening and dna ploidy 
 measurement provide a positive correlation between aneuploid dna and 
 progression of disease ( table 2 ) . furthermore , the percentage of aneuploid cases 
 towards the progression of disease was higher in followup data that supported 
 the prognostic value of aneuploidy as a marker to identify the progressive 
 behavior of lesion . occurrence of aneuploidy was exceptional in patients 
 who had samples with morphology that returned to normal , whereas , patients who 
 had cytological signs of disease progression were found with a significantly 
 greater frequency ( p < .01 , p < .001 ; table 2 ) . high - grade lesions ( according 
 to bethesda classification ) can be detected using cytodiagnosis method with 
 generally good accuracy , but the diagnosis occasionally relies on subjective 
 parameters . moreover , inadequate sampling may result in a mild morphologic 
 change despite early progressive behavior . thus , the measurement of dna content by flow cytometry provides qualitative 
 information and presence of aberrant aneuploid cells in cytological samples , 
 which could serve as a useful parameter along with cytology with good 
 specificity and sensitivity [ 37 , 38 ] . if patients at risk could be identified earlier 
 by analysis of parameter that reflects the true biological properties of 
 lesion , treatment regimes could be given selectively to improve the current 
 poor survival rate of cancer cervix cases . flow cytometric analysis of dna 
 ploidy may provide a strategic diagnostic tool for early detection of carcinoma 
 cervix [ 8 , 25 ] . therefore , 
 we propose the concept of an hpv screening with reflex cytology in combination 
 with dna flow cytometry to detect progressive lesions with the greatest 
 possible sensitivity and specificity .