thyroid nodules and thyroid cancer are more common in women [ 1 , 2 ] .
the incidence rate of thyroid cancer ( tc ) is increasing , which could be due to better surveillance techniques .
nevertheless , other factors are probably involved because the incidence rates of tumors of all sizes increased .
as the increase in incidence of tc appears to be higher in women , sex hormones might have an etiological role .
reproductive factors , as history of uterine fibroids , greater number of live births , greater number of reproductive years , and greater estimated number of ovulatory cycles , were associated with an increased risk of tc . also , during pregnancy , thyroid nodules were shown to increase in number and size [ 7 , 8 ] , and tc has been shown to affect one in a thousand pregnant women , being the second most common type of cancer in pregnancy .
several studies have shown thyroid cells growth in response to estrogen [ 1013 ] , as reviewed recently , and some data suggest a decrease in differentiated function by this hormone in thyroid cells [ 11 , 15 ] .
progesterone might have a protective effect on the thyroid because the use of oral contraceptives and menopausal hormone therapy , with the combination of estrogen and progesterone , were not associated with thyroid cancer risk , but irregular menstrual bleeding , usually due to anovulatory cycles , has been associated with it .
although progesterone could exert its effects through genomic or nongenomic mechanisms [ 17 , 18 ] , its primary action is to regulate the expression of target genes through binding directly to its cognate sequence in the genes , called progesterone responsive elements .
as pr have been variably described in thyroid follicular cells [ 2023 ] , the aim of the present study was to evaluate the effects of progesterone on gene expression in a model of normal human thyroid cells in primary culture .
tissue samples were obtained from patients submitted to thyroidectomy as part of treatment for differentiated tc . after evaluation by two pathologists , normal tissue that would have been discharged
the project was submitted to , and approved by , the research ethics committee of the hospital de clnicas de porto alegre , porto alegre , rs , brazil ( cep : 09 - 454 ) , which waived a written informed consent . in accordance with the resolution hcpa 02/97 , based on the resolution cns 196/96 of the national health council , brazil , and the guideline 9 of the international ethical guidelines for biomedical research involving human subjects ( cioms , who , geneva , 1993 ) , there was no need to obtain informed consent of the patients , because only after the surgical procedure the researchers would know if a tissue sample would be available , they would not know the identity nor had access to the files of the patients , and the tissue samples included would have been discharged by the pathologists .
the authors signed the  statement of commitment to use of data ,  which states that no data that could identify patients would be used and also that their anonymity would be preserved in any publication of the results .
briefly , thyroid tissue was cut in fragments of about 1  mm and digested by 3  mg / ml type i collagenase ( gibco , grand island , ny , usa ) . the suspension of cells was sequentially filtered through nylon meshes ( 250 , 150 , and 60  m pore size ) and the filtered fraction , containing epithelial thyroid cells , was resuspended and seeded in a 35  mm petri dish at a density of 1  10  cells / cm .
thyroid cells were cultured initially in ham 's f-12 coon 's modification medium supplemented with 10% fetal bovine serum , 10  g / ml insulin , 5  g / ml transferrin , 1  mu / ml tsh , and 100  u / ml kanamycin at 37c with 5% co2 ; after two changes , the concentration of serum was decreased to 5% ( 3h medium ) .
five or 7 days later , cells were deprived of tsh for 48 hours ( 3h medium minus tsh : 2h medium ) ; subsequently , they were treated with 2h medium and progesterone , mifepristone , or vehicle ethanol , as follows : group 1 : 2h medium ; group 2 : 2h medium + 20  u / ml tsh ; group 3 : 2h medium + 20  u / ml tsh + 10  nm progesterone , and group 4 : 2h medium + 20  u / ml tsh + 10  nm progesterone + 100  nm mifepristone , for 4  h or 48  h , before total rna isolation .
total rna was isolated using the trizol reagent ( invitrogen life technologies , carlsbad , ca , usa ) .
concentration and purity were assessed by the nanodrop nd-1000 spectrophotometer ( nanodrop technologies , rockland , de , usa ) and stored at 80c .
1  g total rna was transcribed into cdna by superscript iii reverse transcriptase ( invitrogen life technologies ) and stored at 20c .
expression of ki-67 , thyroglobulin tg , thyroperoxidase tpo , and sodium - iodide symporter nis genes was assessed by quantitative reverse transcriptase polymerase chain reaction ( rt - qpcr ) on applied biosystems stepone real - time pcr system using kit platinum sybr green qpcr supermix - udg ( invitrogen life technologies ) .
the primers used for the specific amplification of tg , tpo , and nis were described previously .
the sequences of the primers for progesterone receptor were 5-acccgccctatctcaactacc-3 and 5-aggacaccataatgacagcct-3. annealing temperature of 60c was used for amplification ; dissociation curves were performed by running a gradient of 6095c to confirm the specificity of the pcr amplification .
all samples were amplified in triplicate , and cdna standard curves were constructed using the threshold cycles with five successive 10-fold dilution points of a pool of cdna samples .
rt - qpcr experiments were performed in duplicate and repeated in three ( nis gene ) or four ( ki-67 , tg , and tpo genes ) independent experiments , with cells isolated from different patients .
differences in the means of the treatment groups were evaluated with the generalized estimating equation ( gee ) .
after gee , post hoc bonferroni test was used to identify which means were different .
all statistical analyses were performed with spss software , version 18.0 ( spss , chicago , il ) , and considered significant when p was less than 0.05 .
thyroid follicular cells viability and differentiation were assessed by characteristic morphology and by staining tpo protein in immunocytochemistry . as showed in figure 1(a ) , the thyroid follicular cells isolated in our model present a cuboid shape , with the presence of many vacuoles around the nucleus as observed in previous studies of primary culture of follicular cells thyroid . also , as other epithelioid cells , these cells grow as islands and form a monolayer with extensive cell - to - cell contact . in figure 1(b ) , tpo protein staining , characteristic of thyroid follicular cells , is shown . the specific thyroid genes expression ( tg , tpo , and nis )
tsh increased significantly the nis gene expression ( 2.08 times ) after 4  h ; it also increased significantly the tg ( 2.39 times ) , tpo ( 1.58 times ) , and ki-67 ( 1.87 times ) genes expression , after 48  h , as shown in figure 2 . to evaluate the effect of progesterone treatment on the expression of nis ,
gene expression of progesterone receptor was present in all thyroid cells cultures used in this study ( data not shown ) .
when thyroid cells were treated with 20  ui / ml tsh plus 10  nm progesterone , the mean expression of nis mrna after 4  h , and the mean expression of tg , tpo , and ki-67 mrna after 48  h , increased , respectively , by 1.75 , 1.77 , 1.68 , and 1.95 times , when compared to the group treated with tsh ( figure 3 ) .
in the present study , for the first time , an effect of progesterone on thyroid cells was demonstrated .
genes involved in the differentiated protein expression of thyroid cells were upregulated by progesterone , as well as ki-67 , a marker for growth ; these effects were abolished by mifepristone .
isolated human follicular cells derived from normal thyroid tissue have been studied previously [ 27 , 28 ] , with high functional correspondence with the original follicular cells . in our model ,
tg , tpo , and nis mrna were detected even when the cells were deprived of tsh for 48  h , which could be due to other components of the medium and its supplements , such as insulin , or constitutive activation of thyroid cells .
it is known that thyroid cell regulation varies in different species , and , sometimes , the cells can have different phenotypes and responses to tsh [ 29 , 30 ] . in our model , normal human thyroid follicular cells in primary culture were shown to be differentiated and responsive to tsh .
the response of gene expression to tsh has been shown to vary with the gene and time after treatment .
as no direct effect of progesterone on thyroid cells has been described until now , we have no other studies to compare our results with . as progesterone increased the expression of genes involved in growth and differentiated function of thyroid cells , its effect on the thyroid gland could be protective .
observed an increase in free t4 levels in progesterone - treated women , and a tendency towards lower tsh levels during progesterone treatment , in a randomized controlled progesterone trial .
our results suggest that progesterone effects on human thyroid cell occur via a transcriptional level through its nuclear receptor , since a specific antagonist , mifepristone , abolished it .
the ability of mifepristone to abolish progesterone effect in thyroid cells suggests that these receptors are functional in these cells , although its concentration in normal thyroid has been described as low or undetectable [ 22 , 23 ] . in a recent study
pr expression was seen in 75.8% of patients with papillary thyroid cancer . in our study , pr gene expression was identified in all cell cultures . as mifepristone blocks also the action of glucocorticoids and some synthetic progestogens , as medroxyprogesterone , are ligands for the glucocorticoids receptor ( gr ) ,
the effects described in the present study could have been mediated by these receptors . nevertheless , the ability of progesterone to bind to gr is disputable .
although the model used in the present study has contributed to the understanding of thyroid cell growth regulation , the increase in gene expression does not always result in a proportional increase in functional proteins .
besides , our monolayer model does not reproduce the complex organization of thyrocytes in the follicle , so functional and growth studies are necessary to better understand a possible role of progesterone in thyroid cells .
this work provides evidences that progesterone has a direct effect on thyroid cells , through its receptor , upregulating genes involved in thyroid function and growth .
 thyroid cancer and thyroid nodules are more prevalent in women than men , so female sex hormones may have an etiological role in these conditions . 
 there are no data about direct effects of progesterone on thyroid cells , so the aim of the present study was to evaluate progesterone effects in the sodium - iodide symporter nis , thyroglobulin tg , thyroperoxidase tpo , and ki-67 genes expression , in normal thyroid follicular cells , derived from human tissue . 
 nis , tg , tpo , and ki-67 mrna expression increased significantly after tsh 20  ui / ml , respectively : 2.08 times , p < 
 0.0001 ; 2.39 times , p = 0.01 ; 1.58 times , p = 0.0003 ; and 1.87 times , p < 
 0.0001 . in thyroid cells treated with 20  ui / ml tsh plus 10  nm progesterone , rna expression of nis , tg , and ki-67 genes increased , respectively : 1.78 times , p < 0.0001 ; 1.75 times , p = 0.037 ; and 1.95 times , p < 0.0001 , and tpo mrna expression also increased , though not significantly ( 1.77 times , p = 0.069 ) . 
 these effects were abolished by mifepristone , an antagonist of progesterone receptor , suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone . 
 this work provides evidence that progesterone has a direct effect on thyroid cells , upregulating genes involved in thyroid function and growth . 
1. Introduction
2. Methods 
3. Results
4. Discussion
5. Conclusion