hormonal changes at the moment of parturition may constitute an important regulatory mechanism for myometrial contraction . in some species ( e.g. , sheep ) , sudden changes in circulating cortisol ,
oestrogen , and progesterone appear to be important factors for the onset of labour , whereas in humans , these signs remain unknown [ 110 ] . therefore , we investigated the concentration of oestrogen receptors ( ors ) and progesterone receptors ( prs ) in pregnant human women because local modifications of these receptors may be associated with the myometrial conditions responsible for the mechanism of parturition [ 110 ] .  
prior studies on hormone concentrations during labour have failed to demonstrate significant changes indicating their direct involvement in labour ; however , local changes in hormone receptors are believed to be responsible for parturition [ 4 , 5 , 11 ] .
close to term , the effect of progesterone may be inhibited by a genetic mechanism involving one or more agents not associated with receptors [ 1 , 2 ] .
this genetic mechanism may lead to the attenuation of progesterone 's effects by , for example , a specific gene that interrupts its action at a molecular , biochemical , and/or cellular level .
corroborating these studies in a review , mendelson postulates that an increase in inflammatory response occurs as a result of the activation of the nuclear factor ( kappa ) beta , leading to uterine contractility in two ways : ( 1 ) the direct activation of contractile genes ( e.g. , cox-2 , oxytocin receptors , and connexin 43 ) and ( 2 ) a blockade of the ability of progesterone receptors ( prs ) to maintain uterine quiescence . according to previous reports
, a decrease in the concentration of prs is observed in patients during labour [ 412 ] .
nonetheless , the inhibition of the effects of gestagenic agents does not result in the immediate onset of labour , although this inhibition sensitises the cells of the myometrium to agents that induce uterine contractility .
in contrast , oestrogen enables the myometrium to prepare for parturition by inducing oxytocin receptors , which is perhaps the first step of parturition [ 510 , 12 ] .
recent studies have also suggested that the withdrawal of progesterone is the initiator of parturition , and they have demonstrated the importance of the concentrations of prs a and b and their relationship in the mechanism of parturition [ 510 , 12 ] .
currently , other aspects are also known to be required for the onset of parturition .
cervical changes , for example , are associated with the expression of receptors that modify local mechanisms such as the remodelling of collagen , the migration of inflammatory cells , and changes in the extracellular matrix as a whole [ 9 , 1315 ] . to quantify the concentrations of ors and prs in the myometrium during labour ,
a prospective study including 21 patients was designed to evaluate the concentrations of these receptors on the surfaces of myometrial cells in tissue biopsies collected during a caesarean section of full - term pregnant women in relation to the presence or absence of labour .
myometrial tissue was obtained by biopsies performed during caesarean sections of 21 full - term pregnant women ( 37 to 41 weeks ) .
six women were in labour ( mean 39 weeks , range 37 to 41 ) , and 15 were not in labour ( mean 39 weeks , range 37 to 41 weeks ) .
the study was evaluated and approved by the research ethics committee of the federal university of so paulo ( unifesp ) .
gestational age was confirmed by the date of the last menstrual period and/or by ultrasonography in the first and/or second trimesters .
women were considered parturient based on the assessment of the signs and/or symptoms of labour : a bishop score greater than or equal to 5 , two or more uterine contractions lasting more than 40 seconds in 10 minutes , cervical dilation of 2  cm in primiparous women and 3  cm in multiparous women , and insinuated foetal presentation .
the indications of cesarean section in patients during the labour were as follows : two or more previous cesarean sections , fetal distress observed during the evolution of the labour , and primiparous women in breech .
the exclusion criteria were patients with obstructed labour , clinical , and/or obstetrical complications , and patients undergoing induction / conduction of labour with oxytocin or prostaglandin , using medication except multivitamins . during the caesarean section ( after placental expulsion ) , a small piece of the myometrium was removed at the upper middle portion of the incision ( hysterotomy ) by the researcher ( jsa ) , always using the same technique : a resection of 0.5  cm of the myometrium with the aid of a scalpel , excluding the endometrium and visceral peritoneum , and then proceeding to a normal suture of the hysterectomy . the material obtained from each biopsy was placed in 10% buffered formaldehyde for a maximum of 48 hours and subsequently processed in an autotechnicon ( autotechnicon 2 , chauncey , ny , usa ) for 12 hours ( overnight ) , using alcohol , xylene , and paraffin .
after processing , the specimens were placed in cassettes and embedded in paraffin ( soldan cytological products ltd . , porto alegre , rs , brazil ) .
the paraffin blocks containing the histological processed materials were cooled in a freezer and cut on a rotary microtome to obtain 4-micron - thick histological sections using disposable razors ( leica ) suitable for this purpose .
the paraffin ribbons obtained from the microtome sectioning process were carefully placed in a histological water bath , and the sections were deposited on precleaned slides coated with a special adhesive that improves tissue adhesion ( ver - sa plus , euriegas ) .
( 1 ) preparation of the histological sections : washing in xylene at room temperature for 15 minutes , twice , followed by 100% ethanol ( 3 times ) for 30 seconds each , 95% ethanol for 30 seconds , 80% ethanol for 30 seconds , and 70% ethanol for 30 seconds , with a final washing in running and distilled water . ( 2 )
antigen retrieval : the slides were incubated in moist heat and incubated in citrate buffer ( 10  nm , ph 6.0 ) in a steamer ( t - fal ) for 20 minutes . the immunohistochemical visualisation consisted of the following steps in a reaction using the streptavidin - biotin peroxidase complex ( streptabc ) .
( 1 ) the slides were incubated with a specific antibody ( monoclonal er ( 1b)/pr ( 2b ) ) for 30 minutes in a moist chamber .
( 2 ) the slides were washed in tris - buffered saline ( dako tbs ) , and the buffer was exchanged every 3 minutes .
( 3 ) the slides were then incubated with biotinylated secondary antibody ( anti - immunoglobulin from the animal species from which the specific antibody was obtained ) in a moist chamber for 30 minutes at 37c . ( 4 ) the slides were again washed in buffer with three exchanges , once every 3 minutes .
( 5 ) revelation and mounting : the slides were incubated in a substrate solution ( diaminobenzidine ; dab , 60  mg% ) for 3 minutes at 37c in the dark .
the positive control slides were then observed through the microscope for the development of a golden - brown precipitate , the final product of the reaction ; the slides were washed in running and distilled water for 3 minutes , counterstained with harris haematoxylin for 30 seconds , and washed in running and distilled water .
the slides were then dehydrated in 50% ethanol , 80% ethanol , 95% ethanol , and 100% ethanol ( 3x ) followed by xylene ( 3x ) , mounted in entellan cover slips ( merck 1.07961 ) and examined under an optical microscope by three pathologists to confirm the suitability of the tissue sample .
the pathologists also verified whether scarring from previous caesarean sections would interfere with the results , which was avoided by observing specific areas for study .  
the quantitative analysis of the presence of ors and prs was performed using a computerised image - analysis program ( imagelab ) . for the quantification of the areas occupied by the two hormone receptors ,
microscopic fields of 422  m  322  m were captured at a magnification of 100x while maintaining several standardised settings of the optical microscope ( leitz carbelux model 6mbh ) . a leitz wetzlar-160/0.17 plan
10/0.45 lens was used at maximum light intensity with the maximum condenser aperture at a distance of 6.5  cm from the slide .
the digitalised fields had a horizontal resolution of 640 pixels and a vertical resolution of 480 pixels in 24-bit colour ( 16 million colours ) , and the same range of colours for the area to be quantified was used in each of the fields .
the colour range was adjusted for isolation in areas that represented the ors and prs in the image .
the averaged values obtained from both analysed fields in each slide were determined by considering two parameters : the percentage of the area occupied by hormone receptors and the optical density of the light reflected by each of the receptors . to avoid possible subjectivity ,
all examined materials were identified by each patient 's medical record number , and the collected data and the results obtained were compared only at the end of the study .
the pathological anatomy laboratory technician numbered the patients according to the order of entry of the biopsy material in the laboratory , enabling the statistical analysis of patients numbered 1 to 21 .
the name and number of the medical record were retained , allowing for easy identification .
therefore , the patients in labour and not in labour could readily be compared at the end of the study following the examination of the slides .
redmond , wa , usa ) spreadsheet and analyzed by the statistical package for the social sciences ( spss ) program , version 19.0 for windows ( spss inc . ,
chicago , il , usa ) . the mann - whitney test was used to compare the pregnant women in each study group with respect to the numbers of ors and prs .
the wilcoxon test was used to compare the numbers of ors with the number of prs in each group .
spearman 's correlation coefficients ( r ) were used to study the possible relationships between ors and prs in each group separately .
the medians of prs and ors in pregnant women in labour ( group i ) were 29.3  2.38 ( range , 24.630.2 ) and 32.3  10.92 ( range , 22.949.0 ) , respectively .
the medians of prs and ors in pregnant women without labour ( group ii ) were 43.6  14.94 ( range , 23.670 ) , and 43.9  13.17 ( range , 18.362.6 ) , respectively .
we did not observe significant differences of the number of ors and prs in both groups by the mann - whitney test ( p = 0.13 and 0.37 , resp . ) .  
the z critical between both groups was 1.96 ( oestrogen - z calculated = 1.56 and progesterone - z calculated = 0.93 ) , proving difference in the concentration of both receptors in the myometrium of lower uterine segment of pregnant women during and without labour ( table 1 ) .
the number of ors was statistically more than that of prs in the myometrium of lower uterine segment of group ii ( group i z calculated = 0.94 versus group ii z calculated = 16.00 ) .
we observed that the prs and ors concentrations were smaller in the myometrium of lower uterine segment of group i , and these concentrations were higher in group ii ( group i r calculated = 0.49 versus group ii r calculated = 0.74 ) ( table 2 ) .
knowledge of the mechanism of parturition should allow patients in preterm labour and patients with postterm pregnancy to be properly treated , minimising the complications of such conditions [ 2 , 3 , 14 , 16 ] .
recent works have suggested that a decrease in the number of progesterone b receptors , favouring an increase in the expression of progesterone receptor a , results in a triggering of the mechanism that initiates labour [ 57 , 9 , 10 ] .
thus , these authors suggest a functional withdrawal of progesterone . the results of the present study suggest that the downregulation of the concentration of prs may be associated with human parturition , or at least with its initiation . because no significant changes were noted in ors numbers in either labour or nonlabour conditions , oestrogen action may be held constant .
a constant level of oestrogen may also maintain the status of both ors and prs , given the well - known upregulation of both hormone receptors by oestrogen and the downregulation of these receptors by progesterone [ 1 , 2 , 4 , 11 ] .
another implication of the sustained or levels may be explained by the observation that oestrogen excites the myometrium , increases the levels of oxytocin receptors , and induces the inflammatory process currently known to be involved in parturition [ 5 , 6 , 9 ] .
the present study confirmed the findings of several previous studies , particularly from the study that served as our model , that is , the upregulation of ors and prs . when pr numbers decrease , there is a less expressive decrease in or numbers , and prs apparently tend to decrease in patients in labour [ 8 , 10 ] .
we note that the present study was conducted in the myometrium of lower uterine segment and , similarly to the studies conducted by rezapour et al .   and
, we observed a low concentration of prs in the myometrium of lower uterine segment .
the study by merlino et al .   required a correction of the receptor analysis due to the small number of myometrial cells in the uterine cervix , which is also consistent with findings of the present study . similarly to the study of winkler et al .
, in the present study , a tendency for a decrease in the number of prs was observed in pregnant women at the beginning of labour with a bishop score equal to or less than 5 . according to study
, the fact that lower concentrations of prs were found may have been due to a missing analysis of a specific receptor type .
the authors showed that the ratio of pr - a to pr - b increased during labour .
they also studied pregnant women subjected to elective caesareans , in both term and preterm pregnancies , although not in labour .
their results showed that a pr - a / pr - b ratio of 0.5 was maintained in the term and preterm pregnancies , whereas during labour , the ratio doubled in favour of pr - a , with a value of 2.5/1 .
these findings are consistent with previous studies and support the hypothesis that human parturition involves a specific increase in the ratio of pr - a / pr - b in the myometrium due to an increased expression of pr - a  .
this result drew special attention to the difference in hormone concentrations in pregnant women who had no signs or symptoms of labour .
significant differences were found between the concentrations of hormone receptors in nonparturient women , whereas no difference was noted in parturient women ; that is , there appeared to exist a constancy in the numbers of steroid receptors when the myometrium was clearly in labour activity , which may be explained by the postulate of mendelson stated above .
the differences in the methodologies used to assess the concentrations of the studied hormone receptors in our study and others allowed for a corroboration of the results obtained in other analyses using different methods .
thus , it is worth noting that the study and quantification of or and pr is less laborious and costly using the methodology developed in the present study .
the similarities between this study   and the present study are the following : the patients were subjected to undergo elective caesarean sections ; that is , they were not in labour ; parturients were subjected to caesarean section due to obstetric requirements and distributed according to clinical criteria for the assessment of the presence or absence of labour ; biopsies were obtained from the myometrium of lower uterine segment , at the upper portion of the hysterotomy incision ; and we attempted to remove the whole tissue that was not myometrium .
however , the authors note that , despite the rigour adopted for myometrial tissue removal , there may have been some contamination with other tissues . in an other study on the myometrium , merlino et al .   investigated the expression of the nuclear progesterone receptor in foetal membranes and decidua at term , before and after the onset of labour .
their results suggested that the decidua , rather than the foetal membranes , is the direct target for the nuclear pr during pregnancy because the receptor was only detected in the decidua .  
our hard criteria to include only pregnant women with spontaneous labour ( group i ) and to exclude the dystocia as indication of caesarean section contributed to this small sample .  
the present study is in accordance with the literature , showing the changes in steroid receptor sites and assisting in understanding the possible mechanism of labour and how they can intervene in this situation if necessary , for example , in preterm and prolonged gestation .
finally , the need for an animal model with a parturition physiology similar to the human system is also required , allowing more detailed studies to be performed in vivo .
 
 objective . to assess the concentration of progesterone ( prs ) and oestrogen ( ors ) receptors of myometrium of full - term pregnant women in the myometrium of lower segment of the uterus in relationship with presence or absence of labour 
 . methods . 
 this was a cross - sectional prospective study with 21 pregnant women , being 6 in labour ( group i ) and 15 without labour ( group ii ) . 
 the biopsy of myometrium was realized during caesarian section , and the excised tissue was stained using immunohistochemical techniques for the quantification of the receptors , and with the aid of image - analysis software , the numbers of receptors for each hormone were determined spectrophotometrically . 
 the mann - whitney test was used to compare the pregnant women in each study group with respect to the numbers of ors and prs . 
 the wilcoxon test was used to compare the concentration of ors and prs in each group separately . 
 results . 
 the mean of gestational age was 39 weeks , ( range , 37 to 41 weeks ) . 
 the medians of prs and ors in pregnant women in labour ( group i ) were 29.3 ( range , 24.630.2 ) and 32.3 ( range , 22.949.0 ) , respectively . 
 the medians of prs and ors in pregnant women without labour ( group ii ) were 43.6 ( range , 23.670 ) and 43.9 ( range , 18.362.6 ) , respectively . 
 we did not observe significant differences of the number of ors and prs in both groups ( p = 0.13 and 0.37 , resp . ) . 
 the number of ors was statistically more than that of prs in group ii ( z calculated  =  16.00 ) . 
 conclusion . 
 the concentrations of prs and ors were similar in the myometrium of the lower uterine segment of pregnant women during and without labour , but the concentration of ors was more than that of prs in the myometrium of the lower uterine segment of pregnant women without labour . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion