the prevalence of spontaneous abortion is about 12 percent which increases in next pregnancy [ 35 ] .
threatened - abortion occurs in 3040 percent of pregnancies and it is identified as vaginal spotting or bleeding under 20 weeks of pregnancy .
the semiallogenic fetus antigens are in direct contact with the mother 's immune system during pregnancy ; therefore , immune suppression has a pivotal role in the maintenance of embryo .
hla - g is a nonclassical hla - ib which has an important role in immune tolerance .
this molecule has seven isoforms including hla - g1 , hla - g2 , hla - g3 , hla - g4 , hla - g5 , hla - g6 , and hla - g7 , where hla - g1 , hla - g2 , hla - g3 , and hla - g4 isoforms are surface molecules and hla - g5 , hla - g6 , and hla - g7 isoforms are soluble . in addition
, soluble hla - g1 originated from surface hla - g1 by metalloproteinase protein activity [ 10 , 11 ] .
in physiologic conditions , expression of hla - g is restricted to fetal trophoblasts at the maternal - fetal interface , cornea , thymic epithelium , pancreatic islets , nail matrix , and the erythroid and endothelial precursors [ 1 , 12 ] .
this molecule is also expressed in malignant transformation , autoimmune and inflammatory diseases , transplantation , and infectious diseases [ 1 , 11 ] .
it has been proven that hla - g plays an important role in pregnancy . during pregnancy
, these molecules can react with nk cells and t cell inhibitory receptors ; thus , it can protect the fetal trophoblast cells from maternal uterine natural killer cells invasion .
totally , human natural killer ( nk ) cells include 1015 percent of circulating lymphocytes and are described phenotypically via their expression of cd56 and lack of cd3 expression .
peripheral blood nk cells can be divided into two major subsets based on neural cell adhesion molecule cd56 ( ncam ) expression including cd56 and cd56 nk cells .
cytolytic response is mostly restricted to the cd56 subset , while cytokine production is usually allocated to cd56 cells [ 13 , 14 ] .
based on peripheral blood nk cells level of cd56 expression and lack of cd3 , nk cells can be divided into two major subsets .
the two groups are defined as cd56 and cd56 nk cells . on the other hand ,
unk cells are defined with high expression of cd56 ( cd56 ) and lake of cd16 ( cd-16 ) and so they are phenotypically and functionally different from peripheral blood nk cells .
nk cells could play an important physiological role in preventing fetal trophoblast invasion and also these cells under circumstances could react with trophoblast cells , leading to abortion . in this study , we evaluated hla - g1 and hla - g5 genes expression and unk cells percentage in vaginal discharge of threatened - abortion women in comparison with normal pregnant women .
in a case - control study , 30 threatened - abortion women and 30 normal pregnant women with the age range of the 1942 years participated in the study ( table 1 ) .
the criteria for threatened - abortion women had been light spotting or vaginal bleeding before 20 weeks of pregnancy . on the other hand , diabetes ,
hyper- and hypothyroidism , immunological disorders , anatomic anomalies , and microbial infections , such as toxoplasmosis , rubella , and chromosomal abnormalities , were exclusion criteria of this study .
these items were evaluated using patients ' history , check - ups , and periodic examinations and collecting information through questionnaires approved by a gynecologist and an obstetrician .
a sample of cervicovaginal discharge was taken from each woman with sterile gynecological collector and was directly transferred to 1  ml rpmi-1640 medium ( sigma , usa ) .
after 5  min centrifuge at 3000  rpm , the superior fluid was transferred to a microtube and kept at 70c for the next elisa analysis .
the cellular suspension of vaginal discharge was added softly to ficoll - histopaque 1.077 ( biosera , uk ) solution in a falcon tube and centrifuged at 400  g ( 2000  rpm ) for 20 minutes .
4  10 vaginal discharge mononuclear cells were labeled with 5   of fitc - anti - cd3 monoclonal antibody and pe - anti - cd56 monoclonal antibody ( ebioscience inc . , san diego , ca , usa ) .
these cells were evaluated by flow cytometry using partec pas iii flow cytometer ( partec gmbh , mnster , germany ) .
the gate was set around the lymphocytes on forward scatter and side scatter dot plot to disclose the other cells of the following analysis .
interference of fl1 and fl2 signals was adjusted by compensation using fitc and pe antibodies .
total rna was isolated from vaginal discharge using cinnapure rna extraction kit ( sinaclon bioscience co. , tehran , iran ) , according to the manufacturer 's procedure .
total rna was reversely transcribed using the accupower cyclescript rt premix ( dn 6 ) kit ( bioneer inc . ,
qpcr was performed using specific primers for hla - g1 , hla - g5 isoforms and elongation factor 1 ( reference gene ) genes ( table 2 ) with sybr green qpcr master mix .
quantitative pcr reactions were done in a 20  l volume including 1x sybr green pcr master mix ( bioneer inc . ,
seoul , south korea ) , 1  l of forward and reverse primers , and 2  l of cdna following the manufacturer 's instructions .
after a primary 10  min at 95c as activation step , 40 cycles comprising of denaturation at 95c , 30  s , annealing at 59c , 30  s , and extension at 72c , 45  s were performed by biorad iq5 multicolor real - time pcr ( bio - rad laboratories , hercules , ca , usa ) detection system .
il-10 level in superior fluid of vaginal discharge cellular suspension was measured by an elisa ready - set - go kit ( ebioscience , usa ) according to the manufacturer 's procedure .
briefly , 100  l capture antibody in coating buffer ( 48  l capture antibody ( 250x ) in 12  ml coating buffer per plate ) per well of a 96-well plate ( corning costar flat - bottom plates , ebioscience , usa ) was incubated overnight at 4c .
each well was then blocked with 200  l of 1x assay diluent ( 1 part 5x concentrated assay diluent with 4 parts di water ) for 1 hour at room temperature , followed by overnight incubation with 100  l of sample at 4c .
then , each well was incubated with detection antibody diluted in 1x assay diluent ( 48  l detection antibody in 12  ml 1x assay diluent ) for 1 hour at room temperature .
each incubation step was followed by 3 washes with wash buffer ( pbs including 0.05% tween 20 ) .
100  l of avidin - hrp diluted in 1x assay diluent was added to each well . following 7 washes with wash buffer
, 100  l of substrate solution was added to each well and the plate was incubated for 15  min at room temperature .
reactions were stopped by addition of 50  l stop solution ( 1  m h3 po4 : phosphoric acid ) .
the p values were characterized in all cases and the p < 0.05 was considered to be statistically significant .
totally , sixty pregnant women participated in this case - control study . out of the 30 threatened - abortion women , 26 pregnant women were followed up during their pregnancy .
53.84 percent ( 14/26 ) of the threatened - abortion women pregnancies resulted in abortion .
the population of nk , nkt , and t cells in vaginal discharge mononuclear cells is presented in figure 1 .
the result showed that unk cells level significantly increased in vaginal discharge of threatened - abortion women in comparison with normal pregnant women ( p = 0.01 ) .
moreover , the level of t cells in threatened - abortion women was significantly lower than control group ( p = 0.003 ) .
there was no significant difference in nkt cells percentage between the two groups ( p = 0.85 ) ( figure 2 ) .
hla - g1 expression was significantly lower in the vaginal discharge of threatened - abortion women in comparison with normal pregnant women ( p = 0.0001 ) . however , threatened - abortion women had a decreased level of hla - g5 in comparison with control group , but this difference was not significant ( p = 0.12 , figure 3 ) .
the concentration of il-10 was determined in the vaginal discharge via elisa as an indicator of cytokine production .
this cytokine production level in threatened - abortion women was lower compared with the normal pregnant control group ( 3.91  1.46 versus 4.76  2.5 ) .
at first , we carried out an evaluation on nk cell count and expression of hla - g isoforms expression ( pan hla - g , hla - g1 , hla - g5 ) in the vaginal discharge of the threatened - abortion women in comparison with control group , which was determined before 20 weeks of pregnancy .
previously , it was observed that cell - bearing hla - g acquires tolerogenic potential to downregulate various immune reactions .
our results showed that hla - g1 and hla - g5 isoforms decreased in the abortion - threatened women ( figure 3 ) .
formerly , we showed that hla - g isoforms levels are different among individuals . in this research
, we found that the hla - g1 expression in vaginal discharge was higher than hla - g5 in healthy pregnant women and hla - g1 levels in vaginal discharge of the threatened - abortion women are significantly higher in comparison with hla - g5 expression in healthy pregnant women .
therefore , it is recommended that the evaluation of hla - g1 expression in vaginal discharge can be used as a normal pregnancy marker .
probably , expression of hla - g1 isoform is controlled by key vital factors for preservation of fetus .
in addition , hla - g1 may be a major isoform in the tolerance of mother 's immune system .
furthermore , it is possible that the low expression of hla - g1 contributed to allogenic nk cell proliferative response in semiallogenic fetus .
on the other hand , hla - g1 and hla - g5 have an important role in the upregulation of t regulatory cells in the pregnant women .
also , our results showed that the high expression of hla - g resulted in an excess of il-10 in the vaginal discharge .
thus , hla - g1 is an inhibitory constituent and may exert a key suppressive effect on t cell proliferation response to semiallogenic fetus with the cooperation of hla - g bearing cells inhibitory cytokine secretion . in this study
, it was observed that the percentage of natural killer cells increased in the vaginal discharge of the threatened - abortion women in comparison with normal pregnant women which could be an indicator of fetus loss .
in addition , low expression of hla - g1 immunotolerance molecule is a risk factor for spontaneous abortion in comparison with control group .
furthermore , our data showed that hla - g1 expression was significantly lower in abortion - threatened women in comparison with the control group .
however , hla - g5 expression level was not significantly different between the two groups , which propose that hla - g5 has inconsiderable characteristic in the mentioned population .
several studies have shown a significant correlation between peripheral blood natural killer cell and hla - g in abortion - threatened women .
in addition , it is reported that nk cell level can be a predictor of abortion in pregnant women [ 17 , 18 ] .
furthermore , there is a strong association between hla - g and nk cell level with spontaneous abortion .
it is suggested that predictive value of hla - g or nk cell is superior to other established prognostic factors for fetal loss .
reported that hla - g can be the most relevant marker for abortion screening due to t cell response . on the other hand ,
hla - g identified as a marker for tolerance and its plasma level was associated with better survival in pregnant women [ 23 , 24 ] .
our results show a significant correlation between vaginal discharge il-10 level with high hla - g and low nk cell count .
these findings show a positive correlation between hla - g1 and hla - g5 expression with il-10 level and a negative correlation between nk cell counts with these cytokines .
according to the results , hla - g molecules contribute to the regulation and control of the immune response . in conclusion
, the results of our study showed that vaginal discharge , nk cell percent , and hla - g1 and hla - g5 expression were correlated with abortion in comparison with control . therefore , uterine nk and hla - g1 and hla - g5 have an important key role in fetal surveillance during pregnancy .
 the immunotolerant human leukocyte antigen - g ( hla - g ) molecules have a major role in fetal - maternal tolerance during pregnancy . 
 interaction between these molecules and uterine natural killer ( unk ) cells inhibitory receptors prevents nk cell invasion against fetus trophoblast cells . the aim of this study was to evaluate the percentages of unk cells and hla - g1 and hla - g5 isoforms expression in vaginal discharge of threatened - abortion women in comparison with control . in a case - control study , we investigated 30 threatened - abortion women with bleeding or spotting less than 20 weeks of pregnancy as compared to 30 normal pregnant women . 
 unk cells percentage was assessed by flow cytometry . 
 furthermore , we evaluated hla - g1 and hla - g5 isoforms expression by real - time pcr in these groups . 
 the results of this study showed that threatened - abortion women had increased unk cells and decreased t cells percentage in vaginal discharge in comparison with normal pregnant women ( p = 0.01 , p = 0.003 , resp . ) . 
 in addition , hla - g1 isoform had lower expression in threatened - abortion women in comparison with control group ( p = 0.0001 ) . 
 the increase of unk cells level with the decrease of hla - g expression in vaginal discharge of threatened - abortion pregnant women is an indicator of mother 's immune dysregulation . 
 it is concluded that hla - g expression level with unk cells percentage can be determined as a diagnostic marker for threatened - abortion women . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions