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 .