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Introduction

In women of reproductive age, the incidence of spontaneous abortion ranges from 5 to 25%. Previous findings have indicated that spontaneous abortion is associated with gene expression, environmental factors, immune tolerance, inflammatory response, microbiological contamination, endocrine disorders, and diseases in reproductive tract diseases (1,2). However, 30–50% of patients suffer from unexplained recurrent spontaneous abortion (URSA), which is generally considered to be caused by a decrease in immune tolerance (3). Maternal circulatory and placental tissue cell abnormalities, as well as abnormal humoral immune function, can affect the regular transfer and development of the embryo, resulting in early abortion. It has been found that regulatory T-cell (Treg) levels, especially those of the CD4+CD25+ subtypes, are significantly decreased in the peripheral blood and decidual tissues of patients with spontaneous abortion, and this decrease aligns well with the timing of the abortion (4).

Natural killer (NK) cells account for 65–80% of the lymphocytes in uterine decidual tissues, and in contrast to peripheral blood populations, uterine NK cells are mainly of the CD56bright CD16dim subtype (5). NK cell activity is regulated by killer cell immunoglobulin-like receptors (KIR). KIR-2DL1, as an inhibitor, can specifically identify and combine with HLA-Cw molecules spread on the surface of fetal trophoblastic cells to transmit inhibitory signals to maintain maternal-fetal immune tolerance (6).

In this study, we aimed to analyze the correlation between URSA and CD4+CD25+Treg and KIR-2DL1 expression levels to identify new potential targets for clinical treatment.

Patients and methods

Patient information

We sequentially selected 76 patients who were diagnosed with URSA at Xiangyang Central Hospital between June 2015 and October 2016. Of these, 35 patients were not pregnant (Group A) and 41 patients suffered early abortion (Group B). We additionally selected 30 patients who received a regular abortion as planned (Group C) and 30 healthy volunteers (Group D). Diagnostic criteria for URSA were as follows: i) patients experiencing spontaneous abortion at least twice; ii) patients with no explicit etiological factors (e.g., abnormal chromosomes, reproductive tract diseases, microbiological infections, endocrine disorders, menstrual disorders, positive test results for antinuclear and anticardiolipin antibodies, negative test results for blocking antibodies, medications, trauma and exercise); and iii) patients whose partners had no semen abnormalities. Written informed consent was obtained from all the subjects.

The average age of Group A was 25.6±6.3 years, the average number of abortions was 2.5±0.4 times, and the average gestational age at abortion was 7.3±1.2 weeks. The average age of Group B was 24.3±5.7 years, the average number of abortions was 2.4±0.5 times, and the average gestational age at abortion was 7.6±0.8 weeks. The average age of Group C was 23.9±5.5 years and the average gestational age at abortion was 6.9±1.6 weeks. The average age of Group D was 23.5±5.9 years. No significant differences in these parameters were observed among groups (P>0.05).

Observation indices

Peripheral venous blood and fresh decidual tissues were obtained from patients to detect CD4+CD25+Treg and Foxp3 transcription factor levels via flow cytometry. KIR-2DL1 mRNA and protein levels were assayed via quantitative PCR and western blot analysis, respectively.

Flow cytometry

Fasting venous blood (5 ml) was collected and subjected to gradient density centrifugation to isolate peripheral blood mononuclear cells. Fresh decidual tissues were also collected and converted into cellular suspensions. Mouse anti-human CD4-FITC (5 µl) and CD 25-PE (5 µl) monoclonal antibodies (Beyotime Biotech Co., Ltd., Jiangsu, China) were added, respectively, to two tubes, which were incubated for 20 min at 37°C in the dark. Buffer (0.5 ml) containing a permeabilization reagent was then added and the tubes were incubated for an additional 30 µl in the dark at 4°C. The samples were then washed using PBS, centrifuged for 5 min at 1,500 × g, and then pelleted cells were resuspended after discarding of the supernatant. The cells were then rinsed using permeabilization buffer and incubated with 1 µl normal mouse serum and 50 µl permeabilization buffer for 15 min in the dark at 4°C. APC mouse IgG2a isotype antibody (10 µl; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) was added to one tube as a control, while 10 µl APC-anti-human Foxp3 antibody (Sigma-Aldrich, St. Louis, MO, USA) was added into the other tube. Both tubes were incubated for 30 min at 4°C in the dark, after which the cells in the two tubes were washed using permeabilization buffer. Cells were finally suspended in PBS for 1 h and detected using a FACS Caliber flow cytometer (BD Biosciences, New Jersey, NY, USA).

qPCR

Regular TRIzol reagent was used to extract total cellular RNA; total concentration and purity were determined via ultraviolet spectrometer and cDNA synthesis was performed using a reverse transcription kit. Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) in accordance with GeneBank sequences: KIR-2DL1 forward, 5′-GCAGCACCATGTCGCTCT-3′ and reverse, 5′-GTCACTGGGAGCTGACAC-3′, 356 bp; GAPDH forward, 5′-CGCGAGAAGATGACCCAGAT-3′ and reverse, 5′-GCACTGTGTTGGCGTACAGG-3′, 226 bp. SYBR-Green I was used to detect cDNA amplification using a Prism 7500 Sequence Detector Fluorescence Quantitative PCR Amplifier (both from ABI, Vernon, CA, USA). Reaction system components were as follows: cDNA 3.0 µl + upstream primer 1.0 µl + downstream primer 1.0 µl + 2X SYBR-Green I Mix 5.0 µl + ddH2O2 10.0 µl. Reaction conditions were as follows: 95°C for 5 min, 30 cycles of 95°C for 30 sec, 58°C for 30 sec, and 72°C for 60 sec, and a final 10 min step at 72°C. PCR products were identified using 2% agarose gel electrophoresis, ultraviolet imaging was performed using a gel imaging analysis system, and gray value analysis was carried out via digital photos. The detection procedures were repeated three times and the results were presented via the 2−ΔΔCq method.

Western blot analysis

Radio immunoprecipitation assay (RIPA kit from R&D Systems, Minneapolis, MN, USA) lysis buffer was added to samples in order to extract total cellular protein, with a preliminary quantitative assay performed via the Coomassie Brilliant Blue method (kit from Bio-Rad Laboratories, Hercules, CA, USA). Total protein (30 µg) was subjected to 8% SDS-PAGE to separate proteins, which were then electrically transferred onto polyvinylidene fluoride membranes (4°C, 100 V, 50 min). The membranes were blocked using 5% skimmed milk at room temperature for 2 h. Mouse anti-human KIR-2DL1 monoclonal antibody (dilution, 1:500; cat. no. sc-53595; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was added onto the membrane, which was incubated with constant agitation at 4°C overnight. The membrane was then washed using Tris-buffered saline and Tween-20 (TBST) and incubated with horseradish peroxidase-labeled rabbit anti-mouse IgG poly-clonal secondary antibody (dilution, 1:2,000; cat. no. A11008; Invitrogen, Carlsbad, CA, USA) for 4 h with constant agitation at 37°C. Following this, the membranes were washed again using TBST and treated with ECL for protein visualization. Results were scanned and preserved, with semi-quantitative analysis performed using Lab Works 4.5 gel imaging software (Media Cybernetics, Rockville, MD, USA). Results were expressed as integral optical density units. Experiments were carried out three times in order to determine the mean.

Statistical analysis

SPSS 20.0 software (IBM SPSS, Armonk, NY, USA) was used for statistical analysis. Measurement data were presented as mean ± standard deviation. Single-factor analysis of variance was used for intergroup comparisons, the least significant difference-test was used for paired comparisons, and the independent t-test was employed for comparisons between the two groups. P<0.05 was used to indicate statistical significance.

Results

CD4+CD25+Treg and Foxp3 levels

We found no statistically significant differences between groups in terms of peripheral venous blood CD4+CD25+Treg/CD4+ and Foxp3+/CD4+CD25+Treg percentage (P>0.05). Group C decidual tissues showed elevated levels of both indices compared to the other groups (P<0.05), but no statistically significant differences were found between Groups A, B, and D (P>0.05; Table I).

Table I. CD4+CD25+Treg and Foxp3 levels (%).

Table I.

CD4+CD25+Treg and Foxp3 levels (%).

Variables	Group A	Group B	Group C	Group D
Peripheral blood
CD4+CD25+Treg	4.77±1.22	4.86±1.32	5.62±1.54	5.33±1.46
Foxp3	19.85±8.63	21.52±8.52	25.69±7.95	20.34±7.84
Decidual tissues
CD4+CD25+Treg	8.65±2.52	12.67±6.49	24.52±12.32	10.34±5.87
Foxp3	35.62±16.24	52.95±25.85	89.69±35.57	46.32±24.64

[i] Treg, regulatory T cell.

KIR-2DL1 mRNA and protein expression levels

Peripheral venous blood KIR-2DL1 mRNA and protein expression levels in Group C were significantly higher than in the other three groups (P<0.05), but there were no statistically significant differences between Groups A, B, and D (P>0.05). In decidual tissues, Group C levels were significantly higher in Groups A, B, and D (P<0.05; Table II).

Table II. KIR-2DL1 mRNA and protein expression levels.

Table II.

KIR-2DL1 mRNA and protein expression levels.

Variables	Group A	Group B	Group C	Group D
Peripheral blood
mRNA	0.0869±0.0124	0.1127±0.0564	0.2451±0.1324	0.0958±0.0232
Protein	0.07±0.02	0.09±0.03	0.22±0.11	0.07±0.02
Decidual tissues
mRNA	0.1765±0.0856	0.2546±0.1025	0.4659±0.1654	0.1854±0.0965
Protein	0.17±0.08	0.23±0.09	0.44±0.21	0.16±0.05

[i] KIR, killer cell immunoglobulin-like receptor.

Discussion

Research has suggested that CD4+CD25+Treg cells play an extremely important role in cellular immune function. During immunological rejection of organ transplantation, there is a decrease in the amount of CD4+CD25+Treg cells and CD4+CD25+ T cells are activated to generate cytotoxicity, causing immune injuries and assisting B-cell involvement in the antigen-antibody combination reaction (7). After T-cell receptor stimulation and activation, Tregs can suppress the transcription and expression of the IL-2 gene in CD4+ and CD8+ cells in order to inhibit the proliferation and activation of T cells, thus regulating immune tolerance. Foxp3, a characteristic molecule expressed by CD4+CD25+Tregs, can regulate the development and maintain the function of cells. Foxp3 is highly expressed in CD4+CD25high, lowly expressed in CD4+CD25low, and not expressed in CD4+CD25− cells (8). A study by Khattri et al revealed that the elimination of Foxp3+CD4+CD25+Treg cells in the mouse using a specific antibody can cause severe autoimmune responses (9). Aluvihare et al found that Foxp3 expression levels in the uteruses of intrabreeding BALB/c pregnant mice and interbreeding BALB/c and C57BL/6 pregnant mice were 1,000-fold greater than those in non-pregnant mice of the same age (10). However, no statistically significant differences were found in Foxp3 expression level in uteruses between intrabreeding BALB/c pregnant mice and interbreeding BALB/c and C57BL/6 pregnant mice (10). This result suggested that the increase in Foxp3 expression was caused by pregnancy but not correlated with homoantigen stimulation. In the present study, we found no differences in the proportion of peripheral venous blood CD4+CD25+Treg/CD4+ and Foxp3+/CD4+CD25+Treg cells. We also found that Group C decidual tissues showed significantly higher levels of both indices, but no statistically significant differences were found among Groups A, B, and D. This finding suggested that decreased CD4+CD25+Treg levels and Foxp3 expression were closely correlated with the onset of URSA, and CD4+CD25+Treg-mediated immune tolerance was mainly generated in local decidual tissues. However, this result did not coincide with results found in a previous study (11). Local immune regulation in decidual tissues lay the foundation for immune identification and responses between the matrix and the embryo. Formation, uterine settlement, nutrition, and embryo development can significantly affect the local decidual tissues and induce increased levels of CD4+CD25+Treg cells. These, however, seldomly enter the peripheral circulation, where there are insufficient corresponding antigens or receptors to interact with CD4+CD25+Treg (12).

KIR-2DL1, a transmembrane glycoprotein, can specifically identify the 77th amino acid, aspartic acid, and 80th amino acid, lysine, in the HLA-Cw molecule. Immunoreceptor tyrosine-based inhibitory motifs inside the phosphorylated structure can transmit negative regulation signals to inhibit the killing activity of NK cells (13). Ntrivalas et al (14) and Yamada et al (15) have confirmed that in women with recurrent spontaneous abortion, peripheral blood NK expression of KIR-2DL1 was significantly lower than in healthy women. In this study, we found that peripheral venous blood KIR-2DL1 mRNA and protein expression levels in Group C were significantly higher than those in the other three groups, but there were no statistically significant differences among Groups A, B, and D. In decidual tissues, Group C expression levels were significantly higher than those in Groups A, B, and D. This result suggested that a decreased expression of KIR-2DL1 was closely correlated with the onset of URSA, and can occur in both peripheral blood and decidual tissues. NK cells can simultaneously mediate cellular and humoral immunity. In addition to high expression on the surface of fetal trophoblast cells, HLA-Cw is also expressed on the surface of T and B cells (16). In conclusion, CD4+CD25+Treg and KIR-2DL1 may serve as new targets for the early intervention of URSA.

Acknowledgements

The present study was supported by the Natural Science Foundation of Hubei Province of China (grant no. 2014CFB251).

References