Document Type : Original Article
Authors
1 Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Obstetrics and Gynecology, Al-Zahra Hospital, Isfahan University of Medical Sciences, Isfahan, Iran
3 School of Public Health, Isfahan University of Medical Sciences, Isfahan, Iran
4 4Isfahan University of Medical Sciences, Isfahan, Iran
Abstract
Keywords
The combination of numerous factors result in
a successful pregnancy, of which cells and molecules of the immune system may be the most important. Disturbances in regulating these cells and
molecules can result in an aberrant pregnancy such
as recurrent spontaneous abortion (RSA). RSA is
a common problem among couples and is defined
as the occurrence of three or more clinically detectable pregnancy losses that usually occur prior
to 20 weeks of gestation (
Immunological dysfunctions may cause impaired
maternal immune tolerance to the fetus and result
in fetal rejection. Aberrant expression of human
leukocyte antigen (HLA), autoimmune diseases
and autoantibodies, T helper1/T helper2 (Th1/Th2)
imbalance, in addition to varied functions of uterine natural killer cells are examples of immunological dysfunctions (
CD4+
CD25+
FOXP3+
Treg cells are one of the
best-characterized subsets of immune regulatory
cells. The two forms of these cells are natural Treg
cells which are formed in the thymus and inducible Treg cells which are formed in the periphery
during antigen-specific stimulation (
Human CD8+
Treg cells have been studied in
less detail. Regulatory properties of these cells are
similar to CD4+
FOXP3+
Treg cells (
Th17 cells have been described as a subset of Th
cells (CD4+) which play a major role in induction
of inflammation by producing proinflammatory
cytokines such as IL-17A, IL-17F, IL-22, IL-6,
TNF-α, and matrix metalloproteinase. Recent data
have shown a pathogenic effect of these cells in
autoimmunity, transplant rejection and other diseases (
To the best of our knowledge there are few studies on the role of Th17 and Treg cells in unexplained RSA (URSA). This study evaluates and compares the percentage and ratio of Th17 and Treg cells in peripheral blood of women with URSA to healthy (proven fertile) non-pregnant women. We have studied the frequency and ratio of Th17/Treg cells in URSA women who were at least three-months after their last abortion. We propose that URSA may be the result of an irregularity in these cells and their balance before embryo implantation or pregnancy.
In this case control study, a total of 25 URSA women with a mean age of 29.45 years (range: 21- 43 years) who had at least three consecutive first trimester abortions were enrolled. The diagnosis of URSA was made after excluding any definite caus- es such as abnormalities of the uterus or cervix, chromosomal abnormality, infection, endocrine and metabolic diseases, congenital thrombophilias and autoimmune disease. All male partners had normal semen status, according to criteria from the World Health Organization. The control group comprised 35 non-pregnant healthy women with a mean age of 30.5 years (range: 22-42 years) who had at least one successful pregnancy without any disease. Control group women had no history of any still birth, preterm and post-term labor, ectopic pregnancy, preeclampsia and abnormal pregnancy. All participants were at least three months from their last abortion or pregnancy. Blood samples were taken from both groups. After sampling, subjects were assigned to either of two groups, secretory or proliferative phase. These subjects had regular menstrual cycles of 26-31 days. Women in the last 14 days of their menstrual cycles comprised the secretory group, whereas those prior to the last 14 days of their menstrual cycles were considered to be the proliferative group. Women with menses were removed from the study. Clinical characteristics of URSA and control group women are summarized in table 1.
Clinical characteristics of subjects
Clinical characteristics | URSA | Normal |
---|---|---|
Age (Y) | 29.9 ± 5.7 | 30.3 ± 5.8 |
Proliferative phase (Day of menstrual cycle) | 10.0 ± 1.2 (n=13) | 10.8 ± 2.1(n=15) |
Secretory phase (Day of menstrual cycle) | 20.7 ± 4.7 (n=12) | 21.1 ± 3.4 (n=20) |
Number of abortions | 3.7 ± 1.2 | 0.0 ± 0.0 |
Results were expressed as the mean ± SD.
N; Number of subjects in the phases and URSA; Unexplained recurrent spontaneous abortion.
A total of 8 ml heparinized venous blood and 2 ml without anticoagulant were taken for the ELISA test from the case and control groups. Sera were separated using centrifugation and stored at -80˚C until use.
Peripheral blood mononuclear cells (PBMC) were separated from freshly isolated heparinized venous blood by centrifugation on a Ficoll-Hypaque (Lymphoprep, Sigma, USA) density gradient. Cells at the interface were harvested, washed twice, and resuspended in phosphate buffered saline (PBS). Viable cells were counted by the trypan blue staining method. More than 95% of the cells were viable.
For analysis of Th17 cells, PBMC (2×106 cells/ml) were suspended in complete culture
medium that contained RPMI 1640 with L-glutamine, penicillin (100 U/ml), streptomycin (10 mg/ml) and 10% fetal bovine serum
(FBS). This cell suspension (2×106
cells/ml)
was transferred to the 24-well plates. Phorbol
12-myristate 13-acetate (PMA, 150 ng/ml)
and ionomycin (1 μM) were added to each
well in the presence of monensin (500 ng/
ml) for 12 hours (all purchased from Sigma,
USA). Then, cells were incubated at 37˚C in
a humidified 5% CO2
atmosphere. After 12
hours, cells were washed with PBS at 1500
rpm for 5 minutes. After stimulation of PBMC
For CD4+ Treg staining cell, PBMC (2×106 cells/ml) were incubated with PerCP anti-human CD4. In order to stain CD8+ Treg cells, PBMC were incubated with FITC anti-human CD8. After surface staining, the cells were fixed and permeabilized with BD Cytofix/ Cytoperm solution and stained with PE antihuman FOXP3.
Isotype controls were given to enable correct compensation and antibody specificity onfirmation. All antibodies were purchased from BD Pharmingen, USA. As seen in figure 1, the percentages of Th17 or Treg cells were determined using a flow cytometer (Partec, Germany). A total of 50000 cells were analyzed for detection of Th17 and Treg cells. We used Flowmax software for data analyses.
Representative flow cytometry dot plots of TH17 (CD4+ IL-17A+ ) and Treg (CD4+ FOXP3+ ) in unexplained recurrent spontaneous abortion (URSA) and healthy, normal women. Plots shown were gated on CD4+ lymphocytes. The percentage of cells falling into the respective quadrants is indicated in each plot.
*; Considered significant compared with the control group according to independent sample t test and p=0.001
The serum concentrations of IL-17A, TGF-β1 and IL-10 were measured using an ELISA kit in accordance with the manufacturer’s protocol (Boster Biological Technology Co., Wuhan, China). The ELISA kits had a sensitivity of 1 pg/ml for TGFβ and IL-17 and 0.5 pg/ ml for IL-10. All samples were measured in duplicate.
Analyses with Levene’s test for Treg cells and Th17 cells showed equal variances. Therefore the data distribution was normal. We used the independent sample t test to analyze the significance of difference in the mean percentage of Th17, Treg cells (both CD8+ and CD4+ ), and the Th17/CD4+ Treg ratio between the case and control groups. Levene’s test for cytokine analyses showed unequal variances (not normal distribution). Therefore we used non-parametric statistics. The Mann-Whitney U test was used to compare cytokines in the proliferative and secretory phases of URSA and with control group women. SPSS software was used.
The protocol for this study was approved by the Ethics Committee of Isfahan University of Medical Sciences (Isfahan, Iran). Informed consent was obtained from all subjects who participated in this study.
Table 2 shows that the percentage of Th17 cells (CD4+ IL17+ ) was significantly higher in women with URSA than healthy non-pregnant women. Results also revealed a higher percentage of TH17 cells in the proliferative and secretory phases of menstrual cycles in women with URSA compared to healthy non-pregnant women.
Percentages of CD4+ Treg, CD8+ Treg, and CD4+ Th17 cells in peripheral blood from unexplained recurrent spon- taneous abortion (URSA) and healthy, normal women in the proliferative and secretory phases of the menstrual cycle and without considering phase
Proliferative phase | Secretory phase | Without considering phase | |||||||
---|---|---|---|---|---|---|---|---|---|
Cell subsets | URSA | Normal | P value | URSA | Normal | P value | URSA | Normal | P value |
CD4+IL-17A+ | 1.6 ± 1.5* | 0.6 ± 0.4 | 0.004 | 2.0 ± 1.3* | 0.5 ± 0.4 | 0.001 | 1.8± 1.4* | 0.6 ± 0.4 | 0.001 |
CD4+FOXP3+ | 0.9 ± 1.1* | 2.3 ± 1.7 | 0.010 | 1.3 ± 1.2 | 1.4 ± 1.5 | 0.410 | 1.1±1.1* | 1.9 ± 1.7 | 0.030 |
CD8+FOXP3+ | 0.2 ± 0.1 | 0.2 ± 0.1 | 0.110 | 0.1 ± 0.1 | 0.3 ± 0.3 | 0.09 | 0.1± 0.1* | 0.3 ± 0.2 | 0.040 |
Results are expressed as mean ± SD. Values for cells are expressed as percentages.
*; Considered significant compared with the control group (p≤0.05).
Results showed a significantly lower percentage of
CD4+
FOXP3+
Treg cells in the URSA group compared
to the control group. However, the decreased percentage of Treg was detected in the proliferative phase but
not in the secretory phase (
Figure 2 shows a higher ratio of Th17/CD4+
Treg in the URSA group compared with the
control group. An increased ratio of Th17/CD4+
Treg cells was observed in the proliferative and
secretory phases of URSA patients (
TH17/CD4+ Treg ratio in RSA and normal women. *; Considered significant in comparison with control (used sta- tistical test is independent sample t test and p value=0.001).
TH17/CD4+ Treg ratio in proliferative and secretory phase in RSA and normalwomen.
*; Considered significant in comparison with control (the used statistical test is independent sample t test and p value in secretory phase=0.008 and in proliferative phase, p value=0.06).
According to table 3 the IL-17 concentrations
in sera of women with URSA were significantly
higher compared to the control group. However,
the levels of TGF-β1 and IL-10 in women with
URSA were significantly lower than those of the
control group. Results also revealed a higher IL-
17 concentration in the proliferative and secretory
phases of the menstrual cycle. TGF-β1 and IL-
10 concentrations were lower in the proliferative
phase in women with URSA compared to the con-
trol group (
Concentration of cytokines in serum from unexplained recurrent spontaneous abortion (URSA) and healthy, normal women in the proliferative and secretory phases of menstrual cycles
Proliferative phase | Secretory phase | Without considering phase | |||||||
---|---|---|---|---|---|---|---|---|---|
Cytokine (pg/ml) | URSA | Normal | P value | URSA | Normal | P value | URSA | Normal | P value |
IL-17A | 13.5 ± 1.2* | 12.4 ± 1.6 | 0.030 | 14.6 ± 1.8* | 12.4 ± 1.1 | 0.004 | 14.0 ± 1.5* | 12.4 ± 1.4 | 0.032 |
TGF-β1 | 20.4 ± 13.5* | 50.4 ± 8.9 | 0.002 | 55.6 ± 16.2 | 51.6 ± 22.1 | 0.090 | 36.6 ± 23.0* | 50.8 ± 7.0 | 0.002 |
IL-10 | 9.3 ± 2.3* | 33.0 ± 12.5 | 0.040 | 14.9 ± 8.7 | 15.9 ± 5.8 | 0.090 | 11.9 ± 6.6* | 26.2 ± 13.3 | 0.040 |
Results are expressed as mean ± SD.
*; Considered significant compared with the control group (p≤0.05).
In this study, we evaluated the percentage and
ratio of TH17 and Treg cells in peripheral blood
of women with URSA and compared them with
healthy (proven fertile) non-pregnant women. Results of the present study showed a significantly
lower percentage and number of Treg cells (CD4+
Foxp3+
and CD8+
FOXP3+) in the peripheral blood
of URSA women compared to the control group.
Sasaki et al. showed CD4+
CD25 cells in women
with spontaneous abortion were equal with nonpregnant women (
The results of the current study showed a low
level of CD4+
FOXP3+
Treg cells in peripheral
blood of URSA women in the proliferative or follicular phase of the menstrual cycle compared to the
same phase in non-pregnant women. However, no
significant change in the number of Treg cells was
observed in the secretory or luteal phase (
There are a few studies on the role of CD8+
FOXP3+
Treg cells in pregnancy (
Treg cells exert part of their function by producing immunoregulatory cytokines such as
TGF-β1 and IL-10 (
This study demonstrated that the percentage
and number of Th17 cells (CD4+
IL-17A+) af-
ter stimulation of PBMC
Studies have demonstrated the elevation of Th17
cells in acute tissue rejection (
A recently published study on JEG-3 human
choriocarcinoma cells demonstrated that IL-17
increased progesterone secretion by JEG-3 cells
(
Results of the present study also showed an increased ratio of Th17/CD4+
Treg cells in periph-
eral blood of URSA women compared to the control group. This finding was consistent with recent
studies that reported increased ratios of Th17/
CD4+
Treg in URSA women (
It seems reasonable to conclude that URSA and
infertility may result from failure of pre-implantation immune mechanisms. It has been shown that
elevation of Treg cells before implantation may be
caused by processing and presentation of paternal alloantigen that is present in seminal fluid by
dendritic cells in endometrial and cervical tissues
(
Th17 cells at their physiologic level may be necessary for successful implantation, whereas Treg cells prevent excessive Th17 response and inflammation. Therefore, any factor that causes irregularity between these cells and their balance may induce embryo rejection. Overall, the immunological homeostasis is likely disturbed in URSA women and consequently the regular processes in the menstrual cycle are disorganized. The causes of disturbed homeostasis are not clear. Further studies are needed to clarify the factors that can affect immunological homeostasis in URSA women.