The two-way communication between the mother and the fetus is accomplished by immune cells. CD8+ T cells of normal pregnant (NP) women express progesterone receptor (PR). Binding of PR to progesterone (P) and the production of progesterone-induced blocking factor (PIBF) can aid immune escape, which is an important factor in the maternal immune response. We detected the proportion of CD8+ T cells and the expression of the surface costimulatory molecules BTLA, TIGIT, ICOS, and PD-1 in peripheral blood and decidual tissues of women with unexplained recurrent spontaneous abortion (URSA) and in NP women. All patients were at 8 -10 weeks of gestation. The results showed that there was no change in the proportions of CD8+ T cells in peripheral blood and decidual tissues of URSA patients compared to those of NP women. In peripheral blood, compared with the NP group, the URSA group showed decreased expression of BTLA + CD8+ T cells and the difference was statistically significant, but there was no difference between the groups in terms of TIGIT + CD8+, PD-1 + CD8+, and ICOS + CD8+ T cells. There was no change in the levels of TIGIT + CD8+, PD-1 + CD8+, ICOS + CD8+, and BTLA + CD8+ T cells in decidual tissue. These data confirm that the number of CD8+ T cells in peripheral blood and decidual tissue is not the main factor leading to the pathogenesis of URSA, and other immune cells may play an important role in URSA, but this hypothesis needs further exploration and research.
Recurrent spontaneous abortion (RSA) is an increasingly serious worldwide problem, and its incidence is increasing year by year. Statistics show that 1–5% of all women are affected by RSA, which is usually defined as three or more miscarriages before 20 weeks of pregnancy [1]. An increasing number of people have reached a consensus on the definition of RSA as the occurrence of two or more spontaneous abortions [2]. There are many causes of RSA, including uterine malformations, parental chromosome abnormalities, endocrine abnormalities, thrombosis, and immune disorders [3]. Unfortunately, in 50% of cases, the etiology of RSA is still unknown, and this entity is known as unexplained RSA (URSA) [2, 4–6]. However, an increasing number of studies have found that the immune cell function of patients with URSA is abnormal [7–9].
Normal pregnant CD8+ T cells express progesterone receptor (PR). When the level of progesterone (P) is sufficient, it binds to PR to induce the production of a hydrophilic protein, that is, progesterone-induced blocking factor (PIBF), which provides the conditions necessary for embryo implantation into the endometrium [10, 11]. Therefore, CD8+ T cells play a very important role in maintaining normal pregnancy. Some scholars have found that the endometrial T cell coreceptor CD8 and tissue-resident marker CD69 are significantly decreased in women with URSA compared with normal pregnant women [12]. Shihua Baoa found that there was no change in the proportion of CD3 + CD8+ T cells in the peripheral blood of women with recurrent pregnancy loss (RPL) and premature delivery [13].
After T cell receptor (TCR) antigen recognition, T cells can activate, secrete cytokines and express cytokine receptors in the presence of a second signal (costimulatory molecule) [14]. CD8+ T cells express many costimulatory molecules. Costimulatory molecules can induce positive and negative actions according to their functions. Costimulatory molecules can promote or terminate T cell activation through only the binding of receptors and ligands [15]. TIGIT, BTLA, and PD-1 are T cell surface receptors that induce a negative costimulatory effect and can thus inhibit the activation and proliferation of T cells after binding to the corresponding ligands [16–18]. ICOS is an inducible costimulatory molecule that can promote the proliferation and activation of T cells [19].
It is controversial whether there is a correlation between URSA and the number of CD8+ T cells, and the expression of costimulatory molecules has not been studied. In this study, the proportion of CD8+ T cells and the expression of the costimulatory molecules ICOS, BTLA, PD-1, and TIGIT in peripheral blood and decidual tissues of URSA patients and NP women were studied to provide a theoretical basis for the clinical diagnosis and treatment of URSA.
Materials and methods
Patients
All patients in this study presented with pregnancy losses from 8 to 10 weeks. All cases were selected from the Department of Family Planning and the Department of Reproductive Medicine in the Second Affiliated Hospital of Soochow University. Healthy early pregnancies that were artificially terminated for nonmedical reasons were eligible to be in the normal pregnancy (NP) group. In this study, termination of pregnancy in all NP and URSA groups occurred between 8 and 10 gestational weeks.
All URSA patients were admitted to the study if they met all of the following requirements: (1) The subjects had a history of two or more spontaneous abortions before the 20th week of gestation. (2) All the women had regular menstruation. (3) The women had undergone examinations using ultrasound, hysteroscopy, and conventional blood analysis and had been diagnosed with URSA. Patients with uterus-related abnormalities, endocrine disorders, infectious diseases, chromosomal abnormalities, or autoimmune diseases were excluded. The exclusion criteria for all women in the NP group were as follows: (1) uterine malformation by pelvic examination and ultrasound; (2) chromosome abnormalities; (3) endocrine or metabolic diseases; (4) infectious diseases; and (5) other known causes.
This study was approved by the ethics review board of the Second Affiliated Hospital of Soochow University, and informed consent was obtained after the study had been explained in detail to all study participants (No.JD-LK-2018-030-03).
Flow cytometry
We collected 3 ml of peripheral blood per person from NP patients and patient with URSA, stored it in EDTA anticoagulant tubes, centrifuged it at 1500 rpm for 5 min, and removed the plasma supernatant. We mixed equal volumes of PBS and the remaining blood, transferred 50 µl of each sample to a centrifuge tube, and added the designated antibodies to each tube: FITC anti-human CD3, PE/Cy7 anti-human CD8, PE anti-human CD279 (PD-1), PE anti-human TIGIT, PE anti-human BTLA, and PE anti-human ICOS (BioLegend, USA). The samples were incubated at room temperature for 30 min, combined with 1 ml of PBS per tube, and centrifuged at 1500 rpm for 5 min. The supernatant was removed, mixed with 150 µl of Optilyse C Lysing Solution (Beckman, USA), and placed at room temperature for 5 min. Next, 1 ml of PBS was added to each sample, and all specimens were centrifuged at 1500 rpm for 5 min. The supernatant was removed and combined with 300 µl of PBS; flow cytometry was then used to acquire the data, and FlowJo 7.6 was used for the analysis.
The decidual tissue was placed in PBS to clear blood clots and cut into fragments of 0.5 × 0.5 mm2 with stainless steel ophthalmic scissors. DMEM/F12 medium (GIBCO, USA) containing 1 mg/ml type IV collagenase (Sigma, Japan) and 150 U/ml DNase I (Sigma, Japan) was added to the fragments, which were allowed to digest at 37 °C for 30 min and centrifuged at 1600 rpm for 5 min. PBS was added, and the samples were centrifuged twice, filtered with a nylon membrane, and mixed with the same quantity of PBS. Next, we placed 50 µl of each sample in a separate tube, added antibodies (FITC anti-human CD45, APC anti-human CD3, PE/Cy7 anti-human CD8, PE anti-human CD279 (PD-1), PE anti-human TIGIT, PE anti-human BTLA, and PE anti-human ICOS, (Biolegend, USA)) and incubated the samples at room temperature away from light for 30 min. We then added 1 ml of PBS per sample and centrifuged all tubes at 1500 rpm for 5 min. After centrifugation, the supernatant was removed, and 300 µl of PBS was added to each tube immediately for flow cytometry analysis.
Statistical analysis
We tested for statistically significant differences using GraphPad Prism 8.3 software. For measurement data that followed a normal distribution, pairwise comparisons were performed with an independent samples t-test. For data that did not follow a normal distribution, we used SPSS v24.0 software to logarithmically transform the data; if the data still did not follow a normal distribution, we used the Wilcoxon rank-sum test. Differences were considered statistically significant at P < 0.05.
Results
The proportion of CD8+ T cells and the expression of their costimulatory molecules TIGIT, ICOS, BTLA, and PD-1 in peripheral blood
In peripheral blood (Figure 1A and B), compared with the NP group, the URSA group had a slightly decreased proportion of CD8+ T cells, but the difference was not statistically significant (P > 0.05) (Figure 2A). There was no change in the expression of the costimulatory molecules ICOS, PD-1, and TIGIT (P > 0.05) (Figure 2B,C,D), but the expression of BTLA was decreased significantly in the URSA group compared with the NP group, and the difference was statistically significant (P < 0.01) (Figure E).
The proportion of CD8+ T cells and the expression of their costimulatory molecules TIGIT, ICOS, BTLA, and PD-1 in decidual tissue
In decidual tissue (Figure 1C and D), compared with the NP group, the URSA group had a slightly decreased proportion of CD8+ T cells, but the difference was not statistically significant (P > 0.05) (Figure 3A). The expression of the costimulatory molecules ICOS, PD-1, and BTLA increased slightly (P > 0.05) (Figure 3B, D, E) while that of TIGIT decreased slightly in the URSA group compared with the NP group, but the difference was not statistically significant (P > 0.05) (Figure 3C).
Discussion
The reproduction of mammalian species is a complex phenomenon and involves the dynamic interaction between molecules and cells of the mother and fetus. The fetus is like a semi-allograft, [2]. However, fetal development is not impaired. To some extent, pregnancy preservation involves improving immune tolerance throughout pregnancy. At present, several mechanisms of immune tolerance related to pregnancy have been proposed. The complex events of maternal and fetal tolerance involve T cells that respond to the father's antigens [20, 21]. At present, the specificity of paternal antigens has not been well determined, but the determination of pregnancy failure is related to T helper lymphocytes [22]. The balance of Th1/Th2 cytokines is related to pregnancy outcome. PIBF in pregnant women promoted the synthesis of Th2 cytokines, a decrease in Th1 cytokines and a decrease in the Th1/Th2 cytokine ratio [23]. The production of PIBF is related to progesterone and PR. When there is sufficient P, it combines with PR on the surface of γ δ T and CD8+ T cells to induce the production of a hydrophilic protein, that is, PIBF [24], which can aid immune escape. Therefore, abnormal CD8+ T cells may affect the expression of PR and PIBF, which can further lead to an imbalance in the Th1/Th2 cytokine ratio and pregnancy failure. Clarket al. found that CD3 + CD8+ T cells play an important role in the process of implantation. These cells secrete colony stimulating factor (CSF), which is necessary for implantation [25]. Yang et al. found that CD3 + CD8+ T cells can also inhibit B cell-mediated humoral immunity, CD3 + CD4 + -mediated delayed hypersensitivity and proliferation to prevent embryo rejection [26].
Figure 2.
The proportion of CD8+ T cells and the expression of their costimulatory molecules TIGIT, ICOS, BTLA, and PD-1 as determined by flow cytometry in peripheral blood of URSA patients and NP women. NP group, n = 30. URSA group, n = 32. NS = no significance, **P = 0.01. The data are expressed as the mean ± SD. Logarithmic transformation was used to obtain normally distributed data, and the variance was homogeneous.
Many scholars have found that CD8+ T cells are associated with pregnancy failure. Psarra et al. found that, compared with an NP group, the URSA group had a slightly increased percentage of CD3 + CD4+ cells and a slightly decreased percentage of CD3 + CD8+ lymphocytes, but the difference was not statistically significant [27]. Southcombe et al. found that the expression of the T cell coreceptor CD8 in the endometrium was decreased compared with that in normal tissue [12]. Du et al. found that there was no change in the proportion of CD3 + CD8+ T cells in patients with preterm labor and PRL compared with NP women [13]. Therefore, it is uncertain whether URSA is related to CD8+ T cells. In this study, compared with the NP group, the URSA group had a slightly decreased proportion of CD8+ T cells in peripheral blood and decidual tissue, but there was no significant difference.
There are many costimulatory molecules on the surface of CD8+ T cells that are involved in cell proliferation and activation. Although many scholars have studied the relationship between costimulatory molecules and URSA, there has been no study on the correlation between costimulatory molecule receptors on the surface of CD8+ T cells and URSA. Many scholars have found that the expression of OX40 in the peripheral blood of women with URSA is increased and have considered this increase a risk factor leading to URSA [28]; some scholars have also found expression of TIM3 and CTLA4 [29] in the peripheral blood and placental tissue of women with URSA. TIM3 is considered an index for immune evaluation in URSA [30]. However, whether URSA is related to the proportions of OX40 + CD8+, TIM3 + CD8+, and CTLA4 + CD8+ T cells has not been studied. In this study, CD8+ T cells and their costimulatory molecules TIGIT [15], PD-1 [17], ICOS, and BTLA [31] in peripheral blood and decidual tissue were detected in NP and URSA groups from 8 to 10 weeks of gestation. In peripheral blood, it was found that compared with the NP group, the URSA group had decreased levels of BTLA+CD8+ T cells, but the proportions of TIGIT+CD8+, PD-1 + CD8+, and ICOS+CD8+ T cells did not change. In decidual tissues from women with URSA, the proportion of TIGIT+CD8+ T cells increased slightly, while the proportions of PD-1 + CD8+, ICOS+CD8+, and BTLA+CD8+ T cells decreased slightly, but the differences were not significant. Although there were slight changes in costimulatory molecules, the proportion of CD8+ T cells in patients with URSA did not change much, likely because CD8+ T cells express many positive and negative costimulatory molecules that interact with the studied costimulatory molecules to maintain CD8+ T cells.
Figure 3.
The proportion of CD8+ T cells and the expression of their costimulatory molecules TIGIT, ICOS, BTLA, and PD-1 as determined by flow cytometry in decidual tissue of URSA patients and NP women NP group n = 11, URSA group n = 15. NS = no significance. The data are expressed as the mean ± SD.
In conclusion, the evidence we provide suggests that URSA may have little correlation with the proportion of CD8+ T cells in peripheral blood and decidual tissue. The number of BTLA+CD8+ T cells in peripheral blood from patients with URSA was decreased slightly compared with that in NP women, and in decidual tissues, the numbers of PD-1 + CD8+, ICOS+CD8+, and BTLA+CD8+ T cells were decreased slightly and the number of TIGIT+CD8+ T cells was increased slightly in the URSA group compared with the NP group. However, these differences had little effect on the proportion of CD8+ T cells. There are many costimulatory molecules on the surface of CD8+ T cells that are constantly changing. In this study, only four kinds of costimulatory molecules were studied, and other costimulatory molecules might interact with the studied molecules in various ways to maintain the proportion of CD8+ T cells. This study provides a theoretical basis for the clinical diagnosis and treatment of URSA. However, this work still has many shortcomings, such as the small sample size and incomplete detection indexes. In future research, the sample size should be increased and additional immune regulatory molecules should be studied to further provide a theoretical basis for the diagnosis and treatment of URSA.
Conflict of interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
