Background
Recurrent spontaneous abortion (RSA) refers to 2 or more consecutive pregnancy losses [
1]. RSA is pervasive in clinical pathological pregnancy, but its pathogenesis is very complicated. Besides chromosome abnormalities, anatomic abnormality, endocrine dyscrasia, and infection, the etiology and pathogenesis of 40–60% RSA patients are not understood. RSA with unclear causes is called unexplained recurrent spontaneous abortion (URSA) [
2].
From the view of immunology, a normal pregnancy can be considered a type of successful HLA-haploidentical allogeneic transplant. In a normal pregnancy, the mother’s immune tolerance against the father’s alloantigens is very strong, and the embryo can be comparable to an allograft of semi-identical HLA [
3]. It has been reported that special Th2-cell - related phenomena occur in the mother in a normal pregnancy. The Th1 cells are suppressed, and the Th1-type cytokines (such as IL-2, IFN-γ, and TNF-α) are present at an extremely low level or may even be absent. Th2 cells are mainly assigned to mediate humoral immune response, stimulate B-cells to secrete antibodies, and maintain allograft immune tolerance. Th2-type cytokines contribute greatly to a normal pregnancy [
4]. A disequilibrium of the Th1/Th2 balance together with the over-functioning of Th1-cells leads to susceptibility to pathological pregnancy, including abnormal implantation, fetal growth retardation, fetal growth arrest, and URSA [
5,
6].
Tim-3, a subtype of the T-cell immunoglobulin domain and mucin domain (Tim) protein family, which was first identified in 2001 [
7], is selectively expressed on Th1 cells but not on Th2 cells and can induce the apoptosis of Th1 cells after specific binding with its main ligand galectin-9 [
8]. Popovici et al. found that the expression of galectin-9 was much higher in early decidual cells than in non-pregnant endometrium epithelial cells [
9]. Thus,it is likely that allograft tolerance weakens after the Tim/galectin-9 pathway is blocked, for the dismissal of Th1 cell suppression and the enhanced function of Th1 cells, including the upregulated expression of Th1 cytokines and Th1-type immune response overbalance. Inversely, activating the Tim-3/galectin-9 pathway would suppress the immune response of Th1 cells and benefit the immune tolerance of allografts [
5]. Moreover, Tim-3 expressed on natural killer (NK) cells was found to be elevated when they were activated, suppressing the NK-dependent immune response and strengthening immune tolerance [
10].
Tim-3 polymorphism has also been shown to change the interaction between Tim-3 and its ligand, thereby affecting the processes leading to some immune diseases [
11]. Given all these findings, Tim-3 might be an important regulatory molecule that plays a pivotal role in URSA, which might be triggered mostly by Th1/Th2 immune deviation. Whether anomalies in the activation of the Tim-3/gelatin-9 pathway, for example, change in the structure of Tim-3/gelatin-9, their abnormal expression, or mutual interaction lead to URSA, remains unknown.
Tim-3/Tim-3 polymorphisms and their association with URSA in Han Chinese women have not been studied thus far. Here, we examined polymorphisms of rs10053538/rs10515746 loci in the promoter of Tim-3 and their relevance to URSA in Han Chinese women.
Methods
The protocols of this research were approved by the Ethics Committee of Taizhou Hospital. Written informed consent was obtained from all patients engaged in this study at the start of the research. Because all the researchers of this study work for Taizhou Hospital, which is located in Taizhou, Zhejiang, a Han Chinese habitation in China, all subjects selected were Han Chinese women.
Patients and DNA samples
One hundred and forty-eight women with RSA resulting in still birth were enrolled from the Obstetrics and Gynecology Department of Taizhou Hospital to comprise the research group. In addition, 153 patients with normal pregnancy culminating in live birth were selected at random from the Obstetrics and Gynecology Department of Taizhou Hospital to form the control group. They had delivered at least 1 full-term healthy baby without the aid of assisted reproductive technologies and had not experienced miscarriage or pregnancy complications in the past. The abortions included both embryonic and anembryonic losses before 20 weeks of gestational age, which were determined by ultrasound dating and/or dating based on the last menstrual period. All subjects had undergone a comprehensive examination, including determination of detailed medical history, physical examination, beta subunit of human chorionic gonadotropin (BhCG), and transvaginal three-dimensional ultrasonography. They were also tested for 75 g oral glucose tolerance, thyroid functions, anti-cardiolipin antibodies (IgG/IgM), lupus anticoagulant, anti-thrombin III, protein S, and protein C to rule out glucose intolerance, endocrine dyscrasia, thrombophilia factors, urogenital tumor, and congenital reproductive system malformation. Patients with chromosome abnormalities were also excluded from the study. All patients included in this study were genetically unrelated Han Chinese women. The genomic DNA of white blood cells from every sample was extracted according to the recommended procedure of the DNA isolation kit (Generay, SH, China). The extracted genomic DNA was dissolved in sterile double disstilled water. The concentrations and A260/A280 ratios of the DNA solutions were measured using a nucleic acid spectrometer (Bio-Rad, CA, USA). DNA samples with A260/280 ratios ranging from 1.7 to 2.0 were chosen as polymerase chain reaction (PCR) templates and were preserved at -80°C.
rs10515746 genotyping
Allele-specific polymerase chain reaction (AS-PCR) was used to examine the rs10515746 locus, and 3 primers were designed according to the flanking sequence [GenBank: NM_032782]. The primer sequences were as follows: F1, 5′-GGCTTATGCTGGGAGTTGCT-3′; F2, 5′-GGCTTATGCTGGGAGTTGCG-3′; and R1, 5′-GGTGTCTGATTGCCAGTGATTC-3′. F1 and R1 were used to amplify T allele fragments, and F2 and R1 were used to amplify G allele fragments; all the amplified fragments were 499 bp long. Every sample was subjected to PCR with F1/R1 and F2/R1. The reaction mixture included 10× PCR buffer, 2.5 μL; dNTP (10 mM each), 0.5 μL; forward primer, 0.25 μL (20 mmol/L); reverse primer, 0.25 μL (20 mmol/L); DNA template, 0.1 μg; and Taq DNA polymerase, 2 U. The final volume of the mixture was made up to 25 μL with double distilled water. Touchdown PCR was used to amplify the target amplicons. The reaction was heated on a Thermo Cycler (Bio-rad, CA, USA) to 95°C for 2 min, followed by 94°C for 30 s, annealing temperature is progressively lowered from 62°C to 54°C by 1°C every 2 cycles, annealing for 30 s in every cycle, extension for 40 s, followed by 12 additional cycles at 54°C for 40 s, 28 cycles were conducted totally, final extension at 72°C for 10 min. The amplified products were subjected to 1.8% agarose gel electrophoresis, visualized by ethidium bromide (EB) staining, and photographed by FireReader (UVItec, CB, UK).
rs10053538 genotyping
Primers were designed according to the flanking sequence of rs10053538 provided by GenBank [GenBank: NM_032782]. Sequences of forward and reverse Tim-3 primers were as follows: 5′-ATGGTCATTTGCTGGTTATTAGTT-3′ and 5′-ATGTTGGTCAGGCTGTTCTT-3′. The PCR mix components were as follows: 10× PCR buffer, 2.5 μL; dNTP (10 mM each), 0.5 μL; forward primer, 0.25 μL (20 mmol/L); reverse primer, 0.25 μL (20 mmol/L); DNA template, 0.1 μg; and Taq DNA polymerase, 2 U. The final volume was made up to 25 μL with double distilled water. The reaction mixture was subjected to PCR in a thermal cycler (Bio-Rad) under the following conditions: 95°C for 2 min; followed by 35 cycles each of 94°C for 45 s, 59°C for 45 s, 72°C for 1 min; and a final extension step of 72°C for 10 min. Next, 5 μL of the amplified products was used for 1.5% agarose gel electrophoresis. The PCR products were visualized by ethidium bromide (EB) staining and photographed by FireReader. DNA digestion conditions were as follows: PCR products, 1 μg; 10× buffer solution, 2.0 μL; and restriction endonuclease BseLI, 2.5 U. The final volume was made up to 20 μL with double distilled water. PCR products were digested for 16 h at 55°C. The digestion products were subjected to 2.0% agarose gel electrophoresis, visualized by EB staining, and photographed by FireReader.
DNA sequencing
Some of the PCR products were randomly selected and then purified with a PCR purification kit. The purified products were sent to GeneCore BioTechnologies Co. Ltd., Shanghai for DNA sequencing.
Statistical analysis
Statistical analysis was performed using the SPSS 18.0 software package. The weight factors of both the wild-type homozygote GG and mutant-type homozygote TT were 2, while the weight factor of mutant heterozygote GT was 1. The allele frequencies (constituent ratio) of each group were calculated. G(T) allele frequency = (weight factor of homozygote × the number of homozygote GG(TT) + weight factor of homozygote × the number of heterozygote GT)/(2 × the number of patients being studied). The χ2 test was adopted to compare the allele frequencies of each group. Hardy-Weinberg equilibrium test was adopted to examine the demographic representation of the research group and control group. The relative risks were presented as the odds ratios (OR) and their 95% confidential intervals (CI). Significance was defined as P < 0.05.
Discussion
Tim-3, also known as hepatitis A virus cellular receptor 2 (HAVCR2), is a newly discovered surface molecule on T cells, with important immune regulatory functions. In humans,
Tim-3 is located on chromosome 5q33.2. Tim-3 comprises 301 amino acids, including elementary structural domains such as the signal peptide domain, immunoglobulin V (IgV) structural domain, mucin-like structural domain, transmembrane zone, and cytomere domain of phosphorylation site. In the IgV structural domain, there are 2 antiparallel β fragments and a metal ion ligand binding site (MILIBS), which together function as the ligand-binding site of Tim-3 [
12,
13].
In this study, rs10053538 and rs1055746 loci of
Tim-3 were studied. Statistical data showed that in both healthy women and URSA patients, the frequencies of mutant-type homozygote TT on both loci were low. Hence, the data of mutant-type homozygote TT and mutant-type heterozygote GT were merged and re-analyzed by χ
2 test. However, still, no significant statistical difference was found between the distribution frequencies of GT + TT genotype in either group (Table
1). The distribution frequencies of T allele on rs10053538/rs10515746 locus of each group were of no statistical difference either (see Table
2). All these data suggested that the polymorphisms of rs10053538/rs10515746 may not contribute to the URSA in Han Chinese women.
Tim-3 deficiency has been reported to precipitate the aggravation of diseases in which the Th1-type immune response prevails, increasing the magnitude of illness and mortality [
14]. Furthermore, the Th1/Th2-type cytokine balance leans toward a radical overbalance of Th1-type cytokine in URSA patients [
4]. Herein, it can be postulated that
Tim-3/Tim-3 polymorphisms might be closely related to URSA.
Meanwhile, researchers found that the distribution frequencies of +4259 T/G in
Tim-3 in patients with pancreatic cancer or renal cell carcinoma were statistically different from those of healthy people [
15,
16]. The distribution frequencies of +4259 T/G in
Tim-3 in rheumatoid arthritis patients have been shown to be statistically different compared with those of healthy people both in the Han Chinese and Hui Chinese [
17]. According to another report, in Korea, the distribution frequencies of +4259 T/G in
Tim-3 in children with dermatitis or asthma were not statistically different from those of healthy children, but this was not so in Caucasian or Hispanic children [
18].
Given these findings, it might be concluded that Tim-3/Tim-3 polymorphisms and their association with diseases not only pertain to the inclusion criteria of subjects but also to ethnic and regional differences.
Despite the current data don’t complying with the postulation, the polymorphisms of rs10053538/rs10515746 loci in Tim-3 and their relationship with URSA are still not that clear. This is because of several reasons. First, this study included as research subjects only Han Chinese women in Taizhou, a Han Chinese habitation. Second, not all patients in this study had a precise record of miscarriage history. The number of miscarriages was self-reported; therefore, there might have been errors in the precise number of miscarriages. Third, many studies have found that Tim-3 plays an important role in certain immune diseases. Therefore, the polymorphisms in Tim-3 and their association with URSA, such as the relationship between genotype and phenotype and their function in the molecular genetic mechanisms of URSA, warrant further investigation.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YS carried out the molecular genetic studies and drafted the manuscript. CW, DH, and BZ collected all samples and performed the clinical tests. CF, CY, LF, and HLV helped in carrying out the molecular genetic studies and performed the statistical analysis. MZ participated in the study design and coordination and helped draft the manuscript. All authors have read and approved the final manuscript.