Pathogenesis, diagnosis, and treatment of recurrent spontaneous abortion with immune type

Abstract

Recurrent spontaneous abortion (RSA), defined as three or more consecutive pregnancy losses before 20 weeks of gestation, is difficult to treat in the clinical setting. It affects 1%–5% of women of reproductive age. In the investigations of immunopathogenesis, diagnosis, and treatment of RSA since the late 1980s, it was found that RSA was associated with abnormal maternal local or systemic immune response. The pathogenesis of autoimmune RSA was mainly associated with antiphospholipid antibody (APA), while that of alloimmune RSA was due to the disturbance of maternofetal immunological tolerance. Systemic etiological screening process and diagnosis systems of RSA with immune type were developed, and anticardiolipin (ACL or ACA) + anti-β2-GP1 antibody combining multiple assays for effective diagnosis of RSA with autoimmune type was first established. According to the dynamic monitoring of clinical parameters before and during gestation, low-dose, short-course, and individual immunosuppressive therapy and lymphocyte immunotherapy for RSA with immune type were carried out. The outcomes of the offsprings of patients with RSA were followed up, and the safety and validity of the therapies were confirmed. The research achievement leads to great progress in the diagnosis and treatment of RSA in China.

Keywords

spontaneous abortion, recurrent / autoimmune / alloimmune / pathogenesis / diagnosis / immunotherapy

Introduction

Recurrent spontaneous abortion (RSA), defined as three or more consecutive pregnancy losses prior to the 20th week of gestation, occurs in 1%-5% of women of reproductive age. Epidemiological studies suggest that the risk of subsequent pregnancy loss is approximately 70%-80% in RSA patients. The etiology of RSA remains partly unknown, although chromosomal, anatomical, endocrinological, infectious, and autoimmunological abnormalities have been implicated. It has been proposed that unexplained recurrent spontaneous abortion (URSA) is largely associated with the failure of maternofetal immunological tolerance, and it usually occurs in the first trimester of pregnancy.

Since the late 1980s, our research team has been investigating the immunopathogenesis, diagnosis, and treatment of RSA. Systemic etiological screening process and diagnosis systems of RSA with immune type were developed, and clinical monitoring and evaluation parameters for immunotherapy were established. The outcomes of the offsprings of patients with RSA were followed up, and the safety and validity of the therapies were confirmed. The research achievement leads to great progress in the diagnosis and treatment of RSA in China.

Immunopathogenesis of RSA

According to the immunopathogenesis of the disease, RSA can be classified into autoimmune type and alloimmune type. We found that RSA is associated with abnormal maternal local or systemic immune response. The pathogenesis of autoimmune RSA is mainly associated with antiphospholipid antibody (APA), while that of alloimmune RSA, also called URSA, is due to the disturbance of maternal-fetal immunological tolerance.

Pathogenesis of autoimmune RSA

To study the pathogenesis of autoimmune RSA, we detected several autoantibodies in 245 RSA patients. It was shown that the total positive rate of autoantibody was 18.4%. Among them, the positive rate of APA, antinuclear antibody (ANA), and antiextractable nuclear antigen antibody (ENA) was 13.5%, 6.9%, and 2.9%, respectively [1]. Furthermore, we found that autoantibodies, such as APA, activated platelets and coagulation cascade, and vascular thrombosis were developed in decidua, which contributed to the pathogenesis of autoimmune RSA [2,3]. To further investigate the relationship between human leukocyte antigen (HLA)-DQ region gene polymorphism and RSA with anticardiolipin antibody (ACL), polymerase chain reaction-restrictive fragment length polymorphism (PCR-RFLP) was used to type HLA-DQA1 and HLA-DQB1 alleles in 30 cases of RSA with ACL and 90 women with a normal pregnancy history. Our data showed an association between ACL(+) RSA and HLA-DQB1*0303, which suggests that DQB1*0303 may be susceptible gene to autoimmune RSA. Meanwhile, we demonstrated that the linkage phenotype 677C→T+677TT/1298CC in methylenetetrahydrofolate reductase (MTHFR) increased RSA frequency and the protective phenotype 677CC/1298AA decreased RSA frequency among women with a history of RSA compared with controls, with a statistically significant difference. This suggested that MTHFR might be the key thrombophilia gene conferring susceptibility to RSA in Chinese women. Together, the results suggest the possible influence of both acquired and inherited thrombophilia factors on the etiology of RSA [4,5].

Pathogenesis of alloimmune RSA

To study the pathogenesis of alloimmune RSA, we focused on the roles of immune cells, such as T cells, natural killer (NK) cells, and macrophages in the immunopathogenesis of RSA. We found that T cell receptor (TCR) variable β (BV)-chain 2, -3, -6, and -7 were the four most common BV families in deciduas of pregnant women. The women with URSA showed higher frequency of BV15, -19, and -20 and lower frequency of BV4 and BV7 in comparison with controls, suggesting that the specific skewed usages of TCR-BV might be associated with the susceptibility to URSA [6]. We also found that A/G polymorphism in exon-1 of CTLA-4 was associated with the immunopathogenesis of RSA, and it conferred susceptibility to RSA in Chinese population [7,8]. CD4+CD25+ regulatory T (Treg) cells were recently described as a unique subpopulation of T cells, which is known to play a major role in preventing autoimmunity and tolerating allogeneic organ grafts. We demonstrated that the proportions of CD4+CD25+ Treg cells in both decidua and peripheral blood in URSA patients were statistically significantly lower than those in control women using CD4+CD25^bright, CD4+CD25+CD127dim/-, and Foxp3+ as the mark, respectively [9,10]. Our data suggested that decreased number of CD4+CD25+ Treg cells in URSA women may induce maternal lymphocyte activation to the fetal allograft. Meanwhile, allogeneic lymphocyte therapy could enhance the percentage of CD4+CD25+ Treg cells [11] and shift the balance of Th1/Th2 toward Th2 immunity [12,13] in peripheral blood. Furthermore, we investigated the capacity of CD4+CD25+ Treg cells to interfere with the activities of other immune cells, such as CD4+CD25- T cells, CD8+ T cells, NK cells, dendritic cells, and macrophages in URSA women. Our results indicated that human CD4+CD25+ Treg cells-mediated suppression of abovementioned immune cell functions was decreased in URSA patients. Recently, the new Th17 subpopulation of CD4+ effector T cells, distinct from the well-described Th1, Th2, and Treg cells, has been described, and its discovery has substantially advanced our understanding of T cell-mediated immunity. Our recent report first defined the occurrence of Th17 cells in URSA patients and in normal early pregnant women. It showed that Th17 cells were enriched in peripheral blood and decidua in URSA patients, and there was an inverse relationship between Th17 cells and CD4+CD25+ Treg cells, which suggested that these Th17 cells might contribute to the pathogenesis of URSA, and the balance between Th17 cells and Treg cells may be critical to pregnancy outcomes [14]. All of the above data suggested that lack of immunosuppression of T cells in decidua and peripheral blood was associated with URSA.

Abnormalities of the NK activity were observed in most patients with RSA. Our published data indicated that inhibitory killer immunoglobulin-like receptors (KIR) 2DL2 in RSA patients showed increased frequencies [15], and animal experiment showed that remnant NK cell activity in NOD/SCID mice may be beneficial to feto-maternal tolerance during pregnancy [16]. To further extend the understanding of the relationship between TLR3-involved cell signaling and dsRNA-induced embryo resorption, we found that the interaction between dsRNA and TLR3 may be involved in the mobilization of CD45+CD80+ and CD8_α+CD80+ cells, followed by the up-regulation of IL-2 and down-regulation of IL-10 expression at the feto-maternal interface, finally resulting in embryo rejection [17]. As human pregnancy is a complex process, placental development also depends on the function of secretory molecules produced by decidual macrophages (DMPhi). Our previous study showed that the increased expression of CD36 and the decreased expression of TSP1 were found on DMPhi of alloimmune RSA, and the expression of IL-10 in DMPhi of RSA patients was decreased significantly as compared with that of the control. When adding TSP1 into culture medium, there was no change in IFN-gamma expression, while increased IL-10 expression in DMPhi of RSA patients was observed. The results suggested that TSP-1 on DMPhi could influence IL-10 expression as Th2 cytokines and proved the roles of DMPhi in the pathogenesis of RSA [18].

In the study of immunogenetics of alloimmune RSA, our data suggested for the first time that alloimmune RSA is associated with the HLA-DQB1 coding region, and it is not associated with its upstream regulatory region. The DQB1*0604/0605, DQA1*01-DQB1*0604/0605, and QBP6.2-DQB1*0604/0605 haplotypes may confer susceptibility to URSA, while the DQB1*0501/0502 allele may protect women from alloimmune RSA [19].

Diagnosis of RSA with immune type

In 2000, we first developed a systemic etiological screening process, combining the knowledge of immunopathogenesis of RSA patients. RSA was classified into immune type and nonimmune type according to the common causes of RSA: chromosomal, anatomical, endocrinological, infectious, and coagulation pathologies. Nonimmune-type RSA can be subdivided into four categories, including chromosomal abnormality, anatomical abnormality, infectious abnormality, and endocrinological abnormality, while immune-type RSA can be subdivided into autoimmune RSA and alloimmune RSA on the basis of autoantibody detection.

The diagnostic criteria for immune RSA are as follows [1,20-22]: (1) Autoimmune RSA: presence of anti-β2 glycoprotein (GP) 1 or ACL on two or more occasions at least 6 weeks apart. (2) Alloimmune RSA: three or more consecutive spontaneous abortion, without a history of previous living birth or stillbirth; exclusion of chromosomal, anatomical, endocrinological, or infectious abnormalities; exclusion of autoimmune disease.

In the late 1980s, it was reported that some RSA patients with immune type had a positive test result for APA, while others had a negative test result for APA, and those with positive APA showed a reduction of fetal loss rate after immunosuppressive therapy. In view of the large fluctuation of APA in most patients, improving APA detection rate became the key to diagnosis and treatment of RSA with immune type. APA includes lupus anticoagulant (LA), ACL, and anti-β2-GP1. Among them, ACL seems to be the best mark of autoimmune RSA. However, anti-β2-GP1 has been proposed for more specific measurements of antibodies present in RSA recently. In order to investigate the value of ACA and anti-β2-GPI antibodies detection in screening autoimmune RSA and its clinic application in APS diagnosis, we adopt repeated and combined ACA and anti-β2-GP1 antibodies detection in our study. We found that the repeated and combined detection of ACA and anti-β2-GP1 antibodies could raise the positive rate up to 21.8% in comparison with positive for ACA alone (14.1%), positive for anti-β2-GP1 alone (3.1%), and concurrently positive for both ACA and anti-β2-GP1 antibodies (4.6%). In 91 confirmed positive APA patients, through more frequent screening for ACA and anti-β2-GP1 antibodies, more patients with APA were found. The positive rate of five and more screenings was over 81.32%, which was statistically significant (P<0.05), in comparison with that of four or less screenings (68.13%). Our data implied that it would be appropriate to do five or more screenings of combined ACA and anti-β2-GP1 antibodies detection in suspect patients to facilitate the positive diagnostic rate for autoimmune RSA [23].

Immunotherapy for RSA with immune type

In the early 1990s, we began to treat autoimmune RSA patients with low-dose aspirin (25 mg/day) and prednisone (5 mg/day) throughout their pregnancies [1]. Since 2000, we found that some patients presented over- or inadequate-treatments during dynamic monitoring of clinical parameters, such as APA, platelet aggregation test (PAGT), α-granular membrane protein (GMP-140), and D-dimer. To carry out planned pregnancy (by ultrasonically monitoring individual ovarian follicles and guiding sex life), we then initiated low-dose, short-course, and individual immunosuppressive therapy according to the change of above-mentioned clinical parameters. We treat autoimmune RSA patients with prednisone from ovulation time to one month after APA titer turning negative and with aspirin from the fifth day of the cycle to 28 weeks of gestation. Meanwhile, we adjusted the dose of aspirin on the basis of PAGT level. Moreover, in order to avoid over- or inadequate-treatments, low molecular weight heparin (LMWH) was adopted when necessary in the light of D-dimer level. Live birth rate was 95% in our study, which is much higher than that reported in other studies (70%-80%), and complications, such as sodium and fluid retention, electrolyte disturbances, and hypertension were not reported [24].

We also initiated low-dose lymphocyte active immunotherapy for alloimmune RSA patients over the same period. In our randomized controlled study, lymphocytes from husbands, unrelated males, or females at a concentration of (10-20) × 10⁶/mL were given intradermally to URSA patients in the treatment group four times before pregnancy. After the completion of the first treatment course, the patient was advised to conceive within 3 months. The second treatment course was conducted after confirming pregnancy. It proved that the live birth rate in treatment group was significantly higher than that in control group (86.4% vs 41.7%) [22]. Then, we modified the frequency of immunization to twice per course. The live birth rate of two immunizations per course was similar to four immunizations per course (87.5% vs 86.4%). At the same time, we revealed that there was no significant difference in the therapeutic effect between lymphocytes from the husband and those from an unrelated male or female. Thus, the range of blood donors can be extended, and the potential risk of infectious diseases resulting from active immunity can be decreased. Since 2000, we found that some URSA patients presented inadequate treatment by monitoring autoimmune antibodies and coagulation parameters. Thus, we further developed low-dose, short-course, and individual active immune therapy on the basis of planned pregnancy. This means that besides adopting active immunotherapy, a few URSA patients with coagulation disturbance received combined therapy of aspirin and LMWH, and a few URSA patients with positive autoimmune antibodies received low-dose prednisone. It was shown that the total live birth rate was higher in our study than that reported in overseas studies (84.4% vs 70%-80%), and complications, such as hemorrhage and blood-borne infectious diseases, were not reported [24]. Moreover, we conducted a follow-up study on the offspring of the patients who had received active immunotherapy. There was no marked difference in these children and other same-aged children with regard to birth weight, body growth and development, and intelligence. It proved that active immunotherapy was safe and effective [25,26].

Prospect

With the penetration of molecular biology, cell biology, and genetics, the study of RSA with immune type has exhibited attracting prospects in recent years. Regarding autoimmune RSA, the focus of study is on the relationship between hypercoagulable state and APA, the improvement of the sensitivity and specificity of APA detection, and the individual immunosuppressive therapy. With respect to alloimmune RSA, more and more scholars pay attention to immune cell interactions, immune cell-trophoblasts interaction, and the regulation of neuro-endocrine network on immune cells. We believe that we can get more in-depth understanding of the immunological etiology of RSA and develop more effective therapies for RSA in the near future.

References

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