Spontaneous Abortion In Mice Research Articles

EGR1 modulates EPHB4-induced trophoblast dysfunction in recurrent spontaneous abortion†.

Recurrent spontaneous abortion, defined as at least three unexplained abortions occurring before the 20-24 week of pregnancy, has a great impact on women's quality of life. Ephrin receptor B4 has been associated with trophoblast function in preeclampsia. The present study aimed to verify the hypothesis that ephrin receptor B4 regulates the biological functions of trophoblasts in recurrent spontaneous abortion and to explore the upstream mechanism. Ephrin receptor B4 was overexpressed in mice with recurrent spontaneous abortion. Moreover, ephrin receptor B4 inhibited trophoblast proliferation, migration, and invasion while promoting apoptosis. Downregulation of early growth response protein 1 expression in mice with recurrent spontaneous abortion led to ephrin receptor B4 overexpression. Poor expression of WT1-associated protein in mice with recurrent spontaneous abortion reduced the modification of early growth response protein 1 mRNA methylation, resulting in decreased early growth response protein 1 mRNA stability and expression. Overexpression of WT1-associated protein reduced the incidence of recurrent spontaneous abortion in mice by controlling the phenotype of trophoblasts, which was reversed by early growth response protein 1 knockdown. All in all, our findings demonstrate that dysregulation of WT1-associated protein contributes to the instability of early growth response protein 1, thereby activating ephrin receptor B4-induced trophoblast dysfunction in recurrent spontaneous abortion. Our study provides novel insights into understanding the molecular pathogenesis of recurrent spontaneous abortion.

Long Non-Coding RNA ZEB2-AS1 Augments Activity of Trophoblast Cells and Prevents the Development of Recurrent Spontaneous Abortion in Mice Through EZH2-Mediated CST3 Inhibition.

Recurrent spontaneous abortion (RSA) is the most common complication of pregnancy where reduced invasion of trophoblasts plays a major role. This work aimed to explore the effect of abnormally expressed long non-coding RNA (lncRNA) ZEB2-AS1 on the occurrence of RSA. Differentially expressed lncRNAs in trophoblast cells between healthy controls and patients with RSA were screened using the GEO database. Female CBA/J mice were allowed to mate with male DBA/2 mice to establish inbred mice with RSA. ZEB2-AS1 was poorly expressed in placental tissues and trophoblast cells in the condition of RSA. ZEB2-AS1 upregulation augmented proliferation, migration, and invasion of trophoblast cells in vitro. ZEB2-AS1 negatively regulated cystatin C (CST3) expression. Further overexpression of CST3 blocked the activity of trophoblast cells. ZEB2-AS1 recruited enhancer of EZH2 to the promoter region of CST3, which increased H3K27me3 modification to suppress CST3 expression. In vivo, overexpression of ZEB2-AS1 reduced embryo resorption rate and increased the weights of fetuses and placentas in mice with RSA. However, the protective roles of ZEB2-AS1 were blocked upon artificial silencing of EZH2 or upregulation of CST3. Taken together, this study demonstrates that ZEB2-AS1 enhances activity of trophoblast cells and prevents RSA development through reducing CST3 expression in an EZH2-dependent manner.

Congenital anomalies and spontaneous abortion in mice resulting from the use of escitalopram.

Escitalopram (ESC) use during pregnancy has not been associated with teratogenic effects in fetuses. To investigate whether ESC administered during pregnancy in mice induces maternal toxicity and teratogenicity in offspring. Treated mice groups G1 and control G0 (n =15 per group). Administration of ESC (G1) and saline solution (G0) during pregnancy and euthanasia on the 18thday. Pregnant female mice were treated with ESC (20mg/kg, via gavage) or saline solution (control group) from the 5th to the 17thday of gestation, when implantation was consolidated. During intraembryonic development until the day before delivery, the drug had an influence on the development of alterations from its maintenance in the uterine environment and its development to the disturbance causing skeletal or visceral malformations. The intrauterine development parameters that were altered by ESC treatment were: number of resorptions (G0: [0.93±0.24]); G1: [3.33±0.51]), post-implantation loss (G0: [3.95±1.34], G1: [13.75±3.62]) and reduced fetal viability: [97.30±1.00]; G1: [81.09±6.22]). Regarding fetal formation, the treated group had visceral malformations with a significant frequency: cleft palate (G0: [1.0%], G1: [11.86%]) and reduced kidneys (G0: [0%]; G1: [10.17%]). Regarding skeletal malformations, a higher frequency was observed in the following parameters: incomplete supraoccipital ossification (G0: [0%], G1: [15.25]), absence of ribs (G0: [0%], G1 (G0: [0%], G1 [15.25%]) and absence of one or more of the foot phalanges (G0: [1.0%]; 64%]). Results indicate that ESC is an embryotoxic and teratogenic drug. Until further studies are performed, greater caution is necessary in prescribing the drug to pregnant women.

Trophoblast CD200 expression in successful human pregancies and missed abortions

Presence of the CD200 immune check-point inhibitor at the feto-maternal interface is linked to prevention of spontaneous abortion in mice and humans. In human missed abortions (MA), absence of Th17-driven inflammation has been attributed to expression of villus trophoblast CD200 quantified using immunohistochemistry. While rapid aneuploidy (QF-PCR) testing linked low CD200 to pregnancy failure, data showing normal VT CD200 in first trimester normal pregnancy and in abortion of chromosomally abnormal embryos has not been demonstrated. The present report shows normal CD200 in a 7 week gestation termination with normal male QF-PCR and in a 10 week male trisomy 18 MA.

Adoptive transfer of transforming growth factor-?1-induced CD4+CD25+ regulatory T cells prevents immune response-mediated spontaneous abortion.

The effects of adoptive transfer of transforming growth factor (TGF)-β1-induced regulatory T (Treg) cells in preventing spontaneous abortion in mice were investigated. CD4+CD25- cells were isolated from the spleens of pregnant CBA/J mice and induced into Treg cells positive for CD4, CD25 and forkhead box P3 (FOXP3) ex vivo using interleukin (IL)-2 and TGF-β1. CBA/J mice were mated with DBA/2J mice to establish a model of spontaneous abortion and, on the first day of pregnancy, mice were injected intravenously with 2 × 105 either freshly isolated Treg cells or those induced with TGF-β1. After 14 days, the surviving and reabsorbed fetuses in both groups were counted, and serum cytokine concentrations were measured by ELISA. Adoptive transfer of CD4+CD25+ or TGF-β1-induced Treg cells significantly reduced the fetal resorption rate, increased serum IL-10 and TGF-β1 concentrations and decreased interferon-γ levels. In conclusion, the results of the present study indicate that adoptive transfer of TGF-β1-induced Treg cells prevents spontaneous abortion in mice by increasing the secretion of T helper (Th) 2 cytokines and decreasing the secretion of Th1 cytokines.

Abortion‐Prone Mating Influences Alteration of Systemic a2 Vacuolar ATPase Expression in Spleen and Blood Immune Cells

a2 isoform of vacuolar ATPase (Atp6v0a2) is important for maintaining the delicate immunological balance required for successful pregnancy. The objective of this investigation is to study the dynamic changes in spleen and blood that appear during spontaneous abortion in mice. Atp6v0a2 was measured in multiple immune cell populations from spleen and blood recovered from non-abortion-prone and abortion-prone mating combinations. Atp6v0a2 expression was significantly lower (P ≤ 0.01) in the spleen recovered from abortion-prone ♀CBA × ♂DBA mating on days 12 and 16 of pregnancy when compared to non-abortion-prone ♀BALB/c × ♂BALB/c and ♀CBA × ♂BALB/c matings. Flow cytometric studies showed that significantly decreased expression of Atp6v0a2 in splenic CD4(+), CD8(+), CD19(+), and CD14(+) cells directly correlated with the high percentages of fetal resorption observed in abortion-prone mating on days 12 and 16 of pregnancy. In blood, CD4(+), CD8(+), and CD19(+) cells had a significantly reduced expression of Atp6v0a2 in abortion-prone mating compared to the non-abortion-prone mating combinations only on day 12. This deceased expression of Atp6v0a2 in the various immune cell populations of the spleen and blood suggests that the maternal environment is not supportive to fetus and leads to poor pregnancy outcome in the abortion-prone mating model.

Placental ATPase Expression Is a Link Between Multiple Causes of Spontaneous Abortion in Mice

The a2 isoform of vacuolar ATPase (ATP6V0A2 referred to as a2V) plays a pivotal role in successful pregnancy and provides a microenvironment to maintain the delicate immunological balance at the feto-maternal interaction. We studied the expression of a2V mRNA in embryos and placenta of abortion-prone (female CBA × male DBA) murine matings or LPS (lipopolysaccharide)-treated mice. The expression of a2V was significantly higher in the placentas of nonabortion-prone (female BALB/c × male BALB/c and female CBA × male BALB/c) matings compared with the abortion-prone (female CBA × male DBA) mating. The expression of a2V was significantly decreased in the placentas treated with LPS in both female CBA × male DBA and female BALB/c × male BALB/c mating combinations with increased Lif, Il1b, and Tnf expression in the placenta. Decreased expression of a2V in the placenta is directly correlated with high percentages of pregnancy loss in abortion-prone mating (female CBA × male DBA) as well as in LPS-treated animals. The normal expression of placental a2V on Day 16 in the nonabortion-prone matings correlated with higher Mcp1 (monocyte chemotactic protein 1) gene expression, markedly higher infiltration of M1 and M2 macrophages, and no significant polarization patterns (M1/M2 = 1.2-1.6). However, in the abortion-prone mating, decreased placental a2V expression correlated with significantly lower Mcp1 gene expression with less infiltration of M1 and M2 macrophages and with polarization patterns skewed to M1 phenotypes (M1/M2 = 3.9-4.2). These data indicate that the higher expression of placental a2V is associated with dynamic infiltration of M1 and M2 macrophages through the induction of Mcp1 expression. This strengthens our hypothesis that a2V regulates the delicate cytokine and chemokine networks that coordinate the recruitment of macrophages for successful placental development and growth at the feto-maternal interface.

Relationship between expression of chemokine receptors CCR3, CCR5 and CXCR3 on CD4+ T cells and spontaneous abortion in mice

Background Previous studies have shown that local immune cells in the feto-maternal interface are recruited from peripheral blood, and that chemokines and their receptors play an initial and key role in this recruitment process. In this study, we aimed to determine whether spontaneous abortion is associated with the expression of chemokine receptors CCR3, CCR5, and CXCR3 on CD4+ T cells. Methods Peripheral blood, spleen, and thymus were collected from the spontaneous abortion mouse model CBA/J×DBA/2 (SA group, n=14), the normal pregnant mouse model CBA/J×BALB/c (NP group, n=13), and normal non-pregnant CBA/J mice (NNP group, n=11). The number of chemokine receptors CCR3, CCR5, and CXCR3 expressed on CD4+ T cells was measured by double-label flow cytometry (FCM) method. Results In peripheral blood, the SA group had significantly lower CCR3 expression (P <0.01) and higher CCR5 and CXCR3 expression (P <0.01) on CD4+ T cells than did the NP group. But comparing these chemokines between the SA and NNP groups, there was no significant difference (P >0.05). In spleen, the SA group expressed significantly lower CCR3 expression (P <0.01) and higher CCR5 and CXCR3 expression (P <0.05) on CD4+ T cells than did the NP group. When compared with the NNP group, the SA group had significantly higher CCR3 expression (P <0.01), but was not statistically different with regards to the other two chemokines (P >0.05). In thymus, the SA group had significantly lower CCR3 expression (P <0.05) and higher CXCR3 expression (P <0.05) on CD4+ T cells than the NP group, with no significant difference in CCR5 expression (P >0.05). Compared with the NNP group, the SA group had higher CCR3 expression (P <0.01), but there was no statistical difference in CXCR3 and CCR5 expression (P >0.05) between the two groups. Conclusion The abnormal expression of CCR3, CCR5 and CXCR3 on CD4+ T cells may play an important role in the pathogenesis of spontaneous abortion. Chin Med J 2009;122(4):390–395

Up-Regulation of TH2 feto-placental levels could be involved in the protective effect of low-molecular-weight heparin treatment on spontaneous abortion in mice

Up-Regulation of TH2 feto-placental levels could be involved in the protective effect of low-molecular-weight heparin treatment on spontaneous abortion in mice

The Same Immunoregulatory Molecules Contribute to Successful Pregnancy and Transplantation

At least two dendritic cell-associated molecules have been shown to contribute to the successful outcome of organ and tissue allografts in mice, namely CD200 and MD-1. CD200 is up-regulated in rodent transplantation models where successful inhibition of rejection is accomplished, and is believed to signal immunosuppression following engagement of a receptor, CD200R, on macrophages and/or gammadelta T-cell receptor (gammadelta TCR+ cells MD-1 is implicated in controlling expression of costimulatory molecules including CD80/CD86 which induce an immunorejection response, and thus inhibition of MD-1 expression also facilitates increased graft survival MD-1 also stabilizes expression of CD14, part of the receptor complex for LPS. As well as the inhibition of rejection which follows blockade of MD-1 expression and/or augmentation of CD200 expression, an altered polarization in cytokine production is seen, with increased expression of interleukin-4 (IL-4), IL-10 and transforming growth factor-beta (TGF-beta), and decreased IL-2, interferon-gamma (IFN-gamma) and tumor nerosis factor-alpha (TNF-alpha). Successful pregnancy in allopregnant mice also depends upon control of graft rejection mechanisms. Proinflammatory T-helper 1 (Th1) cytokines (TNF-alpha + IFN-gamma + IL-1) have been shown to cause spontaneous abortion in mice by activating a novel prothrombinase, fibrinogen-like peptide (fibroleukin) fgl2, which may promote fibrin deposition in the graft rejection process; expression of IL-10, TGF-beta, and progesterone-induced blocking factor (PIBF) in contrast leads to lowering of abortion rates. Interestingly, the spontaneous abortion rates in abortion-prone CBA x DBA/2 matings and in the low abortion rate CBA x BALB/c matings were lower than the frequency of implantation sites showing fibrin(hi) + fgl2 (mRNA)hi, implying regulation of the pro-abortion consequences of fgl2 expression. We have investigated, by in situ hybridization, CD200, MD-1 and fgl2 expression in implantation sites in different strains of mice, and studied the effects of anti-MD-1, anti-CD200 and CD200Fc immunoadhesin on fetal and allograft survival. The role of indoleamine dioxygenase (IDO) was evaluated. CD200 mRNA expression occurred in the same sites as fgl2 mRNA. Anti-CD200 antibody raised the abortion rate to predicted levels, and infusion of a CD200 immunoadhesin reduced the abortion rate, as did an anti-MD-1 antibody. The latter also improved organ and tissue graft survival. Suppression by antigen-presenting macrophages triggered by CD200 is dependent upon intact IDO activity. Regulation of CD200 and MD-1 expression may control both pregnancy and allograft survival.

Infiltrating decidual natural killer cells are associated with spontaneous abortion in mice

Immunohistochemistry was used to study a murine model which spontaneously aborts at a frequency of 25 to 30%. Our results show that natural killer (NK) cells are not only the predominant infiltrating cells in aborting feto-placental units, but that they also appear in a similar proportion of feto-placental units before abortion is detectable. The frequency of feto-placental units with significantly elevated NK infiltrates corresponds to the subsequent abortion frequency, indicating a causal relationship. Immunization of the mother with BALB/C splenocytes prevents these NK infiltrates and decreases the abortion frequency to normal levels. These results suggest for the first time that maternal NK cells may have an instrumental role in the etiology of spontaneous abortion.

Maternal T cells promote placental growth and prevent spontaneous abortion

Transplantation immunologists have long been intrigued by the natural allograft that results from normal mammalian pregnancy. Its general success contrasts with the rejection problems associated with most artifactual organ transplantation and raises intriguing questions concerning the nature of the mechanisms involved in that success. This area of research has recently taken on added momentum because it is now clear that immunological maneuvers can prevent recurrent spontaneous abortion in mice, horses and humans [1–3]. The purpose of this review is to discuss some of these recent developments, which lead to the surprising conclusion that maternal T cells, rather than being potentially detrimental to the fetal allograft, promote its growth and viability during normal pregnancy. This review will address these questions by considering: (a) the nature of the exposure of fetal alloantigens to the maternal circulation in the chimeric zone of the placenta; (b) the evidence for maternal immune recognition of the fetal alloantigens; and (c) the consequences of that recognition with respect to the prevention of spontaneous abortion. As in other areas of immunology the T cell emerges as the most important component of this immune recognition.

Vaccination against spontaneous abortion in mice by preimmunization with an anti-idiotypic antibody.

CBA/J females mated with DBA/2 display a high level of fetal wastage which can be corrected by anti-Balb/c vaccination. A batch of CBA/J anti-(CBA/J anti-Balb/c) antiserum was raised. This serum was characterized as anti-idiotypic by various techniques, including a solid-phase radioimmunoassay. Such a serum proved to confer protection against resorptions when injected into CBA/J mice mated with DBA/2. However, the kinetics of the effect pointed to the need for administration in early pregnancy for successful protection. The significance of these data, and the possible mechanism(s) by which the serum acts, are discussed.

Immunogenetic studies of spontaneous abortion in mice: II. Antiabortive effects are independent of systemic regulatory mechanisms

CBA/J females (H-2 k) mated with DBA 2/J males (H-2 d) exhibit a high rate of fetal resorption. Fetal survival has been improved by pretreatment of CBA/J females with spleen cells from BALB/c J (H-2 d) (but not from CBA/J or DBA/ 2/J) males. Similarly, three out of nine recombinant inbred strains (recombining BALB/c and DBA 2 genomes at the homozygous state) possess anti-abortive effects like the BALB/c parent. Previous studies showed that BALB/c pretreatment triggers the appearance of suppressor cells in the spleen and of IgG1 anti-H-2 d antibodies in the serum of CBA/J females. Studies of these two immunological parameters after CBA/J preimmunization by the different recombinant strains suggest that local intrauterine immunoregulation is the determinant of success or failure of allopregnancy.

Immunogenetic studies of spontaneous abortion in mice. Preimmunization of females with allogeneic cells.

CBA females (H-2k) mated with DBA2 males (H-2d) exhibit a high rate of fetal resorption (30%) when compared with the CBA female BALB/c male, CBA female/CBA male, DBA2 female/CBA male, DBA2 female/DBA2 male combinations (6 to 8%). Preimmunization of CBA females with spleen cells from DBA2, BALB/c, or CBA males were performed in order to test their effects on CBA maternal tolerance of (CBA X DBA2)F1 fetuses. Only preimmunization with BALB/c male cells was effective in decreasing resorption; cells from BALB/c females had no effect. In order to further test 1) the role of non-MHC-encoded antigens present in the BALB/c male background, 2) the necessity of an additional H-2 difference, and 3) whether or not the phenomenon is H-2d restricted, preimmunizations were performed by using cells from congenic BALB/k (H-2k), BALB/b (H-2b), or BALB/c (H-2d). Only the latter treatment was efficient, which suggests that the paternal H-2d haplotype must be presented in synergy with some non-MHC-encoded antigens in the BALB/c male background. Immunogenetic studies with cells from nine recombinant inbred strains that reassorted DBA2 and BALB/c genomes showed that three of them behave like BALB/c and six like DBA2. This would suggest that the genetic determinism of this phenomenon is simple.

Vaccination against spontaneous abortion in mice

We report here that the high rate of spontaneous resorption observed in CBA/J female mice mated with DBA/2 J males can be dramatically reduced by vaccination with Balb/c male spleen cells, but not by CBA/J or DBA/2 J male spleen cells. This effect correlates with the differential ability of Balb/c spleen cells to induce MLR suppressor activity in CBA/J female mice, and should lead to a better understanding of the immunology of the materno-fetal relationship.