Unexplained Recurrent Spontaneous Abortion Group Research Articles

Study of plasma metabolic markers in unexplained recurrent spontaneous abortion based on non-target metabolomics approach

Objective: To screen plasma metabolic markers in patients with unexplained recurrent spontaneous abortion (URSA) by non-target metabolomics approach. Methods: From September 2022 to May 2023, the plasma of 23 URSA pregnant women with threatened abortion who visited the outpatient clinic of Gansu Provincial Maternity and Child-care Hospital in the first trimester (URSA group) was collected, and the plasma of 22 healthy pregnant women in the first trimester who underwent prenatal examination during the same period (normal control group) was collected. Plasma metabolomics was analyzed by ultra performance liquid chromatography (UPLC) coupled with mass spectrometry (MS), fold change analysis, principal component analysis and partial least square discriminant analysis were applied to screen for differential metabolites, and the metabolites and their pathways associated with URSA were screened using receiver operating characteristic (ROC) curve and pathway enrichment analysis. Results: There were no significant differences in age, body mass index and gestational weeks between URSA and normal control group(all P<0.05). Metabolomics analysis using UPLC-MS showed that a total of 526 metabolites were detected from plasma, of which 33 were found to be differential metabolites associated with URSA based on the screening standards. Six potential metabolites with large area under the curve (AUC) were identified by ROC curve analysis, including phosphatidylethanolamine (AUC=0.972, 95%CI: 0.920-1.000), santene hydrate (AUC=0.902, 95%CI: 0.786-0.982), L-leucine (AUC=0.884, 95%CI: 0.772-0.960), cembrene (AUC=0.881, 95%CI: 0.758-0.956), caffeine (AUC=0.875, 95%CI: 0.756-0.962), and 4-hydroxybenzoic acid propyl ester (AUC=0.864, 95%CI: 0.732-0.946). The AUC for the combined diagnosis of URSA by the six metabolites was 0.983 (95%CI: 0.929-1.000). Pathway enrichment analysis of the differential metabolites showed that the pathogenesis of URSA was associated with a variety of metabolic pathways including caffeine metabolism, glycerophospholipid metabolism, and unsaturated fatty acid biosynthesis. Conclusion: The plasma metabolic profiles of pregnant women with normal pregnancies versus URSA differ in early pregnancy, and six potential metabolites such as phosphatidylethanolamine, santene hydrate, L-leucine, cembrene, caffeine, 4-hydroxybenzoic acid propyl ester, and their metabolic pathways may be involved in the pathogenesis of URSA.

Differential expression of immune checkpoints (OX40/OX40L and PD-1/PD-L1) in decidua of unexplained recurrent spontaneous abortion women

In this study, we aimed to investigate the expression of OX40, OX40L, PD-1 and PD-L1 in patients with unexplained recurrent spontaneous abortion (URSA) compared to normal pregnancies (NP). A total of 50 patients who were diagnosed with URSA and 41 NP were recruited to this study. Real-time polymerase chain reaction (RT-PCR) was used to determine the expression of OX40, OX40L, PD-1 and PD-L1 in decidual tissues; Immunohistochemistry (IHC) was conducted to characterize the distribution of the involved genes in decidual tissues; Double immunofluorescence staining was used to prove the localization of the involved genes in decidual tissues. The concentrations of OX40L and PD-L1 in plasma were measured with enzyme-linked immunosorbent assay (ELISA). The expression of OX40L in the decidua of URSA patients was significantly increased compared to that in the NP group, while the expression of PD-L1 in the URSA group was decreased compared to that in the NP group. Both proteins are localized in the decidual stroma as analyzed by double immunofluorescence staining. The staining results were confirmed at the mRNA level of decidual tissues, while the mRNA level of peripheral blood showed no significant difference. In conclusion, the results suggest that decidual stromal cells may promote the interaction with OX40 on T cells by upregulating the expression of OX40L and reduce the interaction with PD-1 on T cells by downregulating the expression of PD-L1 in URSA patients, which may interfere with the immune tolerance of the maternal-fetal interface, leading to poor pregnancy outcomes.

Activation of SGK1/ENaC Signaling Pathway Improves the Level of Decidualization in Unexplained Recurrent Spontaneous Abortion.

Recurrent spontaneous abortion (RSA) is one of the most common complications during pregnancy and seriously affects women's physical and mental health. About 50% of RSA cases are of unknown etiology. Our previous study found that the decidual tissue of patients with unexplained recurrent spontaneous abortion (URSA) had low expression levels of serum and glucocorticoid-induced protein kinase (SGK) 1. Endometrial decidualization is a key link in the early stage of pregnancy and is crucial to the development and maintenance of pregnancy. Decidualization is the proliferation and differentiation of endometrial stromal cells into deciduals, which involves a complex physiological process such as ovarian steroid hormones (estrogen, progesterone, prolactin, etc.), growth factors, and intercellular signaling. The binding of estrogen and its receptor stimulates the synthesis of endometrial deciduating markers prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP-1), which mediates the occurrence of decidualization. Among them, SGK1/ENaC is a signaling pathway closely related to decidualization. The purpose of this study was to further investigate the expression of SGK1 and decidualization-related molecules in the decidual tissue of URSA patients and to explore the potential mechanism of SGK1's protective effect in URSA patients and in mouse models. Decidual tissue samples from 30 URSA patients and 30 women who actively terminated pregnancy were collected, and a URSA mouse model was established and treated with dydrogesterone. Expression levels of SGK1 and its signaling pathway-related proteins (p-Nedd4-2, 14-3-3 protein and ENaC-a), estrogen and progesterone receptors (ERβ, PR), and decidualization markers (PRLR, IGFBP-1) were assessed. Our study found that SGK1, p-Nedd4-2, 14-3-3 proteins, and ENaC-a expression levels were reduced in the decidual tissue, the SGK1/ENaC signaling pathway was inhibited, and the expression levels of the decidualization markers PRLR and IGFBP-1 were downregulated in the URSA group compared with the controls. Additionally, the concentrations of E2, P, and PRL in the serum of mice were decreased in the URSA group compared with the controls. However, SGK1/ENaC pathway-related proteins, estrogen and progesterone and their receptors, and decidualization-related molecules were upregulated by dydrogesterone. These data suggest that estrogen and progesterone can induce decidualization by activating the SGK1/ENaC signaling pathway; disruption of this pathway can lead to the development of URSA. Dydrogesterone can increase the expression level of SGK1 protein in decidual tissue.

Assessment of the risk of unexplained recurrent spontaneous abortion based on the proportion and correlation of NK cells and T cells in peripheral blood.

Unexplained recurrent spontaneous abortion (URSA) is difficult to diagnose and treat clinically due to its unknown causeOBJECTIVE: Changes in natural killer (NK) cells, T lymphocytes, and Th1(IFNγ)/Th2(IL-4) cytokines were investigated in the peripheral blood of patients with URSA to examine the pathogenesis, clinical diagnosis, and inform potential treatment strategies for this condition. For this study, we selected patients with URSA as the case group and included normal women in the control group.Flow cytometry was performed to detect lymphocytes and cytokines in the peripheral blood of all subjects. The proportion of NK cells, Th1 cells, and the Th1/Th2 ratio were significantly higher in the URSA group compared to the control group; whereas the proportion of CD3+T cells was lower. Pairwise correlation analysis revealed a positive correlation between the percentage of NK cells and CD3+T cells, as well as CD3+CD4+T cells. Canonical correlation analysis indicated a significant correlation between NK cells and T cells, including their subgroups. Patients with URSA have immune balance disorders, characterised by an increased proportion of peripheral blood NK cells, Th1, and Th1/Th2 ratio along with a decreased proportion of CD3+T cells. The proportion of NK cells and CD3+T may serve as predictive factors for URSA, while NK cells are closely related to the regulation of CD3+T cells and their subsets. By regulating the level of IFN-γ, NK cells can influence the proportion of CD3+T cells and induce a Th1 (IFNγ)/Th2 (IL-4) imbalance.

Decreased expression and DNA hypermethylation of syncytin-1 in human villus tissues with unexplained recurrent spontaneous abortion

Recurrent spontaneous abortion (RSA) affects approximately 5 % of women of reproductive age worldwide. The etiology and pathogenesis of approximately 50 % of RSA cases currently remain unclear, which known as unexplained RSA (URSA). Syncytin-1, an envelope protein encoded by HERV-W gene, is essential for human embryonic development. The purpose of this study was to explore the correlation between syncytin-1 expression and URSA occurrence. The villi tissues of URSA patients and patients with voluntary termination of pregnancy for non-medical reasons in early pregnancy (Control group) were collected. Compared with the Control group, syncytin-1 was abnormally low expressed in URSA villus tissues, and the HERV-W gene promoter was hypermethylated. Compared with the control group, the global DNA methylation level and the expression level of DNA methylases in the villus tissues of the URSA group had no significant difference. In addition, compared with the Control group, URSA villus tissue showed obviously abnormal apoptosis. Overexpression of syncytin-1 promoted the proliferation of HTR-8 cells and inhibited their apoptosis; while knockdown of syncytin-1 inhibited cell proliferation and promoted cell apoptosis. URSA villus tissue exhibited hypermethylation of the HERV-W gene and down-regulation of syncytin-1 expression. Syncytin-1 has the potential to be a predictive and diagnostic biomarker for URSA.

IGFBP7 enhances trophoblast invasion via IGF-1R/c-Jun signaling in unexplained recurrent spontaneous abortion.

Insufficient trophoblast invasion at the maternal-fetal interface contributes to abortion-prone pregnancy. Our study shows that decreased levels of IGFBP7 in unexplained recurrent spontaneous abortion (URSA) trophoblast cells inhibit MMP2 and Slug expression as well as trophoblast invasion, suggesting that IGFBP7 should be considered a potential therapeutic protein target in URSA. Insufficient trophoblast invasion at the maternal-fetal interface contributes to abortion-prone pregnancy. Cyclosporine A (CsA) can exert therapeutic effects on URSA by promoting trophoblast invasion. A previous study showed decreased expression of insulin-like growth factor-binding protein 7 (IGFBP7) in the sera of recurrent spontaneous abortion patients. However, the role of IGFBP7 in URSA remains unknown. The aim of this study was to determine whether IGFBP7 modulates trophoblast invasion in URSA and the underlying molecular mechanisms. We found that IGFBP7 was expressed at lower levels in villous specimens from URSA patients. Manipulating IGFBP7 expression significantly affected the MMP2 and Slug expression in HTR-8/SVneo cells as well as trophoblast invasion in vitro. Inactivation of IGF-1R by IGFBP7 was observed, and IGF-1R inhibition increased the IGFBP7-induced MMP2 and Slug expression in HTR-8/SVneo cells. Moreover, the level of c-Jun was significantly upregulated in the URSA group. Silencing IGFBP7 increased the binding of downstream c-Jun to the MMP2 and Slug promoter regions in HTR-8/SVneo cells, thus suppressing transcription. In addition, increased expression of IGFBP7 in HTR-8/SVneo cells was observed upon CsA treatment. Knockdown of IGFBP7 inhibited the CsA-enhanced MMP2 and Slug expression in HTR-8/SVneo cells. Our results suggest that in normal pregnancy, IGFBP7 induces MMP2 and Slug expression via the IGF-1R-mediated c-Jun signaling pathway, thereby promoting trophoblast invasion. IGFBP7 depletion in URSA inhibits MMP2 and Slug expression as well as trophoblast invasion. Moreover, IGFBP7 participates in CsA-induced trophoblast invasion, suggesting that IGFBP7 is a potential therapeutic target for URSA.

Associations of serum vitamin D and Fok I polymorphism of receptor gene with unexplained recurrent spontaneous abortion

Abstract Background: To investigate the associations of serum vitamin D and Fok I polymorphism of its receptor (VDR) with unexplained recurrent spontaneous abortion (URSA). Methods: Ninety URSA patients and another 104 healthy pregnant women were selected as URSA and control groups, respectively. 25-Hydroxyvitamin D [25-(OH)D] level was detected by chemiluminescence. VDR gene Fok I polymorphism was analyzed by PCR, and the distribution of genotype frequency was calculated by Hardy-Weinberg equilibrium test. Association between Fok I polymorphism and susceptibility to URSA was investigated by logistic regression analysis. Results: Gestational age, uterine height, waist circumference, 25-(OH)D level and proportions of Fok I FF and Ff genotypes were significantly lower in the URSA group (P&lt;0.05). Compared with ff genotype, risk of URSA reduced for Ff and FF genotypes. Compared with allele f, risk of URSA was lower for allele F. 25-(OH)D level of ff genotype was significantly lower in the URSA group, which was lower than that of FF genotype (P&lt;0.05). Compared with women with 25-(OH)D level &gt;30 ng/mL and F allele (FF+Ff), the risk of URSA increased 2.45-, 2.43- and 5.34-fold for those with 25-(OH)D level &gt;30 ng/mL and ff genotype, with 25-(OH)D level ≤30 ng/mL, and with ff genotype and 25-(OH)D level ≤30 ng/mL, respectively. Conclusions: The 25-(OH)D level of the URSA group was significantly lower than that of normal pregnant women. Probably, VDR gene Fok I polymorphism is associated with URSA occurrence, and allele F decreases the risk. The risk of URSA dramatically increases in women with ff genotype and 25-(OH)D deficiency.

Correlations Between High-mobility Group Box 1 and Th1/Th2 Cytokines in Peripheral Blood of Patients with Unexplained Recurrent Spontaneous Abortion

The researchers aimed to explore the correlations between high-mobility group box 1 (HMGB1) and T helper cell (Th1/Th2) cytokines in the peripheral blood of patients with unexplained recurrent spontaneous abortion (URSA). After treatment, IL-2, IFN-ã and HMGB1 expressions significantly declined, while IL-4 and IL-10 expressions rose in URSA group (P&lt;0.05). HMGB1, IL-2 and IFN-ã expressions were significantly lower and IL-4 and IL-10 expressions were higher in the successful pregnancy group than those in the failed pregnancy group (P&lt;0.05). HMGB1 was positively correlated with IL-2 and IFN-ã, while negatively correlated with IL-4 and IL-10 (P&lt;0.01). Th1 cell percentage and Th1/Th2 ratio were significantly higher and Th2 cell percentage was lower at 12 and 24 hours, than those under stimulation with HMGB1 at 0 hours (P&lt;0.05). URSA is associated with the shift of Th1/Th2 balance toward Th1. HMGB1 may regulate the balance of Th1/Th2 immune response, as a potential target for treating URSA.

Inhibition of HMGB1 Ameliorates the Maternal-Fetal Interface Destruction in Unexplained Recurrent Spontaneous Abortion by Suppressing Pyroptosis Activation.

Recurrent spontaneous abortion (RSA) is a common complication of pregnancy that affects the physical and mental health of pregnant women, and approximately 50% of the mechanisms are unclear. Our previous studies have found that high mobility group box 1 (HMGB1) molecules are highly expressed at the maternal-fetal interface of unexplained recurrent spontaneous abortion (URSA) patients. The purpose of this study was to further detect the expression of HMGB1 and pyroptosis in decidual tissue of URSA patients, and explore the potential mechanism of the protective role of HMGB1 in URSA patients and mouse model. The decidua tissues of 75 URSA patients and 75 women who actively terminated pregnancy were collected, and URSA mouse models were established and treated with HMGB1 inhibitor-aspirin. The expression of HMGB1, and their receptors (RAGE, TLR2, TLR4), pyroptosis-associated proteins (NLRP-3, caspase-1, GSDMD) and NF-κB was examined at the maternal-fetal interface of human and mouse. Our study found that HMGB1, NLRP-3, Caspase-1, GSDMD, RAGE, TLR2 and TLR4 were highly expressed and NF-κB signaling pathway were activated in the decidua tissue of URSA group. Moreover, immune cell disorder and co-localization of HMGB1 and macrophages were found at the maternal-fetal interface of URSA mice. However, HMGB1, TLR2, TLR4, NF-κB, and pyroptosis-associated proteins can be down-regulated by administering low-dose aspirin. These data may indicate that highly expressed HMGB1 was actively secreted by macrophages and then activated pyroptosis through the TLR2/TLR4-NF-κB pathway to cause aseptic inflammation, leading to the occurrence and development of URSA. Moreover, low-dose aspirin can reduce HMGB1 protein levels of serum and decidual in URSA.

Effect and mechanism of TNF-α and etanercept on the invasion ability of extravillous trophoblast cell in URSA patients

Objective: To investigate the effect and mechanism of tumor necrosis factor α (TNF-α) and its inhibitor etanercept (ETA) on the invasion ability of extravillous trophoblast in patients with unexplained recurrent spontaneous abortion (URSA). Methods: (1) Patients were collected from March to June in 2019. They were divided into the URSA group (n=15) and the normal control group (n=15), according to whether diagnosed with URSA or not. The mRNA expression levels of TNF-α in villi tissue of patients in the two groups were detected by quantitative real-time PCR (qRT-PCR). (2) The mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2), Slug and CXC chemokine rceptor 4 (CXCR4) in HTR-8/SVneo cells were detected by qRT-PCR or western blot after being stimulated by exogenous TNF-α (0.2, 2, 20 ng/ml) alone or TNF-α along with ETA, or phosphate buffered saline (PBS) as control. (3) The invasion ability of HTR-8/SVneo cells was investigated by transwell test after stimulating by TNF-α alone or TNF-α along with ETA. (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells, which were stimulated by TNF-α (2 ng/ml) alone after nuclear factor-κB (NF-κB) inhibitor, BAY 11-7028, preconditioning, were detected by qRT-PCR or western blot. Results: (1) The mRNA expression level of TNF-α in villi tissue of URSA group (4.10±0.49) was 4.1 times as much as the normal control group (t=10.51, P<0.05). (2) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells of TNF-α group were significantly lower than those in PBS control group (P<0.05) and those in TNF-α along with ETA group (P<0.05). (3) The invasion ability of HTR-8/SVneo cells in TNF-α group was significantly decreased than PBS group and TNF-α along with ETA group (78±14 vs 373±26 vs 227±44, P<0.05). (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells with BAY 11-7028 preconditioning (mRNA: 1.03±0.10, 1.03±0.06, 1.09±0.08; protein: 1.09±0.03, 1.49±0.03, 1.12±0.03) were significantly higher than without preconditioning after being stimulated by TNF-α (all P<0.05). Conclusions: The expression of TNF-α in the villi of URSA patients is much higher than normal early pregnant women. TNF-α could decrease the capacity of invasion by suppressing the expression of MMP-2, Slug and CXCR4 through NF-κB signaling pathway in extravillous trophoblast cells. While ETA could improve the invasiveness capability of extravillous trophoblast cells through inhibiting the negative effect of TNF-α.

Upregulated HMGB1 levels in maternal–fetal interface of patients with unexplained recurrent spontaneous abortion from different sources

Objective To investigate the expression and sources of high mobility group box 1 (HMGB1) protein in the maternal–fetal interface of patients with unexplained recurrent spontaneous abortion (URSA), and further to verify the role of HMGB1 in the etiology of URSA. Methods 55 women at early pregnancy with URSA and 55 women undergoing selective termination of normal early pregnancy as control were included. The abortion tissues including villi and decidua were collected. The expression of HMGB1, CD45, CK7, and vimentin in abortion tissues was detected, and the localization and sources of HMGB1 were analyzed. Results Infiltrating immune cells and expression of HMGB1 were significantly increased in villi and decidua in URSA group compared with those in the control group. In the URSA group, HMGB1 was colocalized with the CD45-labeled immune cells, and it was more obvious in decidua than in villi; in addition, HMGB1 was colocalized with the vimentin-labeled decidual stromal cells, but not with the CK7- labeled villous epithelial cells. Conclusion High expression of HMGB1 in the maternal–fetal interface in URSA patients was actively secreted by the infiltrating immune cells, and decidual stromal cells may passively release HMGB1 during necrosis.

Significance of the ratio interferon-γ/interleukin-4 in early diagnosis and immune mechanism of unexplained recurrent spontaneous abortion.

To investigate the significance of T helper type 1 (Th1)/Th2 cytokines in the pathogenesis of unexplained recurrent spontaneous abortion (URSA), and reveal the value of single cytokines and their proportions in early diagnosis. A total of 44 URSA patients (URSA group), 51 patients with adverse pregnancy history (ad-pregnancy group), and 42 healthy volunteers with normal pregnancy (pregnancy group) were recruited for a cross-sectional study from July 2018 to April 2019 in the Second Xiangya Hospital. Pregnancies involving chromosomal abnormalities, infection, autoimmune diseases, and anatomical abnormalities were excluded. Flow cytometry was used to determine the level of Th1/Th2 cytokines in peripheral blood. The level of interleukin-6 (IL-6) in the peripheral blood of the ad-pregnancy group was significantly higher than in the other two groups. The ratio of interferon-γ (IFN-γ)/IL-4 in the URSA group was significantly higher than that of the pregnancy group. The area under the curve for IFN-γ/IL-4 was 0.821, with high diagnostic efficiency, and sensitivity as high as 84.09%. Laboratory testing for IL-6 is not recommended for the diagnosis or monitoring of URSA. The variable IFN-γ/IL-4 can be used for the initial diagnosis of URSA to reduce the rate of missed diagnosis. This ratio was more important than the expression of a single cytokine in the Th1/Th2 immune response.

The relationship between semen factors and unexplained recurrent spontaneous abortion

BackgroundThe male factor may contribute to unexplained recurrent spontaneous abortion (URSA). The relationship between semen factors and URSA is largely unknown. The analysis of metabolomics which is broadly acknowledged as the omics closest to the phenotype is suitable for providing assistance in the semen parameters analysis. MethodsWe conducted a study including couples with URSA and controls which was next combined with a meta-analysis, and finally the study included 2352 subjects on semen parameters and URSA. Metabolomics analysis was conducted to detect semen metabolic factors related to URSA in total of 106 samples including seminal plasma and sperm cells. ResultsThe URSA group had significantly lower total sperm count, sperm concentration, motility and normal morphology percentage. Meta-analysis next showed consistent findings. Metabolomics revealed that 4 metabolites and one pathway and 8 metabolites and one pathway were significantly associated with URSA in sperm and seminal plasma, respectively. The combination of ascorbic acid and guanine in seminal plasma and hexadecanedioic acid and pyroglutamic acid in sperm showed ability for URSA prediction. ConclusionWe provided novel insights into semen indices in relation to URSA. Lower sperm number and quality might increase the risk of URSA, and oxidative stress and hormone metabolism in sperm as well as nucleic acid synthesis and oxidative stress in seminal plasma were related to URSA.

Distinct changes of in BTLA, ICOS, PD-1, and TIGIT expression on peripheral blood and decidual CD8+ T cells in women with unexplained recurrent spontaneous abortion†.

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.

Destruction in maternal-fetal interface of URSA patients via the increase of the HMGB1-RAGE/TLR2/TLR4-NF-κB signaling pathway

AimsHMGB1 has been reported to play a crucial role in the physiological and pathophysiological responses during pregnancy. However, it is still unknown whether excessively expressed HMGB1 at the maternal-fetal interface related to Unexplained Recurrent Spontaneous Abortion (URSA). This study was designed to investigate the local capability of HMGB1 in the pathology of URSA, determined the distributions and characteristics of HMGB1, its receptors (RAGE/TLR2/TLR4) and important signaling molecule NF-κB p65 expression at the maternal-fetal interface，as well as compared the differences of HMGB1 expression between the URSA group, control group and aspirin treatment group. Material and methodsH&E staining, Western blot analysis, immunofluorescence assay and immunohistochemical staining were applied to determine the effect of HMGB1 and its receptors at the maternal-fetal interface. ELISA was utilized to detect the concentration of HMGB1 in plasma. Key findingsOur study demonstrated that the activation of the HMGB1-RAGE/TLR2/TLR4-NF-κB pathway at the maternal-fetal interface may have occurred in the URSA group. HMGB1 concentration in plasma was higher in the URSA group than the control group. Furthermore, the levels of HMGB1 of subjects with URSA could be reduced by administrating low doses of aspirin (ASPL). SignificanceThis is the first report indicating the roles of HMGB1 at the maternal-fetal interface of URSA patients and broadening the horizons for clinical treatment of URSA. HMGB1-RAGE/TLR2/TLR4-NF-κB signaling pathway may be activated at the maternal-fetal interface in URSA and account for its pathogenesis. HMGB1 have the potential to be promising biomarkers in prevention and therapy of URSA.

Decreased Toll-like Receptor (TLR) 2 and 4 Expression in Spermatozoa in Couples with Unexplained Recurrent Spontaneous Abortion (URSA).

Studies have shown that toll-like receptors (TLRs) play some important roles in reproductive processes such as ovulation, spermatogenesis, sperm capacitation, fertilization, and pregnancy to the best of our knowledge, no study has evaluated the expression and role of these molecules and their impairment in spermatozoa; accompanied by pregnancy complications such as recurrent spontaneous abortion (RSA). Therefore, this study investigates the alteration of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) expression in spermatozoa in men whose spouse have unexplained RSA. Fifteen fertile couples and fifteen couples with unexplained recurrent spontaneous abortion (URSA) were included in this study. The level of TLR2 and TLR4 expression in untreated and lipopolysaccharide (LPS) or PAM3CYS in treated spermatozoa were examined by flow cytometry. The results showed reduced expression of TLR4 in untreated spermatozoa and decreased LPS or PAM3CYS levels in treated spermatozoa in the URSA group compared to the control group. No significant differences were found in TLR2 expression of untreated spermatozoa in RSA and control groups. After the treatment of spermatozoa with LPS, the TLR2 expression was decreased in both groups. After the treatment of spermatozoa with PAM3CYS, the level of TLR2 expression was significantly increased in the URSA group; while no significant differences were shown in the control group in comparison to untreated spermatozoa.We have concluded that decreased TLR4 expression and a differently increased TLR2 expression in response to ligand treatment in spermatozoa is associated with URSA.

Genome-wide identification of microRNAs in decidual natural killer cells from patients with unexplained recurrent spontaneous abortion.

This study revealed miRNA regulation and functional microarray-based profiles of miRNAs in the natural killer (NK) cells of the decidual tissue obtained from patients with unexplained recurrent spontaneous abortion (URSA). Patients with URSA were categorized based on the occurrence of at least two or more successive spontaneous abortions between 7th and 10th gestational week. Total RNA was isolated from the NK cells of the decidual tissue obtained from patients with induced abortion at about the 8th gestational week. The potential contribution of regulatory miRNAs to a genetic predisposition to URSA was characterized by comparison with healthy and fertile controls and bioinformatics analyses. Analysis of the miRNA expression profiles identified 50 miRNAs that were differentially expressed, including one down-regulated and 49 up-regulated miRNAs in the URSA group. MiRNA-Gene-Network, miRNA-GO-Network and miRNA-Gene-TF-Network were constructed. The key miRNAs, genes, GOs and core TFs in the network were determined. Our results suggest that a close relationship exists between the aberrant miRNAs and URSA. Furthermore, these findings support the notion that altered expression of miRNAs may contribute to the clinical diagnosis of URSA and the potential to develop novel strategies for therapeutic targets against URSA.

Expression of Tim-3 in peripheral blood mononuclear cells and placental tissue in unexplained recurrent spontaneous abortion

The expression of T-cell immunoglobulin domain, mucin domain-3 (Tim-3) in unexplained recurrent spontaneous abortion (URSA) was investigated.Tim-3 mRNA expression in peripheral blood mononuclear cells (PBMCs) of URSA and control groups was assayed by fluorescent quantitative real-time polymerase chain reaction. Tim-3 protein expression intensity and localization in placental villi and uterine decidua were determined using immunohistochemical assay. The CD4+Tim-3+/CD4+ cell ratio in PBMCs was determined by flow cytometry.Tim-3 mRNA expression in PBMCs was significantly higher in URSA than in normal controls (1.32 ± 0.25 vs 1.20 ± 0.12, P < .05). Tim-3 was expressed in placental tissue from both URSA patients and normal pregnant females (controls); however, the expression intensity was higher in the URSA group (0.54 ± 0.31 vs 0.35 ± 0.22, P < .05). CD4+Tim-3+/CD4+ cell ratio in PBMCs was significantly higher in the URSA group than that in the control group (4.53 ± 1.66% vs 1.28 ± 0.71%, P < .05).Increased Tim-3 expression in PBMCs and placental tissue of URSA might affect maternal-fetal immune tolerance. Tim-3 was involved in the pathogenesis of URSA, which was expected to serve as an indicator for the immune evaluation of URSA.

Association of coagulation factor V gene polymorphism with unexplained recurrent spontaneous abortion among ethnic Hans from Wenzhou area

To assess the association of coagulation factor V gene polymorphisms with unexplained recurrent spontaneous abortion (URSA) among ethnic Han Chinese from Wenzhou area. Ninety-six patients with URSA and 103 females with a history of normal pregnancy were recruited. Genotypes of coagulation factor V gene were determined through target sequence capture and high-throughput sequencing. The results were confirmed with a MassARRAY system. Allelic and genotypic frequencies between the two groups were compared. Nineteen single nucleotide polymorphism (SNPs), except coagulation factor V Leiden, were identified in the two groups. The frequencies of rs9287090 allele A, rs1046712 allele T and rs1800594 allele G of the URSA group were lower than those of the control group (6.77% vs. 16.50%, 3.12% vs. 13.11%, 10.94% vs. 18.45%, respectively). After Bonferroni and false discovery rate correction, rs9287090 and rs1046712 were significantly associated with URSA (corrected P<0.05). Although genotypic distribution of rs9287090 and rs1046712 also differed between the two groups, the corrected P value showed no significance (corrected P>0.05). A complete linkage disequilibrium (r2=1, D'=1) of rs6022 and rs6029 was observed for the haplotype block rs6022-rs6029-rs6028. The frequencies of rs6022 allele A and rs6029 allele T were higher in the URSA group with corrected insignificance (75.00% vs. 65.53%, corrected P>0.05). Furthermore, significantly more A-T-T haplotype was found in the URSA group (75.00% vs. 65.50%, OR=1.578, 95%CI:1.021-2.438, χ2=4.248, P<0.05). The decreased rate of rs9287090 allele A, rs1046712 allele T, and rs1800594 allele G may contribute to the susceptibility to URSA among ethnic Han Chinese from Wenzhou area. The rs6022 allele A and rs6029 allele T may also predispose to URSA.

Decreased expression of WNT2 in villi of unexplained recurrent spontaneous abortion patients may cause trophoblast cell dysfunction via downregulated Wnt/β-catenin signaling pathway.

WNT2 has been reported to be important for placental development, especially for the proper vascularization of the placenta. However, its precise role in first-trimester trophoblast cells is still unknown. WNT2 expression in the villous tissues of unexplained recurrent spontaneous abortion (URSA) patients was compared with that of healthy women by Western blot. The function of WNT2 in HTR-8/SVneo trophoblast cells was evaluated by altering the cellular WNT2 level through overexpression and shRNA knockdown. The molecular mechanism of the effect of WNT2 on trophoblast cells was investigated. The association of WNT2 with the Wnt/β-catenin signaling pathway was studied through Western blot and immunofluorescence. Results showed that WNT2 protein expression was significantly decreased in villi of the URSA group compared with the control group. In vitro studies showed that WNT2 could promote human trophoblast cell proliferation and migration through activating the Wnt/β-catenin signaling pathway. Moreover, upon the knockdown of WNT2, trophoblast cell proliferation and migration were significantly suppressed. In conclusion, our study indicated that WNT2 plays an important role in trophoblast function. WNT2 insufficiency might cause impaired trophoblast cell proliferation and migration via downregulation of Wnt/β-catenin signaling pathway.