HOTAIR/miR-1277-5p/FBN2 signaling axis is involved in recurrent spontaneous abortion by regulating the growth, migration, and invasion of HTR-8/SVneo cells†.

OBJECTIVE: This study aimed to explore the specific pathways by which HOX transcript antisense intergenic RNA contributes to the pathogenesis of unexplained recurrent spontaneous abortion. METHODS: Real-time quantitative PCR was employed to assess the differential expression levels of HOX transcript antisense intergenic RNA in chorionic villi tissues from unexplained recurrent spontaneous abortion patients and women with voluntarily terminated pregnancies. HTR-8/SVneo served as a cellular model. Knockdown and overexpression of HOX transcript antisense intergenic RNA in the cells were achieved through siRNA transfection and pcDNA3.1 transfection, respectively. Cell viability, migration, and invasion were evaluated using cell counting kit-8, scratch, and Transwell assays, respectively. The interaction among the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 axis was predicted through bioinformatics analysis and confirmed through in vitro experiments. Furthermore, the regulatory effects of the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 signaling axis on cellular behaviors were validated in HTR-8/SVneo cells. RESULTS: We found that HOX transcript antisense intergenic RNA was downregulated in chorionic villi tissues from unexplained recurrent spontaneous abortion patients. Overexpression of HOX transcript antisense intergenic RNA significantly enhanced the viability, migration, and invasion of HTR-8/SVneo cells, while knockdown of HOX transcript antisense intergenic RNA had the opposite effects. We further confirmed the regulatory effect of the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 signaling axis in unexplained recurrent spontaneous abortion. Specifically, HOX transcript antisense intergenic RNA and fibrillin 2 were found to reduce the risk of unexplained recurrent spontaneous abortion by enhancing cell viability, migration, and invasion, whereas miR-1277-5p exerted the opposite effects. CONCLUSION: HOX transcript antisense intergenic RNA promotes unexplained recurrent spontaneous abortion development by targeting inhibition of miR-1277-5p/fibrillin 2 axis.

Decreased thrombospondin-1 impairs endometrial stromal decidualization in unexplained recurrent spontaneous abortion†.

Inappropriate endometrial stromal decidualization has been implied as an important reason of many pregnancy-related complications, such as unexplained recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction. Here, we observed that thrombospondin-1, an adhesive glycoprotein, was significantly downregulated in endometrial decidual cells from patients with unexplained recurrent spontaneous abortion. The immortalized human endometrial stromal cell line was used to investigate the possible THBS1-mediated regulation of decidualization. In vitro experiments found that the expression level of THBS1 increased with the normal decidualization process. Knockdown of THBS1 could decrease the expression levels of prolactin and insulin-like growth factor binding protein-1, two acknowledged human decidualization markers, whereas THBS1 overexpression could reverse these effects. The RNA sequencing results demonstrated that the extracellular regulated protein kinases signaling pathway was potentially affected by the knockdown of THBS1. We further confirmed that the regulation of THBS1 on decidualization was achieved through the ERK signaling pathway by the treatment of inhibitors. Moreover, knockdown of THBS1 in pregnant mice could impair decidualization and result in an increased fetus resorption rate. Altogether, our study demonstrated a crucial role of THBS1 in the pathophysiological process of unexplained recurrent spontaneous abortion and provided some new insights into the research of pregnancy-related complications.

KLF4 regulates trophoblast function and associates with unexplained recurrent spontaneous abortion.

BACKGROUND: Recurrent spontaneous abortion (RSA) is defined as two or more consecutive spontaneous abortions before 20 weeks with the same spouse [1]. However, approximately 50% of RSA cases of unknown cause are classified as unexplained recurrent spontaneous abortion (URSA). Potential factors include decreased trophoblast cell migration and invasion, leading to impaired placental implantation and maintenance of the normal maternal-fetal interface. However, the mechanism of this pathogenesis remains unknown. In this study, we investigated the potential role and mechanism of KLF4 in regulating URSA by influencing the invasion and migration ability of trophoblast cells. METHODS: We firstly identified 817 differentially expressed genes by performing a difference analysis of the dataset GSE121950 [2] related to recurrent abortion, and intersected the top 10 genes obtained respectively by the three algorithms: DMNC, MNC, and EPC using Venn Diagram.To detect the expression levels of core genes, villi samples were obtained from normal pregnant women and patients with URSA. RT-qPCR analysis revealed a significant difference in KLF4 mRNA expression and KLF4 was then analyzed. Trophoblast cell lines HTR8 and JEG3 were used to investigate the effect of KLF4 on trophoblastic function. Wound healing and transwell assays was performed to detect the invasion and migration of trophoblast cells. The expression of epithelial-mesenchymal transition(EMT) molecules were detected by RT-qPCR and western blot. Promoter detection and epigenetic modification were detected by chromatin immunoprecipitation (ChIP) assay. Molecular nuclear localization was detected by immunofluorescence and subcellular fractionation. Miscarried mice model was used to study the effects of KLF4 on URSA induced by reduced trophoblast invasion and migration. RESULTS: KLF4 is highly expressed in the villi of patients with URSA. KLF4 inhibits the expression level of H3R2ME2a in trophoblast cells by regulating the transcriptional level and nuclear translocation of PRMT6, thereby inhibiting the possible regulatory mechanism of trophoblastic invasion and providing a potential treatment strategy for URSA in vivo. CONCLUSIONS: The KLF4/PRMT6/H3R2ME2a axis regulates mechanisms associated with unexplained recurrent spontaneous abortion by regulating trophoblast function.

Association between urinary polycyclic aromatic hydrocarbons and unexplained recurrent spontaneous abortion from a case-control study.

Polycyclic aromatic hydrocarbons (PAHs) have been reported to be associated with adverse pregnancy outcomes. However, there is limited knowledge regarding the effects of single or mixed PAHs exposure on unexplained recurrent spontaneous abortion (URSA). This study aimed to investigate the association between monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) and URSA in a case-control study. The results showed that 1-NAP, 2-NAP, 9-FLU, and 1-PYR were detected in 100% of the subjects among measured all sixteen OH-PAHs. Compared with those in the lowest quartiles, participants in the highest quartiles of 3-BAA were associated with a higher risk of URSA (OR (95%CI) = 3.56(1.28-9.85)). With each one-unit increase of ln-transformed 3-BAA, the odds of URSA increased by 41% (OR (95%CI) = 1.41(1.05-1.89)). Other OH-PAHs showed negative or non-significant associations with URSA. Weighted quantile sum (WQS) regression, Bayesian kernel machine regression (BKMR), and quantile-based g-computation (qgcomp) analyses consistently identified 3-BAA as the major contributor to the mixture effect of OH-PAHs on URSA. Our findings suggest that exposure to 3-BAA may be a potential risk factor for URSA. However, further prospective studies are needed to validate our findings in the future.

Associations between urinary monohydroxylated polycyclic aromatic hydrocarbons and unexplained recurrent spontaneous abortion and health risk assessment in a large case-control study in China.

Polycyclic aromatic hydrocarbons (PAHs) are a group of environmental endocrine disruptors with known carcinogenic, reproductive, and developmental toxicity. Important knowledge gaps remain regarding the relationship between PAH exposure and unexplained recurrent spontaneous abortion (URSA). In the present study, twelve monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) were measured in the urine of 413 URSA cases and 434 controls. The main OH-PAHs measured in this study were monohydroxy metabolites of naphthalene, followed by fluorene and phenanthrene. After the creatinine correction, the median concentration of urinary OH-PAHs in the control group (17.4 µg/g Creatinine) was higher than that in the case group (14.2 µg/g Creatinine). There was no positive relationship between PAH exposure and URSA using binary logistic regression analysis. Among 847 Chinese women of childbearing age, residential environment, type of drinking water, and education level were the influencing factors of PAH exposure. The health risk assessment showed that over 98% of women had a carcinogenic risk with carcinogenic risk values above the acceptable level (10-6). Although this large-scale case-control study did not observe an association between PAH exposure and URSA, more attention should be paid to the high carcinogenic risk due to PAH exposure in women of reproductive age.

Temporal changes in cyclinD-CDK4/CDK6 and cyclinE-CDK2 pathways: implications for the mechanism of deficient decidualization in an immune-based mouse model of unexplained recurrent spontaneous abortion.

BACKGROUND: Deficient endometrial decidualization has been associated with URSA. However, the underlying mechanism is poorly understood. This study aimed to investigate the temporal cytokine changes and the involvement of CyclinD-CDK4/6 and CyclinE-CDK2 pathways in the regulation of the G1 phase of the cell cycle during decidualization in a murine model of URSA. METHODS: Serum and decidual tissues of mice were collected from GD4 to GD8. The embryo resorption and abortion rates were observed on GD8 and the decidual tissue status was assessed. In addition, PRL, Cyclin D, CDK6, CDK4, Cyclin E, CDK2 expression in mice were measured. RESULTS: URSA mice showed high embryo resorption rate and PRL, Cyclin D, Cyclin E CDK2, CDK4, CDK6 down-regulation during decidualization. The hyperactivated Cyclin D-CDK4/CDK6 and cyclin E/CDK2 pathways inhibit the decidualization process and leading to deficient decidualization. CONCLUSION: Insufficient decidualization is an important mechanism of URSA. which is related to the decrease of Cyclin D、Cyclin E、 CDK2、CDK4 and CDK6 in decidualization process of URSA.

Polarization disorder of decidual NK cells in unexplained recurrent spontaneous abortion revealed by single-cell transcriptome analysis.

BACKGROUND: Unexplained recurrent spontaneous abortion (URSA) is one of the most common diseases in pregnancy and is mainly caused by immune disorders. The foetus is similar to semiallogeneic maternal tissue, so the balance of immune tolerance must be dynamically maintained during pregnancy. Decidual natural killer (dNK) cells primarily mediate the immune tolerance microenvironment at the maternal-fetal interface. By using single-cell RNA sequencing (scRNA-seq) and high-throughput transcriptome sequencing analysis, we explored the characteristic distribution of dNK cells in URSA patients. METHODS: Control maternal-fetal interface tissue (from normal pregnant women, n = 3) and case maternal-fetal interface tissue (from patients with URSA, n = 3) samples were analysed by scRNA-seq and high-throughput transcriptome sequencing. RESULTS: By scRNA-seq, we demonstrated the maturation process of the transition of dNK cells from cytotoxic characteristics to immune tolerance in transcriptome analysis. Moreover, compared with normal pregnant women, serious disturbances in the polarization process of dNK cells were found in URSA. Simultaneously, the transcriptional level of the extracellular matrix (ECM) in URSA patients showed a significant decrease. The dNK cells interacted with extravillous trophoblasts to achieve immune-tolerant polarization. CONCLUSIONS: Insufficient expression of KIRs during dNK cell differentiation might be a key reason why polarized dNK cells still had high cytotoxic reactivity in URSA patients. Abnormal expression of ECM may affect the interaction of dNK cells with EVTs, making dNK cells immature. Both resulted in maternal immune intolerance to the foetus during pregnancy.

Levels and health risks of urinary phthalate metabolites and the association between phthalate exposure and unexplained recurrent spontaneous abortion: a large case-control study from China.

Phthalate acid esters (PAEs) are environmental endocrine disruptors that can interfere with endocrine processes and cause adverse reproductive outcomes. The link between PAE exposure and unexplained recurrent spontaneous abortion (URSA) remains unknown. In this study, nine urinary metabolites of PAEs (mPAEs) were measured in 594 URSA cases and 569 healthy controls. The measured mPAEs were ubiquitously detected and present at higher levels (median: 203 ng/mL) in the URSA cases than in the controls (median: 161 ng/mL). Multiple logistic regression analysis showed that URSA was associated with higher concentrations of mono (2-ethyl-5-hydroxyhexyl) phthalate (mEHHP), mono (2-ethylhexyl) phthalate (mEHP), and mono-ethyl phthalate (mEP) and lower concentrations of mono-isobutyl phthalate (miBP). Moreover, a quantile-based g-computation (QGC) model revealed a positive association between mPAEs mixture and URSA. The URSA cases showed significantly higher concentrations of di-(2-ethylhexyl) phthalate (DEHP) than the controls. This was consistent with the health risk assessment, which suggested that DEHP is the main contributors to potential non-carcinogenic risk. DEHP accounted for over 80% of total risk. The large case-control study results suggest that PAE exposure may increase the risk of URSA, and that policy-makers and public health experts should pay more attention to DEHP exposure.

Association of emerging and legacy per- and polyfluoroalkyl substances with unexplained recurrent spontaneous abortion.

Emerging per- and polyfluoroalkyl substances (PFAS) alternatives are increasingly used in daily life. Although legacy PFAS have been associated with miscarriage in previous studies, it remains unknown whether exposure to emerging and legacy PFAS has any impact on the risk of unexplained recurrent spontaneous abortion (URSA). We conducted a case-control study with 464 URSA cases who had at least 2 unexplained miscarriages and 440 normal controls who had at least one normal livebirth. Concentrations of 21 PFAS in plasma, including three emerging PFAS alternatives, eight linear and branched PFAS isomers, four short-chain PFAS, and six legacy PFAS, were measured by ultra-performance liquid chromatography coupled with a tandem mass spectrometry (UPLC-MS/MS). Multiple logistic regression was applied to evaluate the relationship between PFAS and URSA risk. Perfluorooctanoic acid (PFOA, median: 6.18 ng/mL), perfluorooctane sulfonate (PFOS, median: 4.10 ng/mL), and 6:2 chlorinated perfluoroalkyl ether sulfonic acid (6:2 Cl-PFESA, median: 2.27 ng/mL) were the predominant PFAS in the controls. Exposure to 6:2 Cl-PFESA [adjusted odds ratio (aOR) = 1.18 (95% CI: 1.00, 1.39)] and hexafluoropropylene oxide dimer acid (HFPO-DA) [aOR = 1.35 (95% CI: 1.15, 1.59)] were significantly associated with increased risks of URSA. Women with older age (>30 years old) had a stronger association between PFAS and URSA. Our results suggest that emerging PFAS alternatives may be an important risk factor for URSA.

Alteration of vaginal microbiota in patients with recurrent miscarriage.

The aim of this study was to characterise the structure of vaginal microbiota in unexplained recurrent miscarriage (RM). The vaginal bacterial communities of 16 patients with RM and 20 healthy volunteers were sampled. Then, the microbiomes of bacterial profiles of RM patients and healthy volunteers were compared by sequencing the V3-V4 regions of the bacterial 16S ribosomal RNA gene using the Illumina MiSeq platform (Illumina, San Diego, CA). Taxonomic analysis demonstrated that abundance of Lactobacillus and Gardnerella were significantly different between the RM and control groups. Furthermore, at the genus level, Lactobacillus was the most dominant genus in the two groups. Statistically significant differences were observed in three genera between RM and control groups. In the control group, two bacterial taxa were significantly more abundant (Lactobacillus and Gardnerella), while only one taxon was overrepresented in the RM group (Atopobium). These present findings provide experimental evidence supporting vaginal microbiota dysbiosis in women with RM.Impact statementWhat is already known on this subject? Currently, bacterial vaginosis is thought to be mainly due to the vaginal dysbacteriosis, which can induce unexplained recurrent miscarriage, premature rupture of membranes, low birth weight premature birth, premature birth, chorioamnionitis and series of diseases.What do the results of this study add? The current study demonstrated that Lactobacillus and Gardnerella were significantly decreased in RM patients compared to healthy control, while Atopobium was overrepresented in the RM group.What are the implications of these findings for clinical practice and/or further research? Clinically, women with RM might benefit from vaginal microbiota treatment, adjuvant therapy with Lactobacillus-based live biotherapeutics.

Reduced frequency and functional defects of CD4+CD25highCD127low/- regulatory T cells in patients with unexplained recurrent spontaneous abortion.

BACKGROUND: Unexplained recurrent spontaneous abortion (URSA) is defined as two or more consecutive pregnancy losses, generally of unknown cause; it is related to a failure of fetal-maternal immunological tolerance. Regulatory T cells (Tregs) exert immunosuppressive effects, which are essential to maintain fetal-maternal immunological tolerance and regulate immune balance. In this study, we used the specific cell-surface phenotype of CD4+CD25highCD127low/- Tregs to investigate the number and suppressive function of Tregs isolated from the peripheral blood of patients with URSA with the aim of expanding our understanding of their role in URSA. METHODS: We isolated a relatively pure population of peripheral CD4+CD25highCD127low/- Tregs and CD4+CD25- responder T cells (Tresps) from the patients with URSA and normal fertile nonpregnant control women via fluorescence-activated cell sorting. We compared the frequency, suppressive capacity, and forkhead box transcription factor P3 (FOXP3) expression of Tregs in the peripheral blood between patients with URSA and normal controls. RESULTS: The frequency of CD4+CD25highCD127low/- Tregs in the peripheral blood was lower in URSA patients than in the controls (P < 0.05). The mean fluorescence intensity of FOXP3 and FOXP3 mRNA expression in Tregs was also significantly lower in the URSA patients (P < 0.01). Tregs suppressed the activity of autologous Tresps stimulated with anti-CD3/CD28 beads in a concentration-dependent manner, with the strongest suppression occurring in cocultures with a 1:1 Treg:Tresp ratio in both groups; however, patient-derived Tregs exhibited a poorer capacity to suppress the proliferation of autologous Tresps than the Tregs from normal controls (P < 0.01). Moreover, Tregs isolated from URSA patients inhibited the proliferation of Tresps from normal controls less potently than the Tregs from normal controls (P < 0.01), and Tresps from URSA patients were less effectively suppressed by autologous Tregs than by those from normal controls (P < 0.01). Tresp activity were intact in both groups. CONCLUSIONS: We observed a lower frequency of peripheral CD4+CD25highCD127low/- Tregs with lower FOXP3 expression in the peripheral blood of URSA patients. In addition, highly purified Tregs from patients with URSA exhibited impaired suppressive effects. The defect in immune regulation in URSA patients appears to be primarily related to impaired Tregs, and not to increased resistance of Tresps to suppression. Our findings reveal a potential novel therapeutic target for URSA.

Influence of forkhead box protein 3 polymorphisms (rs2232365, rs3761548) with the outcome of pregnancy: A meta-analysis.

Dysfunction of regulatory T cells (Tregs) may contribute to certain immune-related pregnancy complications. Forkhead box protein 3 (FOXP3) is the key transcription factor of Treg. We performed a systematic review and meta-analysis to evaluate the possible association between FOXP3 polymorphisms -924A/G (rs2232365) and -3279C/A (rs3761548) and immune-related pregnancy complications. After reviewing 78 fully published studies, 10 studies fulfilled previously defined eligibility criteria and were used for meta-analysis. Two single nucleotide polymorphisms showed a significant correlation with increased or reduced risk for immune-related pregnancy complications. For rs3761548, women with allele A were significantly at a higher risk than women carrying allele C (odds ratio = 1.29, 95% confidence interval: 1.20-1.38; p = 0.001). For rs2232365, women with GG or AG genotype were at a higher risk than women with genotype AA, thereby, allele G was significantly associated with a higher risk than allele A. Our meta-analysis supports the notion that immune-related pregnancy complications might be linked to genetic variations in the FOXP3 gene.

[Association of arsenic with unexplained recurrent spontaneous abortion: a case-control study].

Objective: To explore the association of arsenic with unexplained recurrent spontaneous abortion (URSA). Methods: A case-control study was conducted to select URSA patients who were admitted to the Beijing Maternal and Child Health Care Hospital affiliated to Capital Medical University from April to October 2018 as a case group. Women who had a normal pregnancy in the Family Planning Department of the hospital but volunteered to have an abortion were selected as a control group. The case and control group were paired in a 1: 1 ratio. The inclusion criteria of the case group were patients with newly diagnosed recurrent spontaneous abortion who had clinically confirmed more than 2 spontaneous abortions and had 20 weeks prior to pregnancy, excluding patients with recurrent spontaneous abortion caused by abnormal blood coagulation (anti-phospholipid antibody positive), abnormal physiological anatomy (B-ultrasound), abnormal immune factors (anti-nuclear antibody positive, anti-cardiolipin antibody, etc.), genetic chromosomal abnormalities (karyotype analysis) and pathogenic microbial infection. The control group was matched according to the age of the case group (±3 years old) and the gestational age (±2 weeks) to exclude adverse pregnancy outcomes such as stillbirth, congenital malformation, premature delivery and low birth weight infants. A total of 192 subjects were included. Questionnaires were used to collect information of all subjects, and 12 ml of peripheral venous blood was collected to detect blood arsenic levels. Blood arsenic levels were divided into low concentration group (<1.00 µg/L), medium concentration group (1.00-1.50 µg/L) and high concentration group (>1.50 µg/L). The multivariate conditional logistic regression was performed to analyze the relationship between blood arsenic exposure and URSA and explore the influencing factors of blood Arsenic. Results: The geometric mean values of blood arsenic level in the cases group and control group were 1.68 (1.50-1.86) µg/L and 1.26 (1.17-1.37) µg/L, respectively. The blood arsenic level in the case group was significantly higher than that in the control group (P<0.05). The results of multivariate conditional logistic regression analysis showed that after adjusting for tobacco exposure during pregnancy, pre-pregnancy body mass index and the effects of residential decoration in past five years, the risk of URSA was higher in the high-concentration group compared with the low-concentration group (OR=2.56, 95%CI:1.06-6.24). Conclusion: Blood arsenic may increase the risk of URSA in women of childbearing age.

Association between LRH-1 single nucleotide polymorphisms and unexplained recurrent spontaneous abortion in Chinese Han couples.

To explore the genetic relationships between LRH-1 (rs2816948), CYP19 (rs727479 and rs700518), and P450scc (rs4077582) as a potential mechanism behind unexplained recurrent spontaneous abortions in a Chinese Han population. A case-control study was used and featured two groups: Patients with unexplained recurrent miscarriage (n = 82, abortion group) and those who voluntary surrendered of a normal early pregnancy (n = 97, control group). Abortion villi samples were obtained from all patients. Genomic DNA was later extracted and sequenced, after which statistical analyses performed to assess the relationship between single nucleotide polymorphisms and unexplained recurrent spontaneous abortions. There were significant differences in the genotypic and allelic distribution (p < .05) for CYP19 (rs727479) between the abortion and the control groups. There were no significant differences in the genotypic or allelic distributions (p > .05) for either the LRH-1 (rs2816948) or CYP19 (rs700518). There were also significant genotypic differences (p < .05) for P450scc (rs4077582), but no significant differences for its allelic distribution (p > .05). There was a significant correlation between the occurrence of unexplained recurrent spontaneous abortion and CYP19 (rs727479) single nucleotide polymorphisms.

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

A study on phthalate metabolites, bisphenol A and nonylphenol in the urine of Chinese women with unexplained recurrent spontaneous abortion.

Humans are widely exposed to phthalates, bisphenol A and nonylphenol owing to the ubiquitous use of these chemicals in consumer products. Increasing attention has been paid to exposure to phthalates, bisphenol A and nonylphenol because of their potential adverse effects on human fertility. A validated method was developed to investigate the three classes of environmental estrogen, mentioned above, in the urine of Chinese women of Nanjing area with unexplained recurrent spontaneous abortion. Solid-phase extraction coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used. In this method, amounts of bisphenol A (BPA), nonylphenol (NP) and four phthalate metabolites, mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), mono-benzyl phthalate (MBzP) and mono-2-ethylhexyl phthalate (MEHP), along with their isotope labeled internal standards, were measured using UPLC-MS/MS operated in negative electrospray ionization multiple reaction monitoring mode. The limits of detection were 0.3ng/mL for the four phthalate metabolites, and 0.5ng/mL for bisphenol A and nonylphenol. For women with unexplained recurrent spontaneous abortion, the mean concentrations of MBP, MiBP, MBzP, MEHP, BPA and 4-n-NP were 6.52±6.04, 5.51±4.19, 0.53±0.42, 10.12±4.16, 7.13±7.42, 0.41±0.49ng/mL (mean±SD), respectively. For the control group, the mean concentrations of the corresponding analytes were 4.15±3.57, 2.96±3.30, 0.46±0.49, 6.50±2.81, 4.43±2.23,0.48±0.43ng/mL (mean±SD), respectively. Levels of MiBP and MEHP were significantly different between the two groups, using Wilcoxon rank sum tests. This method can be applied in epidemiological studies to explore the association between exposure to environmental estrogens and relevant adverse outcomes.

Correlation between protein expression of FOXP3 and level of FOXP3 promoter methylation in recurrent spontaneous abortion.

AIM: The aim of this study was to investigate the level of Forkhead box P3 (FOXP3) promoter methylation and protein expression in recurrent spontaneous abortion and to elucidate the pathogenesis of unexplained recurrent spontaneous abortion (URSA). METHODS: We assessed a total of 56 URSA patients with a normal embryo, 24 recurrent spontaneous abortion (RSA) patients with an abnormal embryo (as control group 1), and 39 normal pregnant women (as control group 2). The expression of FOXP3 protein in deciduas was assessed through Western blot, and the level of FOXP3 promoter methylation was detected using bisulfite-assisted genomic sequencing polymerase chain reaction. RESULTS: The expressing quantity of FOXP3 protein in the URSA group was significantly lower than that in control groups 1 and 2, both with a P-value < 0.05. By contrast, no statistical difference was observed in the expressing quantity of FOXP3 protein of the two control groups (P = 0.212). The FOXP3 promoter methylation level in the URSA group was significantly higher than that in the two control groups, both of which exhibited a statistical difference of P-values < 0.05. Meanwhile, no statistical difference was observed in the FOXP3 promoter methylation level of the two control groups (P = 0.141). A negative correlation was found between the FOXP3 promoter methylation level and the expressing quantity of FOXP3 protein (r = -0.861, P < 0.05). CONCLUSION: Increasing FOXP3 promoter methylation levels may cause abnormal immune tolerance through the downregulation expression of the FOXP3 protein, which in turn leads to URSA.

Effectiveness and potential mechanisms of intralipid in treating unexplained recurrent spontaneous abortion.

AIM: Abnormal natural killer (NK) cell activity has been suggested to be a high-risk factor associated with unexplained recurrent spontaneous abortion (URSA). Intralipid, like immunoglobulin, is able to lower the activity of NK cells, which has been reported to be useful for improving URSA outcomes in pregnancy. This study aimed to determine whether intralipid could be used as an alternative treatment to intravenous immunoglobulin (IVIG) which is expensive and has many side-effects. METHODS: A prospective, randomized clinical trial was conducted from December 2010 to December 2012. Eligible participants were matched and sorted randomly into the intralipid and the IVIG group. The primary outcome was the rate of successful pregnancy. In addition, comparisons of peripheral NK cell activities were accessed by flow cytometry. Moreover, the effects of intralipid on trophoblasts were investigated using a Matrigel assay with the JEG-3 cell line. RESULTS: Seventy-six patients in the intralipid group and 78 in the IVIG group completed the trial. There were no statistically significant differences in successful pregnancy rates between the two groups (92.1 vs 88.2 %, P = 0.415). The reduced NK cell concentrations revealed the cytotoxic effects of the treatments in both groups. The invasive ability of JEG-3 cells was inhibited during co-culture with patient PBMCs. However, the inhibitory effect could be alleviated if the patient PBMCs were stimulated with intralipid. CONCLUSIONS: Intralipid can be used as an alternative treatment to IVIG for URSA, and its potential mechanism of action may occur by regulating NK cell function and promoting trophoblast invasion.

Baicalin ameliorates deficient decidualization in URSA by regulating mitochondrial fission induced necroptosis.

Unexplained recurrent spontaneous abortion (URSA) is a common complication of pregnancy that affects the health of pregnant women. Deficient endometrial decidualization has been associated with URSA. However, the underlying mechanism is poorly understood. This study aims to explore the mechanisms of mitochondrial fission induced necroptosis in deficient decidualization in URSA, and explore the regulation of baicalin on this mechanism. Initially, decidual tissues were collected from patients with URSA and health controls. Subsequently, in vitro induced decidualization model of Telomerase-Immortalized Human Endometrial Stromal Cells (T-hESCs) was constructed. Additionally, murine models of URSA (CBA/J × DBA/2) and normal pregnancy (CBA/J × BALB/c) were established, respectively. The level of decidualization, necroptosis, and mitochondrial fission of decidual tissues from clinical samples were detected. The function of mitochondrial fission on necroptosis during decidualization in T-hESCs was assessed by enhancing or inhibiting mitochondrial fission or necroptosis. Finally, CBA/J × DBA/2 pregnant mice were administrated with different doses of baicalin or saline, and the expression of mitochondrial fission, necroptosis, and decidualization markers were verified. The results of the study demonstrated a significant decrease in decidualization markers in the decidual tissues of URSA patients (P < 0.05), along with an increase in the incidence of cell necroptosis (P < 0.05) and hyperactive mitochondrial fission (P < 0.05). In vitro experiments, LPS was induced to trigger necroptosis of T-hESCs during induced decidualization, and decidualization markers IGFBP1 and PRL were subsequently decreased (P < 0.05). Besides, the mitochondrial fission agonist Tyrphostin A9 was found to promote the level of necroptosis (P < 0.05) and induced deficient decidualization (P < 0.05), which could be rescued by mitochondrial fission inhibitor Mdivi-1 and necroptosis inhibitor Nec-1 (P < 0.05). In addition, baicalin was shown to reduce hyperactive mitochondrial fission (P < 0.05), necroptosis (P < 0.05) and ameliorate deficient decidualization in vitro and in URSA murine models (P < 0.05). Collectively, baicalin shows potential in ameliorating deficient decidualization in URSA by inhibiting mitochondrial fission-triggered necroptosis.

Prediction of Unexplained Recurrent Miscarriages Using Thromboelastography.

Objective: This study investigates the thromboelastography (TEG) changes in patients with unexplained recurrent spontaneous abortion (URSA) to identify effective diagnostic markers for URSA. Methods: We retrospectively analyzed 160 URSA patients from the Gynecology Department of the First People's Hospital of Lianyungang (June 2017 - June 2020) and compared them with 190 healthy, fertile women without adverse pregnancy histories (control group). TEG parameters were assessed using logistic regression, applying stepwise selection for model optimization. Model performance was evaluated using Receiver Operating Characteristic (ROC) curves, determining sensitivity and specificity. The Youden index identified optimal cut points for predictive probabilities. Results: Significant differences were observed between the URSA and control groups in coagulation reaction time (R), clot formation time (K), clot formation rate (Angle-α), and maximum clot strength (MA) (P<0.05). Multivariable logistic regression identified R, Angle-α, and MA as independent URSA risk factors. The model demonstrated excellent discrimination (AUC: 0.940; 95% CI: 0.918-0.962). The optimal cut point of predictive probability (Youden index) was P=0.355, yielding a sensitivity of 0.925 and specificity of 0.795. Conclusion: URSA patients exhibit a hypercoagulable state even when not pregnant. More research is needed to validate our findings and explore the potential clinical implications of anticoagulants in treating URSA.