Pathogenesis Of Recurrent Spontaneous Abortion Research Articles

In situ detection of miR-93/VEGFR2 by nanosensor and inhibition of trophoblast apoptosis

We investigated the pathogenesis of recurrent spontaneous abortion (RSA) and evaluated the therapeutic effect of Pill Nourishing Kidney and Fetus (PNKF). A sensitive DNA sensor was used to detect miR-93 expression and vascular endothelial growth factor receptor 2 (VEGFR2). We testing the penetration capacity and physicochemical properties of the DNA sensor and found that the sensor had higher penetration and good stability and effectively detected miR-93 and VEGFR2 at different reaction temperatures and mediums with similar fluorescence signals. The sensor detected higher miR-93 expression, increased apoptosis, and negative regulation of VEGFR2 in patients with RSA. Treatment with PNKF of cultured trophoblast cells collected from patients with RSA were treated with PNKF significantly inhibited trophoblast cell apoptosis and promoted trophoblast cell proliferation by reducing miR-93 expression and promoting VEGFR2 expression. In conclusion, PNKF can improve the proliferation and inhibit apoptosis of trophoblast cells by suppressing miR-93 and promoting VEGFR2. Furthermore, the miR-93/VEGFR2 sensor demonstrated high sensitivity that has potential applications in clinical practice.

IGF2BP3 participates in the pathogenesis of recurrent spontaneous abortion by regulating ferroptosis

The aberrant invasive capability of trophoblast cells is widely acknowledged as a primary mechanism underlying RSA. Recently, IGF2BP3 has been implicated in various cancers due to its influence on cellular invasion and migration. However, whether IGF2BP3 involve in the occurrence of RSA and the specific functions it assumes in the development of RSA remain elusive. In our study, we firstly collected villous tissues from RSA and those with normal pregnancies individuals to performed Protein sequencing and then detected the expression of IGF2BP3 through Western blot, qRT-PCR and immunohistochemistry. Secondly, we analyzed the single-cell data (GSE214607) to assess the expression of IGF2BP3 in invasive EVT trophoblasts. Thirdly, we utilized lentivirus technology to establish HTR-8/SVneo cell lines with stable IGF2BP3 knockdown and RNA-seq analysis was employed to investigate the GO functional pathway enrichment of IGF2BP3. Meanwhile, the effect of IGF2BP3 knockdown on trophoblast cells apoptosis, migration, and ferroptosis was evaluated through functional experiments. Additionally, LPS-induced abortion animal model was constructed to evaluate IGF2BP3 expression in placental tissues. A significant downregulation of IGF2BP3 was observed in the villous tissues of RSA patient, a finding corroborated by subsequent single cell sequencing results. Furthermore, it suggested that IGF2BP3 may be involved in the migration and apoptotic processes of trophoblast cells. Mechanistic research indicated that IGF2BP3 knockdown could compromise GPX4 mRNA stability, leading to the promotion of ferroptosis. Finally, our investigation observed the down-regulation of IGF2BP3 expression in placental villous tissues of an LPS-induced abortion animal model. Our findings revealed that IGF2BP3 was downregulated in the villous tissues of RSA patients. Mechanically, down-regulation of IGF2BP3 may induce RSA by promoting GPX4-mediated ferroptosis and inhibiting trophoblast invasion and migration. Our study may provide new targets and research directions for the pathogenesis of RSA.

A preliminary study unveils CISD2 as a ferroptosis-related therapeutic target for recurrent spontaneous abortion through immunological analysis and two-sample mendelian randomization

Recurrent spontaneous abortion (RSA) affects approximately 1 % of women striving for conception, posing a significant clinical challenge. This study aimed to identify a prognostic signature in RSA and elucidate its molecular mechanisms. Prognostic gene impacts were further assessed in HTR-8/SVneo and human primary extravillous trophoblast (EVT) cells in vitro experiments. A total of 6168 differentially expressed genes (DEGs) were identified, including 3035 upregulated and 3133 downregulated genes. WGCNA pinpointed 8 significant modules and 31 ferroptosis-related DEGs in RSA. Optimal clustering classified RSA patients into three distinct subgroups, showing notable differences in immune cell composition. Six feature genes (AEBP2, CISD2, PML, RGS4, SRSF9, STK11) were identified. The diagnostic model showed high predictive capabilities (AUC: 0.966). Mendelian randomization indicated a significant association between CISD2 levels and RSA (OR: 1.069, P-value: 0.049). Furthermore, the downregulation of CISD2 promotes ferroptosis in HTR-8/SVneo and human primary EVT cells. CISD2 emerged as a pivotal gene in RSA, serving as a ferroptosis-related therapeutic target. The diagnostic model based on gene expression and Mendelian randomization provides novel insights into the pathogenesis of RSA.

Kisspeptin prevents pregnancy loss by modulating the immune microenvironment at the maternal-fetal interface in recurrent spontaneous abortion.

Immune factors are crucial in the development of recurrent spontaneous abortion (RSA). This study aimed to investigate whether kisspeptin regulates immune cells at the maternal-fetal interface and whether G protein-coupled receptor 54 (GPR54) is involved in this process, through which it contributes to the pathogenesis of RSA. Normal pregnancy (NP) (CBA/J × BALB/c) and RSA (CBA/J × DBA/2) mouse models were established. NP mice received tail vein injections of PBS and KP234 (blocker of kisspeptin receptor), whereas RSA mice received PBS and KP10 (active fragment of kisspeptin). The changes in immune cells in mouse spleen and uterus were assessed using flow cytometry and immunofluorescence. The expression of critical cytokines was examined by flow cytometry, ELISA, Western blotting, and qPCR. Immunofluorescence was employed to detect the coexpression of FOXP3 and GPR54. The findings revealed that the proportion of Treg cells, MDSCs, and M2 macrophages in RSA mice was lower than that in NP mice, but it increased following the tail vein injection of KP10. Conversely, the proportion of these cells was reduced in NP mice after the injection of KP234. However, the trend of γδT cell proportion change is contrary to these cells. Furthermore, FOXP3 and GPR54 were coexpressed in mouse spleen and uterus Treg cells as well as in the human decidua samples. Our results suggest that kisspeptin potentially participates in the pathogenesis of RSA by influencing immune cell subsets at the maternal-fetal interface, including Treg cells, MDSC cells, γδT cells, and M2 macrophages.

Study on the clinical value of Vitamin D in recurrent spontaneous abortion: Revisiting the Importance of Vitamin D.

This study explores the possible pathogenesis of recurrent spontaneous abortion (RSA) caused by vitamin D (VD), provides evidence-based bases for prevention and treatment of RSA, improves female reproductive health. This study randomly selected 305 patients without spontaneous abortion (SA0), 216 patients with a spontaneous abortion (SA1) and 200 patients with RSA from 1421 women of childbearing age who visited the RSA specialty clinic of Hangzhou First People's Hospital from January 2021 to June 2023 to conduct a prospective clinical study. Then, we collected the data of clinical diagnosis and treatment, conducted intervention and follow-up, and finally executed statistical analysis. (1) RSA patients were significantly older than the other two groups. (2) The rates of VD deficiency in SA1 and RSA patients were significantly higher than those in SA0. (3) When BMI<20 or>24kg/m2 , there were abnormal increase in VD and increased number of spontaneous abortions. (4) The bilateral S/D of the VD-sufficient, VD-insufficient and VD-deficient groups gradually increased with statistical significance (p ≤ .018). (5) Among the 65 cases undergoing embryo chromosome examinations, chromosomal abnormalities accounted for 55.38% and 69.05% in RSA patients. (6) Among 186 patients with abnormal ACA, there was a certain negative correlation between ACA and VD, which was stronger among RSA patients. Moreover, ACA significantly decreased (p<.001) after effectively supplementing VD, and the miscarriage rate of re-pregnancy also decreased. The rate of VD deficiency is higher in RSA patients. VD deficiency may be related to the age of women of childbearing age and too low or high BMI, and may cause abnormal plasma antiphospholipid antibodies, increased uterine artery resistance and abnormal chromosomal division during fertilization, leading to spontaneous abortion and even RSA. The improvement of VD deficiency may reduce the risk of RSA occurrence.

Mechanisms underlying the therapeutic effects of Semen cuscutae in treating recurrent spontaneous abortion based on network pharmacology and molecular docking.

Background: This paper aims to analyse the active components of Semen cuscutae (SC) by network pharmacology and screen the most stable compounds with tumour necrosis factor-alpha (TNF-α) by molecular docking to explore the mechanisms of SC treatment of recurrent spontaneous abortion (RSA) and provide a theoretical basis for drug development. Methods: The active compounds of SC and the potential inflammatory targets of RSA were obtained from the Traditional Chinese Medicine Systems Pharmacology database and GeneCards, respectively. The RSA-SC target gene interaction network was obtained and visualized using the STRING database and Cytoscape software. GO and KEGG pathway enrichment analyses were obtained from DAVID to further explore the RSA mechanism and therapeutic effects of SC. Interactions between TNF-α and drugs were analysed by molecular docking. Treatment of human trophoblast cells with sesamin and TNF-α was carried out to detect their proliferative and apoptotic abilities, and WB assay was carried out to detect EGFR, PTGS2, and CASP3 protein expression. Results: Ten compounds and 128 target genes were screened from SC, of which 79 overlapped with RSA target inflammatory genes, which were considered potential therapeutic targets. Network pharmacological analysis showed that sesamin, matrine, matrol, and other SC compounds had a good correlation with the inflammatory target genes of RSA. Related genes included PGR, PTGS1, PTGS2, TGFB1, and CHRNA7. Several signalling pathways are involved in the pathogenesis of RSA, such as the TNF-α signalling pathway, HIF-1 signalling pathway, oestrogen signalling pathway, proteoglycans in cancer cells, and FoxO signalling pathway. Molecular docking results suggested that sesamin was the most suitable natural tumour necrosis factor inhibitor (TNFi). Sesamin can promote proliferation and inhibit apoptosis in human trophoblasts by downregulating EGFR and CASP3 expression and upregulating PTGS2 expression. Conclusion: Our findings play an important role and basis for further research into the molecular mechanism of SC treatment of RSA and drug development of TNFi.

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.

FKBP5 regulates trophoblast-macrophage crosstalk in recurrent spontaneous abortion through PI3K/AKT and NF-κB signaling pathways

FK506-binding protein 5 (FKBP5) contributes to many diseases; However, it remains unclear whether FKBP5 is relevant to recurrent spontaneous abortion (RSA) and the mechanisms by which it is involved in maternal-fetal immunological tolerance. Placental tissue was collected in women with normal pregnancy and RSA and examined for FKBP5 expression. Human trophoblast cell lines and THP-1-derived M0 macrophages were used to explore the role of FKBP5 in RSA and its mechanism. The role of FKBP5 on pregnancy outcomes was assessed using a mouse model of miscarriage. This study found that upregulation of FKBP5 at the placental interface is involved in the pathogenesis of RSA by depressing trophoblast function and promoting M1-type macrophage polarization. First, FKBP5 expression was upregulated in the villi of RSA, and FKBP5 regulated trophoblast function by inhibiting HAPLN1 expression through suppression of PI3K/AKT signaling. In addition, FKBP5 inhibited trophoblast IL-6 secretion by suppressing PI3K/AKT signaling, thereby promoting macrophage polarization toward the M1 phenotype. Meanwhile, FKBP5 was significantly elevated in decidual macrophages from patients with RSA and promoted M1 macrophage polarization via ROS/NF-κB signaling and further inhibited trophoblast function. Finally, FKBP5 inhibitors improved embryo resorption rate in miscarried mice. In conclusion, FKBP5 is essential in maintaining pregnancy and trophoblast-macrophage crosstalk in the maternal-fetal interface, which may be a potential target for diagnosing and treating RSA.

Omega-3 fatty acid supplements and recurrent miscarriage: A perspective on potential mechanisms and clinical evidence.

Recurrent miscarriage (RM) affects approximately 1%-5% of couples worldwide. Due to its complicated etiologies, the treatments for RM also vary greatly, including surgery for anatomic factors such as septate uterus and uterine adhesions, thyroid modulation drugs for hyperthyroidism and hypothyroidism, and aspirin and low molecular weight heparin for antiphospholipid syndrome. However, these treatment modalities are still insufficient to solve RM. Omega-3 fatty acids are reported to modulate the dysregulation of immune cells, oxidative stress, endocrine disorders, inflammation, etc., which are closely associated with the pathogenesis of RM. However, there is a lack of a systematic description of the involvement of omega-3 fatty acids in treating RM, and the underlying mechanisms are also not clear. In this review, we sought to determine the potential mechanisms that are highly associated with the pathogenesis of RM and the regulation of omega-3 fatty acids on these mechanisms. In addition, we also highlighted the direct and indirect clinical evidence of omega-3 fatty acid supplements to treat RM, which might encourage the application of omega-3 fatty acids to treat RM, thus improving pregnancy outcomes.

MITA Promotes Macrophage Proinflammatory Polarization and Its circRNA-Related Regulatory Mechanism in Recurrent Miscarriage.

MITA (also called STING), a master regulator of DNA-mediated innate immune activation, is a potential therapeutic target for viral infection and virus-related diseases. The circRNA-mediated ceRNA network plays important roles in gene regulation and may contribute to many human diseases. However, the relationship between MITA and recurrent miscarriage (RM) and its circRNA-related regulatory mechanisms remain unclear. In this study, we validated that the decidual M1/M2 ratio was upregulated in RM patients, suggesting the vital roles of decidual macrophages in the pathogenesis of RM. We found that MITA was highly expressed in decidual macrophages of RM patients and validated that MITA could promote apoptosis and macrophage proinflammatory polarization in THP-1-derived macrophage (TDM) cells. Using circRNA sequencing and bioinformatic analysis, we screened out a novel circRNA (circKIAA0391) that is overexpressed in decidual macrophages of RM patients. Mechanistically, we found that circKIAA0391 could promote the apoptosis and proinflammatory polarization of TDM cells by sponging the miR-512-5p/MITA axis. This study provides a theoretical basis for further understanding the impact of MITA on macrophages and its circRNA-related regulatory mechanisms, which may have a crucial immunomodulatory function in the pathophysiology of RM.

SHP2 participates in decidualization by activating ERK to maintain normal nuclear localization of progesterone receptor.

The establishment and maintenance of embryo implantation and pregnancy require decidualization of endometrial stromal cells. This paper reveals that SHP2 ensures the correct subcellular localization of progesterone receptor, thereby safeguarding the process of decidualization. Decidualization is the process of conversion of endometrial stromal cells into decidual stromal cells, which is caused by progesterone production that begins during the luteal phase of the menstrual cycle and then increases throughout pregnancy dedicated to support embryonic development. Decidualization deficiency is closely associated with various pregnancy complications, such as recurrent miscarriage (RM). Here, we reported that Src-homology-2-containing phospho-tyrosine phosphatase (SHP2), a key regulator in the signal transduction process downstream of various receptors, plays an indispensable role in decidualization. SHP2 expression was upregulated during decidualization. SHP2 inhibitor RMC-4550 and shRNA-mediated SHP2 reduction resulted in a decreased level of phosphorylation of ERK and aberrant cytoplasmic localization of progesterone receptor (PR), coinciding with reduced expression of IGFBP1 and various other target genes of decidualization. Solely inhibiting ERK activity recapitulated these observations. Administration of RMC-4550 led to decidualization deficiency and embryo absorption in mice. Moreover, reduced expression of SHP2 was detected in the decidua of RM patients. Our results revealed that SHP2 is key to PR's nuclear localization, thereby indispensable for decidualization and that reduced expression of SHP2 might be engaged in the pathogenesis of RM.

Understanding the potential immunogenetic role of TNFα-308 polymorphism in the pathogenesis of recurrent miscarriage

BackgroundRecurrent miscarriage (RM) represents the spontaneous termination of two or more successive pregnancies. TNFα is a proinflammatory cytokine that is often considered harmful for embryonic development when expressed beyond normal levels. AimThe study was conducted to assess the association between TNFα-308 polymorphism and RM pathogenesis. MethodsSamples of blood were obtained from patients and controls through venipuncture. The levels of TNFα in serum were measured by ELISA. TNFα gene promoter-associated single-nucleotide polymorphism was investigated with polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques with precise primers and the restriction endonuclease, NcoI. ResultsSerum TNFα levels in patients were considerably high (p < 0.05) than controls. The genotype and allele frequencies for TNFα gene polymorphism differs significantly (p = 0.0089; p = 0.0043 respectively) between patients and controls. The TNFα-308 SNP exhibited a link with higher RM risk in heterozygous (GG vs. GA; OR: 3.086, 95% CI: 1.475–6.480; p: 0.0027), dominant (GG vs. GA + AA; OR: 2.919, 95% CI: 1.410–6.056, p: 0.0038), and allelic/codominant (G vs. A; OR: 2.449, 95% CI: 1.313–4.644, p: 0.0064) models. However, this SNP showed an insignificant association with higher and lower RM risk in homozygous (GG vs. AA; OR: 1.915, 95% CI: 0.3804–10.99, p: 0.6560) and recessive (AA vs. GA + GG; OR: 0.6596, 95% CI: 0.1152–3.297, p: >0.9999) models, respectively. Further, the TNFα-308G/A genotype frequencies were in concord with HWE both in the controls (χ2 = 3.235; p = 0.1985) and the patients (χ2 = 0.0117; p = 0.9942). ConclusionThe serum TNFα levels were significantly higher in the patients than the controls. The genotyping analysis also demonstrated that TNFα-308G/A SNP significantly increases the overall risk of RM, suggesting that the SNP modulates the TNFα gene expression and thereby increases serum TNFα levels that adversely affect the pregnancy outcome.

Research progress in the mechanism of action of NK cell-related receptors during recurrent spontaneous abortion

Recurrent spontaneous abortion (RSA) refers to multiple spontaneous abortions with the same sexual partner; In 2020, Chinese expert consensus suggested that it should be defined as the loss of the embryo or fetus before 28 weeks of gestation with the same sexual partner for two or more times in a row, including the biochemical pregnancy that occurred several times in a row. The pathogenesis of RSA is complex and diverse. At present, the pathogenesis factors have been widely recognized by the industry, including genetic factors, endocrine factors, anatomical abnormalities, infectious factors and immune factors. Among them, the role of immune factors such as T lymphocytes, dendritic cells and natural killer cells (NK) in the occurrence and development of RSA has attracted wide attention, especially NK cells. NK cells are an important part of human immune system. The specific surface molecules of Decidual natural killer cell (dNK) play an immunomodulatory role in the invasion of trophoblast cells and the remodeling of uterine spiral artery during pregnancy, which is particularly important in the early gestation period. The function of dNK cells is tightly regulated by a balance between activation and inhibition signals transmitted by the various family of receptors (NK cell receptors, NKRS) that DNK cells express. In this paper, the existing research progress of decidual NK cells on the mechanism of its receptors in the process of recurrent spontaneous abortion will be reviewed as follows.

Deletion of Insulin-like growth factor II mRNA-binding protein 3 participates in the pathogenesis of recurrent spontaneous abortion by inhibiting IL-10 secretion and inducing M1 polarization.

Insulin-like growth factor II mRNA-binding protein 3 (IGF2BP3) has been proved to affect trophoblast function and embryonic development, but its role and potential mechanism in recurrent spontaneous abortion (RSA) are not clear. RSA is a complex reproductive disease, causing physical and mental damage to patients. In recent years, many studies have found that immune microenvironment is vital to maintain successful pregnancy in the maternal fetal interface. Therefore, this study aims to explore the role of IGF2BP3 in affecting macrophage polarization and its possible mechanism. In this article, we found that IGF2BP3 expression was decreased in placental villous samples of human and RSA mouse model, and knockdown of IGF2BP3 in HTR8/SVneo cells promotes M1 Mφ polarization. Combining with RNA sequencing analysis, we found that IGF2BP3 may regulate the Mφ polarization by affecting the expression of trophoblast cytokines, especially IL-10 secretion. Further mechanistic studies showed that knockdown of IGF2BP3 decreased expression of IL-10 by activating NF-κB pathway. Moreover, we found that M2 Mφ promote trophoblast invasion not IGF2BP3 dependent. Our study reveals the interaction between trophoblast cells and macrophages at the maternal-fetal interface of RSA patients, and will provide theoretical guidance for its diagnosis and treatment of RSA patients.

Immunogenetic Role of IL17A Polymorphism in the Pathogenesis of Recurrent Miscarriage.

Interleukin-17A (IL17A) is a proinflammatory cytokine and is assumed to play an important role in fetal rejection. In order to evaluate the potential role of IL17A polymorphism in the pathogenesis of recurrent miscarriage (RM), serum IL17A levels were estimated by ELISA. Single-nucleotide polymorphism was assessed by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) using gene-specific primers and the EcoNI restriction enzyme. Serum IL17A levels were nonsignificantly (p &gt; 0.5) low in RM patients compared with the control group. IL17A gene amplification by PCR yielded the undigested product of 815 bp, and its digestion with EcoNI enzyme produced 815, 529, 286, and 270 bp fragments for the GG genotype; 529, 286, and 270 bp fragments for the GA genotype; and 529 and 286 bp fragments for the AA genotype. The genotype frequency between the RM and control groups exhibited a significant difference (p = 0.001), whereas no significant difference was observed between allele frequencies in the two groups (p = 0.0954). These data suggest that the IL17A gene polymorphism exhibits no significant effect on IL17A gene expression. However, it significantly decreases and increases RM risk in the homozygous and recessive models, suggesting its potential pregnancy-protecting and -harming roles in the AA and GA + GG genotypes, respectively.

Silencing of LncRNA SNHG6 protects trophoblast cells through regulating miR-101-3p/OTUD3 axis in unexplained recurrent spontaneous abortion.

Recurrent spontaneous abortion (RSA) is a gestational disease with complex pathogenesis, and trophoblast cells are closely involved in the pathogenesis of RSA. This study aimed to explore the regulatory effects and mechanisms of SNHG6 on trophoblast cells. The expression of SNHG6, miR-101-3p, and OTUD3 were detected in villous tissues from patients with unexplained RSA and normal pregnant women with induced abortion by qRT-PCR. The target relationships between miR-101-3p and SNHG6/OTUD3 were confirmed by dual-luciferase reporter assay. The viability, migration, and apoptosis of trophoblast cells were measured by MTT, wound healing, and flow cytometry assays, respectively. Western blot was performed to detect the protein expression of OTUD3, Ki-67, Bax, and Bcl-2. The results showed that SNHG6 and OTUD3 were up-regulated, and miR-101-3p was down-regulated in RSA patients. MiR-101-3p was a target of SNHG6, and OTUD3 was a target of miR-101-3p. There were negative correlations between the expression of miR-101-3p and OTUD3/SNHG6 in RSA patients. In addition, both SNHG6 silencing and miR-101-3p overexpression could increase cell viability and migration, decrease cell apoptosis, up-regulate Ki-67 and Bcl-2, and down-regulate Bax in HTR-8/SVneo cells. The effects of SNHG6 silencing on HTR-8/SVneo cells were reversed by miR-101-3p silencing or OTUD3 overexpression. To sum up, silencing of SNHG6 enhanced the viability and migration, and inhibited the apoptosis of trophoblast cells through regulating miR-101-3p/OTUD3. SNHG6/miR-101-3p/OTUD3 may be potential targets for the prevention of unexplained RSA.

O-252 Next generation sequencing analysis of the seminal microbiome in male partners of women with idiopathic recurrent pregnancy loss: results of a prospective cohort study

Abstract Study question Is there an association between the semen microbiome, seminal reactive oxygen species (ROS) and DNA fragmentation in men with recurrent pregnancy loss (RPL)? Summary answer This pilot study outlines the subtle role that microbiota play in influencing ROS and sperm DNA damage for male partners of women with RPL What is known already RPL is defined as the loss of two or more consecutive pregnancies. This devastating condition impacts approximately 1% of couples. Paternal causes are not routinely screened for and an underlying cause is not found in up to 50% of cases. Recent studies have reported an association between elevated seminal ROS and sperm DNA fragmentation in the male partner, and RPL. We hypothesised that seminal microbiota contribute to increased ROS and sperm DNA damage. To test this, we investigated the relationship between seminal bacterial composition and ROS levels in men with proven fertility versus men with a history of RPL. Study design, size, duration We conducted a prospective, case-control study and recruited participants between November 2018 and March 2020 at Imperial College Healthcare NHS Trust. A total of 109 men participated in the study; 46 men with RPL and 63 men with proven fertility and no history of RPL. Each participant attended for a single study visit which consisted of a full medical history, assessment of testicular volume, height, weight, blood samples and production of a semen sample. Participants/materials, setting, methods Routine semen analysis (WHO) and endocrine and lipid profiles were performed for all patients. Semen ROS and DNA fragmentation were performed (luminol and TUNEL methodologies, respectively). ROS were classified as high (&amp;gt;3.77 RLU/s) or low (&amp;lt;3.77 RLU/s). Metataxonomic profiling of samples was performed using Illumina Miseq-based sequencing of the V1-V2 hypervariable regions of bacterial 16S rRNA gene amplicons. Multivariate and univariate modelling was performed to explore associations between metataxonomic profiles, ROS levels and clinical metadata. Main results and the role of chance Men with RPL had higher mean semen volume (p = 0.02) and increased prevalence of high ROS (p = 0.02, Fisher's exact) compared with controls; but other clinical characteristics were similar between groups. A total of 3,700,136 high quality sequence reads were generated for the dataset with an average of 33,946 reads/sample. Hierarchical clustering of bacterial genera relative abundances identified 4 distinct microbial signatures characterised by high relative abundance of 1. Streptococcus, 2. Lactobacillus and Gardnerella, 3. polymicrobial (including Prevotella), and 4. Corynebacterium and Finegoldia. Prevalences of these groups were similar in control and RPL groups (p = 0.11). Additionally, no association between the bacterial genera groups and elevated ROS, DNA fragmentation, or clinical factors such as age, ethnicity, or semen volume were observed (chi-square tests). At species taxonomy level relative abundance of L. crispatus was higher in controls compared to RPL, but did not withstand false discovery rate correction for multiple testing (p = 0.006, q = 0.67). Higher relative abundance of Microbacterium was detected in semen samples with high DNA fragmentation (p = 8.7E-4, q = 0.08). This relationship was even stronger within the RPL cohort (p = 2.8E-5, q = 0.002). No significant enrichment of specific taxa was observed between high or low ROS samples however, low ROS was associated with Corynebacterium relative abundance &amp;gt;20%. Limitations, reasons for caution More patients are required to enhance statistical power. Duplicate sample collection may establish the robustness of seminal compositions observed. Time since last sexual intercourse samples may affect the analysis. Concomitant analysis of the vaginal microbiome of female partners may improve our understanding of how partners may affect each other’s fertility. Wider implications of the findings Our data suggests interactions between microbiota composition, ROS and sperm DNA damage which may be implicated in the pathogenesis of recurrent miscarriage. Further studies are needed to determine if seminal microbiota play causal roles in RPL, and whether interventions modifying the seminal microbiome may modify pregnancy outcomes in affected couples Trial registration number not applicable

The haplotypes GCA and ACA in ESR1 gene are associated with the susceptibility of recurrent spontaneous abortion (RSA) in Chinese Han: A case-control study and meta-analysis.

The nonstructured abstract were supplied as following: Estrogen receptor is involved in the pathogenesis of recurrent spontaneous abortion (RSA). The ESR1 and ESR2 genes can mediate nongenomic estrogen responses. This study aimed to assess the genetic association between the ESR1 and ESR2 genes polymorphisms and RSA susceptibility in a Chinese Han population. A total of 258 women who had experienced RSA and 264 unrelated healthy women were recruited. Genotypes of the 6 polymorphisms in the ESR1 (rs9340799, rs2234693, and rs3798759) and ESR2 genes (rs207764, rs4986938, and rs1256049) were analyzed using Snapshot technology. No association was detected between the alleles and genotypes of ESR1 rs9340799, rs2234693, and rs3798759 polymorphims and RSA risk (P > .05). Subjects carrying the haplotype of rs9340799A-rs2234693C-rs3798759A had a significantly increased RSA risk in the case group compared with the control group (P = .0005, Padj = .003, odds ratios [95% CI] = 0.35 [0.19–0.65]). However, subjects carrying the haplotype of rs9340799G-rs2234693C-rs3798759A had a significantly decreased RSA risk in the case group compared with the control group (P = .0005, Padj = .003, odds ratios [95% CI] = 2.99 [1.57–5.70]). In addition, no association was found between the alleles, genotypes, and haplotypes of ESR2 rs207764, rs4986938, rs1256049 polymorphisms and RSA risk (P > .05). In conclusion, the haplotype rs9340799A-rs2234693C-rs3798759A of ESR1 might be a risk factor. And the haplotype rs9340799G-rs2234693C-rs3798759A of ESR1 might be a protective factor for RSA in a Chinese Han population.

Overexpression of long non-coding RNA NEAT1 enhances cell viability and inhibits apoptosis in recurrent spontaneous abortion by targeting the miR-125b/BCL-2 axis.

The current study aimed to investigate the function of the long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) in the pathogenesis of recurrent spontaneous abortion (RSA) and to examine its potential mechanism. The expression of NEAT1, microRNA (miR)-125b and Bcl-2 in the villi of patients with RSAs and women with normal pregnancies was measured by reverse transcription-quantitative PCR. Cell viability was detected by the MTT assay and cell apoptosis was evaluated by flow cytometry. A dual-luciferase reporter assay was performed to verify the associations between NEAT1 and miR-125b. The protein expression of Bcl-2 was detected by western blot analysis. In the present study, the expression of NEAT1 and Bcl-2 was reduced and that of miR-125b was increased in clinical samples of villus tissues from patients with RSAs. In vitro, overexpression of NEAT1 enhanced the viability and suppressed the apoptosis of JEG-3 cells. It was demonstrated that miR-125b acts as a molecular sponge of NEAT1 and its expression was negatively regulated by NEAT1. miR-125b overexpression reduced the viability and promoted the apoptosis of JEG-3 cells. The expression of BCL-2, a target gene of miR-125b, was inversely correlated with that of miR-125b. Overexpression of miR-125b and inhibition of BCL-2 partially reversed the effect of NEAT1 overexpression on the viability and apoptosis of JEG-3 cells. Collectively, it was demonstrated that the NEAT1/miR-125b/BCL-2 axis plays a pivotal role in regulating the viability and apoptosis of JEG-3 cells. The findings of the present study offer new insights into the pathogenesis of RSA and may provide information on RSA treatment.

The Role of Dendritic Cell Subsets in Recurrent Spontaneous Abortion and the Regulatory Effect of Baicalin on It.

Recurrent spontaneous abortion (RSA) is a relevant complication of pregnancy. Aberrant dendritic cell (DC) activities and differentiation have been identified to be involved in RSA, but the underlying mechanisms remain unclear. Baicalin from Radix Scutellariae possesses a wide range of pharmacological and biological activities. However, the effect of baicalin on DC function in RSA has not been investigated. Here, we analyzed the changes of peripheral and maternal-fetal interface DC subsets and function in patients and mice with RSA, respectively. Then, we further treated RSA mice with baicalin and analyzed the therapeutic effect and underlying mechanism. We found that DCs from the peripheral blood and decidua of RSA patients and the maternal-fetal of RSA mice were all polarized to conventional DCs, whose proportion was positively correlated with the mice embryo absorption rate. Moreover, DCs from RSA patients and mice showed increased expression of HLA-DR/MHC-II, CD80, and CD86 but decreased expression of CD274 and 33D1. Importantly, baicalin could alleviate embryo resorption of RSA mice by reversing conventional DCs to plasmacytoid DCs and functional molecule expression via inhibiting the STAT5-ID2 pathway. Our research further proved that DCs play an important role in the pathogenesis of RSA and baicalin might be used for treating RSA.