Discovery of Pathogenic Variants Associated with Idiopathic Recurrent Pregnancy Loss Using Whole-Exome Sequencing.

Idiopathic recurrent pregnancy loss (RPL) is defined as at least two pregnancy losses before 20 weeks of gestation. Approximately 5% of pregnant couples experience idiopathic RPL, which is a heterogeneous disease with various causes including hormonal, chromosomal, and intrauterine abnormalities. Although how pregnancy loss occurs is still unknown, numerous biological factors are associated with the incidence of pregnancy loss, including genetic variants. Whole-exome sequencing (WES) was conducted on blood samples from 56 Korean patients with RPL and 40 healthy controls. The WES data were aligned by means of bioinformatic analysis, and the detected variants were annotated using machine learning tools to predict the pathogenicity of protein alterations. Each indicated variant was confirmed using Sanger sequencing. A replication study was also conducted in 112 patients and 114 controls. The Variant Effect Scoring Tool, Combined Annotation Dependent Depletion tool, Sorting Intolerant from Tolerant annotation tool, and various databases detected 10 potential variants previously associated with spontaneous abortion genes in patients by means of a bioinformatic analysis of WES data. Several variants were detected in more than one patient. Interestingly, several of the detected genes were functionally clustered, including some with a secretory function (mucin 4; MUC4; rs200737893 G>A and hyaluronan-binding protein 2; HABP2; rs542838125 G>T), in which growth arrest-specific 2 Like 2 (GAS2L2; rs140842796 C>T) and dynamin 2 (DNM2; rs763894364 G>A) are functionally associated with cell protrusion and the cytoskeleton. ATP Binding Cassette Subfamily C Member 6 (ABCC6) was the only gene with two variants. HABP2 (rs542838125 G>T), MUC4 (rs200737893 G>A), and GAS2L2 (rs140842796 C>T) were detected in only the patient group in the replication study. The combination of WES and machine learning tools is a useful method to detect potential variants associated with RPL. Using bioinformatic tools, we found 10 potential variants in 9 genes. WES data from patients are needed to better understand the causes of RPL.

Factors affecting the distribution of serum anti-müllerian hormone levels among infertile Asian women: a multi-nation, multi-centre, and multi-ethnicity prospective cohort study.

STUDY QUESTION: How do age, ethnicity, and other characteristics affect serum anti-mullerian hormone (AMH) levels in Asian women undergoing fertility treatment? SUMMARY ANSWER: Age, ethnicity, obesity (BMI ≥ 30 kg/m2), and polycystic ovarian syndrome (PCOS) significantly impacted serum AMH levels, with the rate of decrease accelerating as age increased; a concentration of 4.0 ng/ml was the optimal cut-off for diagnosis of PCOS. WHAT IS KNOWN ALREADY: There are significant differences in ovarian reserve among women from different races and ethnicities, and Asian women often have poorer reproductive outcomes during assisted reproductive treatment cycles. STUDY DESIGN, SIZE, DURATION: A population-based multi-nation, multi-centre, multi-ethnicity prospective cohort study of 4613 women was conducted from January 2020 to May 2021. Infertile women of 20-43 years of age were enrolled. The exclusion criteria included: age <20 or >43, non-Asian ethnicity, and missing critical data. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were Asian women of Chinese, Japanese, Korean, Thai, Vietnamese, Malay, Indian, and Indonesian ethnicities from 12 IVF centres across Asia. These women were all naïve to ovarian stimulation cycles and attended IVF centres for fertility assessment. The AMH measurement was performed using an AMH automated assay on a clinically validated platform. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 4556 infertile Asian women were included in the final analyses. The mean ± SD for serum AMH concentrations (ng/ml) across specific age groups were: overall, 3.44 ± 2.93; age <30, 4.58 ± 3.16; 30-31, 4.23 ± 3.23; 32-33, 3.90 ± 3.06; 34-35, 3.21 ± 2.65; 36-37, 2.74 ± 2.44; 38-39, 2.30 ± 1.91; 40 and above, 1.67 ± 2.00. The rate of AMH decrease was ∼0.13 ng/ml/year in patients aged 25-33 and 0.31 ng/ml/year in women aged 33-43. The highest rates of PCOS were found in Indians (18.6%), Malays (18.9%), and Vietnamese (17.7%). Age (P < 0.001), ethnicity (P < 0.001), obesity (P = 0.007), PCOS (P < 0.001), and a history of endometrioma cystectomy (P = 0.01) were significantly associated with serum AMH values. Smoking status, pretreatment with GnRH agonist (GnRHa) or the oral contraceptive pill (OCP), freezing-thawing of blood samples, and sampling on Day 2 to Day 5 of the menstrual cycle or randomly did not appear to affect serum AMH levels. An AMH concentration of 4.0 ng/ml was the optimal cut-off for PCOS diagnosis with a sensitivity of 71.7% and specificity of 75.8% (AUC = 0.81, CI 95%: 0.79-0.83; P < 0.001). LIMITATIONS, REASONS FOR CAUTION: The incidence of PCOS was supposedly high in this cohort as some IVF clinics were tertiary referral centres for managing specific fertility issues encountered by women with PCOS. Treatment with GnRHa or OCP before AMH testing was regionally and ethnically confined, mostly in Hong Kong SAR and Japan. Moreover, this reference for serum AMH value is limited to Asian women of the ethnicities examined and may not apply to other ethnicities not included in the study. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to collate and construct age-specific reference ranges for serum AMH levels using the same bioassay on Asian women of different ethnicities. The findings of this investigation can assist clinicians to counsel and prognosticate about Asian women's ovarian reserve and reproductive potential, thus providing better strategies for personalized fertility interventions. STUDY FUNDING/COMPETING INTEREST(S): This study was technically supported by Ferring Pharmaceuticals and received no specific grant from any funding agency. All authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER: NCT04203355.

Genetic Correlation of miRNA Polymorphisms and STAT3 Signaling Pathway with Recurrent Implantation Failure in the Korean Population.

The growing prevalence of in vitro fertilization-embryo transfer procedures has resulted in an increased incidence of recurrent implantation failure (RIF), necessitating focused research in this area. STAT3, a key factor in maternal endometrial remodeling and stromal proliferation, is crucial for successful embryo implantation. While the relationship between STAT3 and RIF has been studied, the impact of single nucleotide polymorphisms (SNPs) in miRNAs, well-characterized gene expression modulators, on STAT3 in RIF cases remains uncharacterized. Here, we investigated 161 RIF patients and 268 healthy control subjects in the Korean population, analyzing the statistical association between miRNA genetic variants and RIF risk. We aimed to determine whether SNPs in specific miRNAs, namely miR-218-2 rs11134527 G>A, miR-34a rs2666433 G>A, miR-34a rs6577555 C>A, and miR-130a rs731384 G>A, were significantly associated with RIF risk. We identified a significant association between miR-34a rs6577555 C>A and RIF prevalence (implantation failure [IF] ≥ 2: adjusted odds ratio [AOR] = 2.264, 95% CI = 1.007-5.092, p = 0.048). These findings suggest that miR-34a rs6577555 C>A may contribute to an increased susceptibility to RIF. However, further investigations are necessary to elucidate the precise mechanisms underlying the role of miR-34a rs6577555 C>A in RIF. This study sheds light on the genetic and molecular factors underlying RIF, offering new avenues for research and potential advancements in the diagnosis and treatment of this complex condition.

Effect of Human Peripheral Blood Mononuclear Cells on Mouse Endometrial Cell Proliferation: A Potential Therapeutics for Endometrial Regeneration.

OBJECTIVES: The persistently thin endometrium is a major cause of repeated implantation failure; however, there is no definite treatment for it yet. This study aimed to confirm the potential of human peripheral blood mononuclear cells (hPBMCs) as a therapeutic agent for endometrial regeneration. DESIGN: An experimental study was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: To assess the in vitro effect of hPBMC, the human primary endometrial epithelial cell lines SNU-685 and SNU-1077 were co-cultured with or without 1 × 105 hPBMCs for 24 h. To evaluate the in vivo effect, either 1 × 105 hPBMCs in PBS or PBS alone were injected into the left uterine horn of nonobese diabetic-severe combined immune-deficient mice, and the right untreated uterine horn was used as control. RESULTS: Co-culture with hPBMCs stimulated significant proliferation in both SNU-685 and SNU-1077 cell lines (p = 0.002 and 0.044, respectively). Moreover, treatment with hPBMCs significantly increased the thickness in all parts of the endometrium compared with that in the untreated control uterine horn (proximal: 1.69 ± 0.19 vs. 1.00 ± 0.10, p = 0.009; middle: 1.51 ± 0.14 vs. 1.00 ± 0.12, p = 0.010; distal: 1.72 ± 0.22 vs. 1.00 ± 0.12, p = 0.003, respectively). Compared with the PBS injection group, the hPBMC injection group had significantly thickened endometrium in the middle (p = 0.036) and distal segments (p = 0.002) of the uterine horn. Immunohistochemical analysis revealed the presence of exogenously injected hPBMCs in the uterus of recipient mice. hPBMC-recipient mice had cyclic uterus with normal histology in the endometrium. LIMITATIONS: hPBMCs were not applied directly to a mouse model with thin endometrium, so further study is needed. CONCLUSION: The beneficial effect of hPBMCs on endometrium may suggest their clinical feasibility for the safe treatment of infertile patients with persistently thin endometrium.

Primaquine Inhibits the Endosomal Trafficking and Nuclear Localization of EGFR and Induces the Apoptosis of Breast Cancer Cells by Nuclear EGFR/Stat3-Mediated c-Myc Downregulation.

Triple-negative breast cancer (TNBC) cells overexpress the epidermal growth factor receptor (EGFR). Nuclear EGFR (nEGFR) drives resistance to anti-EGFR therapy and is correlated with poor survival in breast cancer. Inhibition of EGFR nuclear translocation may be a reasonable approach for the treatment of TNBC. The anti-malarial drugs chloroquine and primaquine have been shown to promote an anticancer effect. The aim of the present study was to investigate the effect and mechanism of chloroquine- and primaquine-induced apoptosis of breast cancer cells. We showed that primaquine, a malaria drug, inhibits the growth, migration, and colony formation of breast cancer cells in vitro, and inhibits tumor growth in vivo. Primaquine induces damage to early endosomes and inhibits the nuclear translocation of EGFR. Primaquine inhibits the interaction of Stat3 and nEGFR and reduces the transcript and protein levels of c-Myc. Moreover, primaquine and chloroquine induce the apoptosis of breast cancer cells through c-Myc/Bcl-2 downregulation, induce early endosome damage and reduce nEGFR levels, and induce apoptosis in breast cancer through nEGFR/Stat3-dependent c-Myc downregulation. Our study of primaquine and chloroquine provides a rationale for targeting EGFR signaling components in the treatment of breast cancer.

Association between HOX Transcript Antisense RNA Single-Nucleotide Variants and Recurrent Implantation Failure.

Recurrent implantation failure (RIF) refers to the occurrence of more than two failed in vitro fertilization-embryo transfers (IVF-ETs) in the same individual. RIF can occur for many reasons, including embryo characteristics, immunological factors, and coagulation factors. Genetics can also contribute to RIF, with some single-nucleotide variants (SNVs) reported to be associated with RIF occurrence. We examined SNVs in a long non-coding RNA, homeobox (HOX) transcript antisense RNA (HOTAIR), which is known to affect cancer development. HOTAIR regulates epigenetic outcomes through histone modifications and chromatin remodeling. We recruited 155 female RIF patients and 330 healthy controls, and genotyped HOTAIR SNVs, including rs4759314, rs920778, rs7958904, and rs1899663, in all participants. Differences in these SNVs were compared between the patient and control groups. We identified significant differences in the occurrence of heterozygous genotypes and the dominant expression model for the rs1899663 and rs7958904 SNVs between RIF patients and control subjects. These HOTAIR variants were associated with serum hemoglobin (Hgb), luteinizing hormone (LH), total cholesterol (T. chol), and blood urea nitrogen (BUN) levels, as assessed by analysis of variance (ANOVA). We analyzed the four HOTAIR SNVs and found significant differences in haplotype patterns between RIF patients and healthy controls. The results of this study showed that HOTAIR is not only associated with the development of cancer but also with pregnancy-associated diseases. This study represents the first report showing that HOTAIR is correlated with RIF.

Genetic Polymorphisms in miR-604A>G, miR-938G>A, miR-1302-3C>T and the Risk of Idiopathic Recurrent Pregnancy Loss.

The purpose of this study was to investigate whether polymorphisms in five microRNAs (miRNAs), miR-604A>G, miR-608C>G, 631I/D, miR-938G>A, and miR-1302-3C>T, are associated with the risk of idiopathic recurrent pregnancy loss (RPL). Blood samples were collected from 388 patients with idiopathic RPL (at least two consecutive spontaneous abortions) and 227 control participants. We found the miR-604 AG and AG + GG genotypes of miR-604, the miR-938 GA and GA + AA genotypes of miR-938, and the miR-1302-3CT and CT + TT genotypes of miR-1302-3 are less frequent than the wild-type (WT) genotypes, miR-604AA, miR-938GG, and miR-1302-3CC, respectively, in RPL patients. Using allele-combination multifactor dimensionality reduction (MDR) analysis, we found that eight haplotypes conferred by the miR-604/miR-608/miR-631/miR-938/miR-1302-3 allele combination, A-C-I-G-T, A-C-I-A-C, G-C-I-G-C, G-C-I-G-T, G-G-I-G-C, G-G-I-G-T, G-G-I-A-C, G-G-D-G-C, three from the miR-604/miR-631/miR-938/miR-1302-3 allele combination, A-I-G-T, G-I-G-C, G-I-A-T, one from the miR-604/miR-631/miR-1302-3 allele combination, G-I-C, and two from the miR-604/miR-1302-3 allele combination, G-C and G-T, were less frequent in RPL patients, suggesting protective effects (all p < 0.05). We also identified the miR-604A>G and miR-938G>A polymorphisms within the seed sequence of the mature miRNAs and aligned the seed sequences with the 3'UTR of putative target genes, methylenetetrahydrofolate reductase (MTHFR) and gonadotropin-releasing hormone receptor (GnRHR), respectively. We further found that the binding affinities between miR-604/miR-938 and the 3'UTR of their respective target genes (MTHFR, GnRHR) were significantly different for the common (miR-604A, miR-938G) and variant alleles (miR-604G, miR-938A). These results reveal a significant association between the miR-604A>G and miR-938G>A polymorphisms and idiopathic RPL and suggest that miRNAs can affect RPL in Korean women.

Targeted deletion of Crb1/Crb2 in the optic vesicle models key features of leber congenital amaurosis 8.

The Crb1 and 2 (Crumbs homolog 1 & 2) genes encode large, single-pass transmembrane proteins essential for the apicobasal polarity and adhesion of epithelial cells. Crb1 mutations cause degenerative retinal diseases in humans, including Leber congenital amaurosis type 8 (LCA8) and retinitis pigmentosa type 12 (RP12). In LCA8, impaired photoreceptor development and/or survival is thought to cause blindness during early infancy, whereas, in RP12, progressive photoreceptor degeneration damages peripheral vision later in life. There are multiple animal models of RP12 pathology, but no experimental model of LCA8 recapitulates the full spectrum of its pathological features. To generate a mouse model of LCA8 and identify the functions of Crb1/2 in developing ocular tissues, we used an mRx-Cre driver to generate allelic combinations that enabled conditional gene ablation from the optic vesicle stage. In this series only Crb1/2 double knockout (dKO) mice exhibited characteristics of human LCA8 disease: locally thickened retina with spots devoid of cells, aberrant positioning of retinal cells, severely disrupted lamination, and depigmented retinal-pigmented epithelium. Retinal defects antedated E12.5, which is far earlier than the stage at which photoreceptor cells mainly differentiate. Most remarkably, Crb1/Crb2 dKO showed a severely attenuated electroretinogram at the eye opening stage. These results suggest that human LCA8 can be modeled in the mouse by simultaneously ablating Crb1/2 from the beginning of eye development. Importantly, they also indicate that LCA8 is caused by malfunction of retinal progenitor cells during early ocular development rather than by defective photoreceptor-Muller glial interaction, a mechanism proposed for RP12.

Associations between microRNA (miR-25, miR-32, miR-125, and miR-222) polymorphisms and recurrent implantation failure in Korean women.

BACKGROUND: Recurrent implantation failure (RIF) is the failure of embryos to implant more than two times in a given individual. There is debate about a precise definition for RIF, but we consider more than two implantation failures for individuals who undergo in vitro fertilization-embryo transfer (IVF-ET) to constitute RIF. There are many potential reasons for RIF, including embryonic factors, immunological factors, uterine factors, coagulate factors, and genetic factors. Genetic variation has been suggested as one of the contributing factors leading to RIF, and a number of single-nucleotide polymorphisms (SNPs) have been reported to be associated with RIF. The recent elucidation of miRNA functions has provided new insight into the regulation of gene expression. METHODS: We investigated associations between polymorphisms in four miRNAs and RIF in 346 Korean women: 118 patients with RIF and 228 controls. We determined the genotypes of the miRNAs in the study participants by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis. We analyzed the effects of genotypes, allele combinations, and environmental and clinical factors on the risk of RIF. RESULTS: The miR-25 T/miR-125aT/miR-222G (odds ratio (OR), 0.528; 95% confidence interval (CI), 0.282-0.990; P = 0.044) and miR-25 T/miR-125aT allele combinations were associated with a reduced risk of RIF. The miR-25 T/miR-32C/miR-125aC/miR-222 T allele combination was associated with an increased risk of RIF. The miR-222GT+TT genotypes interacted with high prothrombin time (≥ 12 s) to increase the risk of RIF. CONCLUSIONS: MicroRNA polymorphisms are significantly different between patients that experience RIF and healthy controls. Combinations of microRNA polymorphisms were associated with the risk of RIF. Interactions between environmental factors and genotypes increased the risk of RIF in Korean women.

The FDA-Approved Anti-Asthma Medicine Ciclesonide Inhibits Lung Cancer Stem Cells through Hedgehog Signaling-Mediated SOX2 Regulation.

Ciclesonide is an FDA-approved glucocorticoid (GC) used to treat asthma and allergic rhinitis. However, its effects on cancer and cancer stem cells (CSCs) are unknown. Our study focuses on investigating the inhibitory effect of ciclesonide on lung cancer and CSCs and its underlying mechanism. In this study, we showed that ciclesonide inhibits the proliferation of lung cancer cells and the growth of CSCs. Similar glucocorticoids, such as dexamethasone and prednisone, do not inhibit CSC formation. We show that ciclesonide is important for CSC formation through the Hedgehog signaling pathway. Ciclesonide reduces the protein levels of GL1, GL2, and Smoothened (SMO), and a small interfering RNA (siRNA) targeting SMO inhibits tumorsphere formation. Additionally, ciclesonide reduces the transcript and protein levels of SOX2, and an siRNA targeting SOX2 inhibits tumorsphere formation. To regulate breast CSC formation, ciclesonide regulates GL1, GL2, SMO, and SOX2. Our results unveil a novel mechanism involving Hedgehog signaling and SOX2 regulated by ciclesonide in lung CSCs, and also open up the possibility of targeting Hedgehog signaling and SOX2 to prevent lung CSC formation.

The Antiasthma Medication Ciclesonide Suppresses Breast Cancer Stem Cells through Inhibition of the Glucocorticoid Receptor Signaling-Dependent YAP Pathway.

Ciclesonide is an FDA-approved glucocorticoid used to treat asthma and allergic rhinitis. However, whether it has anticancer and anti-cancer stem cell (CSC) effects is unknown. This study focused on investigating the effect of ciclesonide on breast cancer and CSCs and determining its underlying mechanism. Here, we showed that ciclesonide inhibits breast cancer and CSC formation. Similar glucocorticoids-dexamethasone and prednisone-did not inhibit CSC formation. Ciclesonide-induced glucocorticoid receptor (GR) degradation was dependent on ubiquitination. We showed via GR small interfering RNA (siRNA) that GR plays an important role in CSC formation. We showed via western blot and immunofluorescence assays that ciclesonide reduces the nuclear level of GR. The GR antagonist RU-486 also inhibited CSC formation. Ciclesonide reduced the protein level of the Hippo transducer Yes-associated protein (YAP). GR siRNA induced a decrease in YAP protein expression and inhibited mammosphere formation. The YAP inhibitor verteporfin inhibited CSC formation and transcription of the connective tissue growth factor and cysteine-rich protein 61 genes. The GR/YAP1 pathway regulated breast CSC formation. We showed that the GR/YAP signaling pathway regulates breast CSC formation and revealed a new approach for targeting GR and YAP to inhibit CSC formation.

Coriolic Acid (13-(S)-Hydroxy-9Z, 11E-octadecadienoic Acid) from Glasswort (Salicornia herbacea L.) Suppresses Breast Cancer Stem Cell through the Regulation of c-Myc.

Cancer stem cells have certain characteristics, such as self-renewal, differentiation, and drug resistance, which are related to tumor progression, maintenance, recurrence, and metastasis. In our study, we targeted breast cancer stem cells (BCSCs) using a natural compound, coriolic acid, from Salicornia herbacea L. This compound was isolated by mammosphere formation inhibition bioassay-guided fractionation and identified by using NMR spectroscopy and electrospray ionization mass spectrometry. Coriolic acid inhibited the formation of mammospheres and induced BCSC apoptosis. It also decreased the subpopulation of CD44high/CD24low cells, a cancer stem cell (CSC) phenotype, and specific genes related to CSCs, such as Nanog,Oct4, and CD44. Coriolic acid decreased the transcriptional and translational levels of the c-Myc gene, which is a CSC survival factor. These results indicated that coriolic acid could be a novel compound to target BCSCs via regulation of c-Myc.

Inhibitory Effects of Tangeretin, A Citrus Peel-Derived Flavonoid, on Breast Cancer Stem Cell Formation through Suppression of Stat3 Signaling.

Breast cancer stem cells (BCSCs) are responsible for tumor chemoresistance and recurrence. Targeting CSCs using natural compounds is a novel approach for cancer therapy. A CSC-inhibiting compound was purified from citrus extracts using silica gel, gel filtration and high-pressure liquid chromatography. The purified compound was identified as tangeretin by using nuclear magnetic resonance (NMR). Tangeretin inhibited cell proliferation, CSC formation and tumor growth, and modestly induced apoptosis in CSCs. The frequency of a subpopulation with a CSC phenotype (CD44+/CD24-) was reduced by tangeretin. Tangeretin reduced the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3). Our results in this study show that tangeretin inhibits the Stat3 signaling pathway and induces CSC death, indicating that tangeretin may be a potential natural compound that targets breast cancer cells and CSCs.

6-Methoxymellein Isolated from Carrot (Daucus carota L.) Targets Breast Cancer Stem Cells by Regulating NF-κB Signaling.

The presence of breast cancer stem cells (BCSCs) induces the aggressive progression and recurrence of breast cancer. These cells are drug resistant, have the capacity to self-renew and differentiate and are involved in recurrence and metastasis, suggesting that targeting BCSCs may improve treatment efficacy. In this report, methanol extracts of carrot root were purified by means of silica gel, Sephadex LH-20, and preparative high-performance liquid chromatography to isolate a compound targeting mammosphere formation. We isolated the compound 6-methoxymellein, which inhibits the proliferation and migration of breast cancer cells, reduces mammosphere growth, decreases the proportion of CD44+/CD24- cells in breast cancer cells and decreases the expression of stemness-associated proteins c-Myc, Sox-2 and Oct4. 6-Methoxymellein reduces the nuclear localization of nuclear factor-κB (NF-κB) subunit p65 and p50. Subsequently, 6-methoxymellein decreases the mRNA transcription and secretion of IL-6 and IL-8. Our data suggest that 6-methoxymellein may be an anticancer agent that inhibits BCSCs via NF-κB/IL-6 and IL-8 regulation.

Betavulgarin Isolated from Sugar Beet (Beta vulgaris) Suppresses Breast Cancer Stem Cells through Stat3 Signaling.

Breast cancer is a major health problem that affects lives worldwide. Breast cancer stem cells (BCSCs) are small subpopulations of cells with capacities for drug resistance, self-renewal, recurrence, metastasis, and differentiation. Herein, powder extracts of beetroot were subjected to silica gel, gel filtration, thin layer chromatography (TLC), and preparatory high-pressure liquid chromatography (HPLC) for isolation of one compound, based on activity-guided purification using tumorsphere formation assays. The purified compound was identified as betavulgarin, using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI) mass spectrometry. Betavulgarin suppressed the proliferation, migration, colony formation, and mammosphere formation of breast cancer cells and reduced the size of the CD44+/CD24- subpopulation and the expression of the self-renewal-related genes, C-Myc, Nanog, and Oct4. This compound decreased the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3) and reduced the mRNA and protein levels of sex determining region Y (SRY)-box 2 (SOX2), in mammospheres. These data suggest that betavulgarin inhibit the Stat3/Sox2 signaling pathway and induces BCSC death, indicating betavulgarin might be an anticancer agent against breast cancer cells and BCSCs.

The microRNApolymorphisms inmiR-150 and miR-1179 are associated with risk of idiopathic recurrent pregnancy loss.

RESEARCH QUESTION: Are single nucleotide polymorphisms of microRNAs (miRNAs) and risk of idiopathic recurrent pregnancy loss (RPL) associated? DESIGN: A total 375 patients with idiopathic RPL (age, mean ± standard deviation [SD] 33.02 ± 4.24 years; body mass index [BMI], mean ± SD, 21.57 ± 3.70 kg/m2) and 276 control participants (age, mean ± SD, 33.01 ± 5.27 years; BMI, mean ± SD, 21.58 ± 3.20) were recruited. Pregnancy loss was diagnosed using human chorionic gonadotrophin concentrations, ultrasonography and/or physical examination prior to 20 weeks of gestation. The genotype of the participants was determined by polymerase chain reaction restriction fragment length polymorphism analysis. Statistical analysis was performed to investigate the differences in frequencies between the control and RPL genotypes RESULTS: The miR-150G>A heterozygous genotype was significantly associated with increased risk of RPL (adjusted odds ratio 2.502, 95% confidence interval 1.555-4.025; P = 0.0002). The miR-1179A>T heterozygous genotype was significantly associated with decreased risk of RPL (adjusted odds ratio 0.633, 95% confidence interval 0.454-0.884; P = 0.007). Some allele combinations that included miR-150A or miRNA-1179T resulted in an increase or decrease in risk of RPL, respectively. CONCLUSIONS: The miR-150G>A and miR-1179A>T polymorphisms were more frequently associated with RPL compared with controls.

Genetic polymorphisms of the cobalamin transport system are associated with idiopathic recurrent implantation failure.

PURPOSE: Vitamin B12 (cobalamin, Cbl) plays a role in the recycling of folate, which is important in pregnancy. Transcobalamin II (TCN2) and transcobalamin receptor (TCblR) proteins are involved in the cellular uptake of Cbl. TCN2 binds Cbl in the plasma, and TCblR binds TCN2-Cbl at the cell surface. Therefore, we investigated the potential association between polymorphisms in Cbl transport proteins, TCN2 and TCblR, and recurrent implantation failure (RIF) susceptibility. METHODS: The genotypes of TCN2 67A>G, TCN2 776C>G, and TCblR 1104C>T were determined for RIF patients and healthy controls using a polymerase chain reaction restriction fragment length polymorphism assay. Additionally, statistical analysis was performed to compare the genotype frequencies between RIF patients and controls. RESULTS: The TCN2 67 polymorphism AG type was associated with RIF risk. Some allele combinations that contained the TCN2 67 polymorphism G allele were associated with increased RIF risk, whereas other allele combinations that contained the TCblR 1104 polymorphism T alleles were associated with decreased RIF risk. In genotype combination analysis, two combinations containing the TCN2 67 polymorphism AG type were associated with RIF risk. CONCLUSION: Our study showed that the polymorphisms of TCN2 and TCblR are associated with RIF and are potential genetic predisposing factors for RIF among Korean women. Additionally, our findings support a potential role for TCN2 and TCblR in RIF among Korean women. However, further studies are required to investigate the role of the polymorphisms in those proteins and RIF because the roles of the TCN2 and TCblR polymorphisms in RIF are not clear.

3'-UTR Polymorphisms in the Vascular Endothelial Growth Factor Gene (VEGF) Contribute to Susceptibility to Recurrent Pregnancy Loss (RPL).

Numerous studies have examined the genetic association of vascular endothelial growth factor (VEGF) single nucleotide polymorphisms (SNPs) with recurrent pregnancy loss (RPL). However, of the four known SNPs in the 3'-untranslated region (3'-UTR) of VEGF, three SNPs-namely rs3025040 (1451C>T), rs10434 (1612G>A), and rs3025053 (1725G>A)-remain poorly characterized with regard to RPL. Herein, we evaluated the association between these three SNPs in the VEGF 3'-UTR and RPL susceptibility. We analyzed VEGF 3'-UTR gene variants in with and without RPL using TaqMan allelic discrimination. There were significant differences in the genotype frequencies of 1612G>A (GA: adjusted odds ratio (AOR), 0.652; 95% confidence interval (CI), 0.447-0.951; p = 0.026) and 1725G>A (GA: AOR, 0.503; 95% CI, 0.229-0.848; p = 0.010) in RPL patients vs. controls. Our results indicate that the 1612G>A and 1725G>A polymorphisms in the 3'-UTR of VEGF are associated with RPL susceptibility in Korean women. These data suggest that VEGF 3'-UTR polymorphisms may be utilized as biomarkers for the detection of RPL risk and prevention.

Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice.

BACKGROUND & AIMS: Mesenchymal stem cells (MSCs) mediate tissue repair and might be used to prevent or reduce liver fibrosis. However, little is known about the anti-fibrotic factors secreted from MSCs or their mechanisms. METHODS: Umbilical cord-derived MSCs (UCMSCs) were differentiated into hepatocyte-like cells (hpUCMSCs), medium was collected, and secretome proteins were identified and quantified using nanochip-liquid chromatography/quadrupole time-of-flight mass spectrometry. Liver fibrosis was induced in mice by intraperitoneal injection of thioacetamide or CCl4; some mice were then given injections of secretomes or proteins. Liver tissues were collected and analyzed by histology or polymerase chain reaction array to analyze changes in gene expression patterns. We analyzed the effects of MSC secretomes and potential anti-fibrotic proteins on transforming growth factor ß 1 (TGFß1)-mediated activation of human hepatic stellate cell (HSC) lines (hTert-HSC and LX2) and human primary HSCs. Liver tissues were collected from 16 patients with liver cirrhosis and 16 individuals without cirrhosis (controls) in Korea and analyzed by immunohistochemistry and immunoblots. RESULTS: In mice with fibrosis, accumulation of extracellular matrix proteins was significantly reduced 3 days after injecting secretomes from UCMSCs, and to a greater extent from hpUCMSCs; numbers of activated HSCs that expressed the myogenic marker α-smooth muscle actin (α-SMA, encoded by ACTA2 [actin, alpha 2, smooth muscle]) were also reduced. Secretomes from UCMSCs, and to a greater extent from hpUCMSCs, reduced liver expression of multiple fibrotic factors, collagens, metalloproteinases, TGFß, and Smad proteins in the TGFß signaling pathways. In HSC cell lines and primary HSCs, TGFß1-stimulated upregulation of α-SMA was significantly inhibited (and SMAD2 phosphorylation reduced) by secretomes from UCMSCs, and to a greater extent from hpUCMSCs. We identified 32 proteins in secretomes of UCMSCs that were more highly concentrated in secretomes from hpUCMSCs and inhibited TGFß-mediated activation of HSCs. One of these, milk fat globule-EGF factor 8 (MFGE8), was a strong inhibitor of activation of human primary HSCs. We found MFGE8 to down-regulate expression of TGFß type I receptor by binding to αvß3 integrin on HSCs and to be secreted by MSCs from umbilical cord, teeth, and bone marrow. In mice, injection of recombinant human MFGE8 had anti-fibrotic effects comparable to those of the hpUCMSC secretome, reducing extracellular matrix deposition and HSC activation. Co-injection of an antibody against MFGE8 reduced the anti-fibrotic effects of the hpUCMSC secretome in mice. Levels of MFGE8 were reduced in cirrhotic liver tissue from patients compared with controls. CONCLUSIONS: MFGE8 is an anti-fibrotic protein in MSC secretomes that strongly inhibits TGFß signaling and reduces extracellular matrix deposition and liver fibrosis in mice.

Catechol derived from aronia juice through lactic acid bacteria fermentation inhibits breast cancer stem cell formation via modulation Stat3/IL-6 signaling pathway.

Cancer stem cells (CSCs) as a subpopulation of cancer cells are drug-resistant and radiation-resistant cancer cells to be responsible for tumor progress, maintenance and recurrence of cancer, and metastasis. This study isolated and investigated a new cancer stem cell (CSC) inhibitor derived from lactic acid fermentation products using culture broth with 2% aronia juice. The anti-CSC activity of aronia-cultured broth was significantly higher than that of the control. Activity-guided fractionation and repeated chromatographic preparation led to the isolation of one compound. Using nuclear magnetic resonance and ESI mass spectrometry, we identified the isolated compound as catechol. In this study, we report that aronia-fermented catechol has a novel inhibitory effect on human breast CSCs. Catechol inhibited breast cancer cell proliferation and mammosphere formation in a dose-dependent manner. This compound reduced the CD44high /CD24low subpopulation, ALDH-expressing cell population and the self-renewal-related genes nanog, sox2, and oct4. Catechol preferentially reduced mRNA transcripts and protein levels of Stat3 and did not induce c-Myc degradation. These findings support the novel utilization of catechol for breast cancer therapy via the Stat3/IL-6 signaling pathway. Our results suggest that catechol can be used for breast cancer therapy and that Stat3 expression is a marker of CSCs. Catechol inhibited Stat3 signaling by reducing Stat3 expression and secreted IL-6, a CSC survival factor. These findings support the novel utilization of catechol for breast cancer therapy via Stat3/IL-6 signaling.