To identify predictors of the ovarian response to clomiphene citrate (CC) in infertile patients with polycystic ovary syndrome (PCOS).We performed a prospective cohort study of infertile patients with PCOS. The participants underwent assessments of their physical, endocrine, and metabolic characteristics, and treatment with CC at an initial dose of 50 mg/day and a maximum of 100 mg/day between days 3 and 7 of their menstrual cycles. Participants who ovulated were identified as responders and those who did not as non-responders.Of the 72 participants, 48 (66.7%) were identified as responders and 24 as non-responders. Sex hormone-binding globulin (SHBG) (odds ratio 1.022, 95% confidence interval: 1.000-1.045) was found to be associated with the ovarian response to CC using logistic multivariate regression analysis. Receiver operating characteristic analysis also showed that SHBG was a significant predictor of the response to CC (area under the curve 0.799).We have shown that SHBG is the best prognostic indicator of an ovulatory response to CC. However, larger prospective studies, in which more variables are assessed, are required to confirm this finding and to identify appropriate cut-off values.
Abstract STUDY QUESTION Do distinct subpopulations of decidual stromal cells (DSCs) exist and if so, are given subpopulations enriched in recurrent miscarriage (RM)? SUMMARY ANSWER Three subpopulations of DSCs were identified from which inflammatory DSCs (iDSCs) and glycolytic DSCs (glyDSCs) are significantly enriched in RM, with implicated roles in driving decidual inflammation and immune dysregulation. WHAT IS KNOWN ALREADY DSCs play crucial roles in establishing and maintaining a successful pregnancy; dysfunction of DSCs has been considered as one of the key reasons for the development of RM. STUDY DESIGN, SIZE, DURATION We collected 15 early decidual samples from five healthy donors (HDs) and ten RM patients to perform single-cell RNA sequencing (scRNA-seq). A total of 43 RM patients and 37 HDs were enrolled in the validation cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS Non-immune cells and immune cells of decidual tissues were sorted by flow cytometry to perform scRNA-seq. We used tissue microarrays (TMA) to validate three distinct subpopulations of DSCs. The expression of inflammatory and glycolytic proteins by DSCs was validated by immunohistochemistry (IHC) and multiplex immunohistochemistry (mIHC). Different subsets of decidual NK (dNK) cells and macrophages were also validated by multicolor flow cytometry and mIHC. Cell ligand–receptor and spatial analyses between DSCs and immune cells were analyzed by mIHC. MAIN RESULTS AND THE ROLE OF CHANCE We classify the DSCs into three subtypes based on scRNA-seq data: myofibroblastic (myDSCs), inflammatory (iDSCs) and glycolytic (glyDSCs), with the latter two being significantly enriched in RM patients. The distribution patterns of DSC subtypes in the RM and HD groups were validated by mIHC. Single-cell analyses indicate that the differentiation of iDSCs and glyDSCs may be coupled with the degrees of hypoxia. Consequently, we propose a pathological model in which a vicious circle is formed and fueled by hypoxic stress, uncontrolled inflammation and aberrant glycolysis. Furthermore, our results show that the inflammatory SPP1+ macrophages and CD18+ dNK cells are preferentially increased in the decidua of RM patients. Cell ligand–receptor and mIHC spatial analyses uncovered close interactions between pathogenic DSCs and inflammatory SPP1+ macrophages and CD18+ NK cells in RM patients. LARGE SCALE DATA The raw single-cell sequence data reported in this paper were deposited at the National Omics Data Encyclopedia (www.biosino.org), under the accession number OEP002901. LIMITATIONS, REASONS FOR CAUTION The number of decidual samples for scRNA-seq was limited and in-depth functional studies on DSCs are warranted in future studies. WIDER IMPLICATIONS OF THE FINDINGS Identification of three DSC subpopulations opens new avenues for further investigation of their roles in RM patients. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Strategic Priority Research Program (No. XDB29030302), Frontier Science Key Research Project (QYZDB-SSW-SMC036), Chinese Academy of Sciences; National Key Research and Development Program of China (2021YFE0200600), National Natural Science Foundation of China (No. 31770960), Shanghai Municipal Science and Technology Major Project (No. 2019SHZDZX02, HS2021SHZX001), and Shanghai Committee of Science and Technology (17411967800). All authors report no conflict of interest.
Objective To examine the effect of a peroxisome proliferator-activated receptor(PPAR) γ ligand troglitazone on invasiveness in human hepatic cancer cell line HepG2 and its mechanism.Methods HepG2 cell line was used in the experiments.Invasiveness assay was performed in Transwell chambers.Urokinase-type plasminogen activator(u-PA) and vascular endothelial growth factor(VEGF) mRNA were measured by RT-PCR.Results HepG2 cell invasiveness was significantly suppressed after treatment with troglitazone,which was associated with a reduction of mRNA levels of u-PA and VEGF.Conclusion Troglitazone inhibits invasion of human liver cancer cells,these effects involving down-regulation of u-PA and VEGF.
Can new genetic factors responsible for male infertility be identified, especially for those characterized by asthenospermia despite normal sperm morphology?We identified the novel pathogenetic gene IQ motif and ubiquitin-like domain-containing (IQUB) as responsible for male infertility characterized by asthenospermia, involving sperm radial spoke defects.To date, only a few genes have been found to be responsible for asthenospermia with normal sperm morphology. Iqub, encoding the IQUB protein, is highly and specifically expressed in murine testes and interacts with the proteins radial spoke head 3 (RSPH3), CEP295 N-terminal like (CEP295NL or DDC8), glutathione S-transferase mu 1 (GSTM1) and outer dense fiber of sperm tails 1 (ODF1) in the yeast two-hybrid system.The IQUB variant was identified by whole-exome sequencing in a cohort of 126 male infertility patients with typical asthenospermia recruited between 2015 and 2020. Knockout (KO) and knockin (KI) mouse models, scanning and transmission electron microscopy (TEM), and other functional assays were performed, between 2019 and 2021.The IQUB variant was identified by whole-exome sequencing and confirmed by Sanger sequencing. Iqub KO and KI mice were constructed to mimic the phenotype of the affected individual. After recapitulating the phenotype of human male infertility, scanning and TEM were performed to check the ultrastructure of the sperm. Western blot and co-immunoprecipitation were performed to clarify the pathological mechanism of the IQUB variant.We identified a homozygous nonsense IQUB variant (NM_001282855.2:c.942T> G(p.Tyr314*)) from an infertile male. Iqub KO and KI mice mimicked the infertility phenotype and confirmed IQUB to be the pathogenetic gene. Scanning and TEM showed that sperm of both the mouse models and the affected individual had radial spoke defects. The functional assay suggested that IQUB may recruit calmodulin in lower Ca2+ environments to facilitate the normal assembly of radial spokes by inhibiting the activity of RSPH3/p-ERK1/2 (a nontypical AKAP (A-Kinase Anchoring Protein) forming by RSPH3 and phosphorylation of extracellular signal-regulated kinase 1 and 2 (p-ERK1/2)).Additional cases are needed to confirm the genetic contribution of IQUB variants to male infertility. In addition, because no IQUB antibody is available for immunofluorescence and the polyclonal antibody we generated was only effective in western blotting, immunostaining for IQUB was not performed in this study. Therefore, this study lacks direct in vivo proof to confirm the effect of the variant on IQUB protein level.Our results suggest a causal relation between IQUB variants and male infertility owing to asthenospermia, and partly clarify the pathological mechanism of IQUB variants. This expands our knowledge of the genes involved in human sperm asthenospermia and potentially provides a new genetic marker for male infertility.This work was supported by the National Key Research and Development Program of China (2021YFC2700100), the National Natural Science Foundation of China (32130029, 82171643, 81971450, 82001538, and 81971382) and the Guangdong Science and Technology Department Guangdong-Hong Kong-Macao Joint Innovation Project (2020A0505140003). There are no competing interests to declare.N/A.
Our previous study using suppression subtractive hybridization (SSH), cDNA microarray and semi-quantitative RT-PCR showed that RPS12 was overexpressed in gastric cancer and it was closely related to metastasis. However, the role of RPS12 in gastric cancer is not clear, which led us to conduct the current study to further investigate the effects of RPS12 on the proliferation and migration of gastric cancer cells, and also to explore the underlying molecular mechanisms. RNA interference was used to inhibit the expression of RPS12. The expression of RPS12 and S100A4 in gastric cancer cells was determined using semi-quantitative RT-PCR and western blot analysis. Cell proliferation and migration were detected by MTT and transwell assay, respectively. In addition, the promoter activity of S100A4 was measured by a Dual-Luciferase Reporter Assay System. We found that RNAi‑mediated RPS12 downregulation led to reduced proliferation and migration of BGC823 and SGC7901 gastric cancer cells. Further results showed that RPS12 inhibition led to reduced S100A4 expression and decreased promoter activity of S100A4 in BGC823 cells. We demonstrated that ectopic expression of S100A4 reversed the reduced proliferation and migration ability after RPS12 inhibition in BGC823 cells. Our findings provide the first demonstration that RPS12 plays important roles in regulating the proliferation and migration of gastric cancer cells. S100A4 can mediate the effects of RPS12 as a downstream effector.
Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs.The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability.Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.
This study aimed to explore valuable preconception predictors of gestational diabetes mellitus (GDM) in PCOS patients.A prospective cohort study enrolling infertile Chinese PCOS women treated with ovulation induction was performed. The endocrine, metabolic and physical features of all the patients were collected before pregnancy and then followed up to 6 weeks after delivery. The prevalence of GDM was determined during 24-28 gestational weeks. Logistic regression analysis and receiver operating characteristic (ROC) curves were applied to explore the risk factors and their predictive value for GDM.A total of 94 infertile PCOS women who got singleton pregnancy by ovulation induction were enrolled in the study. Logistic regression analysis showed that the preconception insulin under the curve (IAUC) and sex hormone-binding globulin (SHBG) levels were two most significant risk factors for developing GDM (p = 0.014; p = 0.042, respectively). The area of SHBG and IAUC under the ROC curve were 0.806 (p < 0.001) and 0.775 (p = 0.001), respectively. The optimal cutoff values were failed to be calculated because of the limited group size.Low SHBG level and hyperinsulinism were both strongly associated with the development of GDM and might be two valuable predictors in PCOS patients.
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