Downregulation of FHL2 suppressed trophoblast migration, invasion and epithelial-mesenchymal transition in recurrent miscarriage.

RESEARCH QUESTION: Is four and a half LIM domain 2 (FHL2) involved in trophoblast migration, invasion and epithelial-mesenchymal transition (EMT) in recurrent miscarriage? DESIGN: Villus tissue was collected from 24 patients who had experienced recurrent miscarriage and 24 healthy controls. FHL2 mRNA and protein expression in villus specimens were observed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Small interfering RNA and overexpression plasmid were used to change the FHL2 expression. JAR and HTR8/SVneo cell lines were used to conduct scratch-wound assay and transwell assay to detect trophoblast migration and invasion of FHL2. Downstream molecule expression of mRNA and protein and EMT markers were verified by qRT-PCR and Western blot. RESULTS: Significantly lower FHL2 mRNA (P = 0.019) and protein (P = 0.0014) expression was found in trophoblasts from the recurrent miscarriage group compared with healthy controls. FHL2 knockdown repressed migration (P = 0.0046), invasion (P < 0.001) and EMT, as shown by significant differences in mRNA and protein expression of the EMT markers N-cadherin, E-cadherin, Vimentin and Snail (all P < 0.05) of extravillus trophoblasts. FHL2 overexpression enhanced migration (P = 0.025), invasion (P < 0.001) and EMT of extravillus trophoblasts (all EMT markers P < 0.05). The positive upstream factor FHL2 in the extracellular signal-related kinase pathway induced JunD expression, thereby promoting trophoblast migration and invasion via matrix metalloproteinase 2. CONCLUSIONS: FHL2 is involved in a regulatory pathway of trophoblast migration, invasion and EMT during early pregnancy, and may have a role in recurrent miscarriage pathogenesis, which can serve as a possible target for novel therapeutic development.

Transcriptomic analysis supports collective endometrial cell migration in the pathogenesis of adenomyosis.

RESEARCH QUESTION: Adenomyosis is a common uterine disorder of uncertain causes. Can transcriptomic analyses of the endometrium and myometrium reveal potential mechanisms underlying adenomyosis pathogenesis? DESIGN: Transcriptomic profiles of eutopic endometrium and myometrium from women with and without diffuse adenomyosis and with symptomatic FIGO type 2-5 fibroids in the proliferative phase of the menstrual cycle were assessed using RNA sequencing and bioinformatic analysis. Differentially expressed genes (DEG) and potential pathways were validated by quantitative reverse transcription polymerase chain reaction, immunoblotting and Masson staining, using additional clinical samples. RESULTS: Top biological processes in the endometrium of women with versus without adenomyosis, enriched from DEG, comprised inflammation, extracellular matrix (ECM) organization, collagen degradation and hyaluronan synthesis, which are key in cell migration and cell movement. Top biological processes enriched from DEG in the myometrium of women with versus without adenomyosis revealed ECM organization dysfunction, abnormal sensory pain perception and gamma aminobutyric acid (GABA) synaptic transmission. Dysregulation of prolactin signalling was also enriched in eutopic endometrium and in the myometrium of women with adenomyosis. CONCLUSIONS: Overall, our results support the invasive endometrium theory in the pathogenesis of adenomyosis, in which inflammation induces ECM remodelling resulting in a track for subsequent endometrial collective cell migration and onset of adenomyosis. Moreover, abnormal myometrial GABA synaptic transmission may contribute to dysmenorrhoea in women with adenomyosis and is a possible target for novel therapeutic development. Prolactin signalling abnormalities may serve as another opportunity for therapeutic intervention.

Propylparaben concentrations in the urine of women and adverse effects on ovarian function in mice in vivo and ovarian cells in vitro.

Female reproduction is precisely regulated by hormones, and the ovary is easily affected by environmental endocrine disruptors (EDCs), which are ubiquitous in industrialized societies. Parabens are EDCs that are used as antibacterial preservatives in cosmetics, personal care products (PCPs), medicines, and food. We used ultrahigh-performance liquid chromatography-mass spectrometry to quantitatively detect methyl-, ethyl-, butyl-, and propylparaben (PP) concentrations in urine samples from 74 women of childbearing age. Balb/c mice were subcutaneously injected with 100 mg/kg/day of PP for 21 consecutive days or 100 or 1,000 mg/kg/day of PP during superovulation. Various concentrations of PP (ranging from 1 to 1,000 nM) were added to a human ovarian granulosa tumor-derived cell line (KGN) culture for 24 h. The urinary paraben concentrations of women who used cosmetics and other PCPs within 48 h prior to sample collection were significantly elevated, and the PP concentration was significantly positively correlated with the basal estradiol concentration. After PP injection, the mouse serum estradiol concentrations were significantly increased, estrus cycles were disordered, corpus luteum number was reduced, and number of oocytes retrieved was significantly reduced. In in vitro experiments, PP treatment increased estradiol synthesis and the expression levels of aromatase enzyme (CYP19A1) and steroidogenic acute regulatory protein. This study demonstrates the adverse effects of PP on ovarian estradiol secretion and ovulation, further evaluates the safety of PP as a preservative, and provides guidance for the use of PCPs and cosmetics by women of childbearing age.

Erythropoietin-producing hepatocellular receptor A7 restrains estrogen negative feedback of luteinizing hormone via ephrin A5 in the hypothalamus of female rats.

We have previously shown that systemic injection of erythropoietin-producing hepatocellular receptor A7 (EPHA7)-Fc raises serum luteinizing hormone (LH) levels before ovulation in female rats, indicating the induction of EPHA7 in ovulation. In this study, we aimed to identify the mechanism and hypothalamus-pituitary-ovary (HPO) axis level underlying the promotion of LH secretion by EPHA7. Using an ovariectomized (OVX) rat model, in conjunction with low-dose 17ß-estradiol (E2) treatment, we investigated the association between EPHA7-ephrin (EFN)A5 signaling and E2 negative feedback. Various rat models (OVX, E2-treated OVX, and abarelix treated) were injected with the recombinant EPHA7-Fc protein through the caudal vein to investigate the molecular mechanism underlying the promotion of LH secretion by EPHA7. Efna5 was observed strongly expressed in the arcuate nucleus of the female rat by using RNAscope in situ hybridization. Our results indicated that E2, combined with estrogen receptor (ER)α, but not ERß, inhibited Efna5 and gonadotropin-releasing hormone 1 (Gnrh1) expressions in the hypothalamus. In addition, the systemic administration of EPHA7-Fc restrained the inhibition of Efna5 and Gnrh1 by E2, resulting in increased Efna5 and Gnrh1 expressions in the hypothalamus as well as increased serum LH levels. Collectively, our findings demonstrated the involvement of EPHA7-EFNA5 signaling in the regulation of LH and the E2 negative feedback pathway in the hypothalamus, highlighting the functional role of EPHA7 in female reproduction.

Down-regulation of CCR7 via AKT pathway and GATA2 inactivation suppressed trophoblast migration and invasion in recurrent spontaneous abortion†.

The underlying mechanism of the chemokine-C receptor 7 (CCR7) that leads to aberrant trophoblast migration and invasion in recurrent spontaneous abortion (RSA) remains unknown. CCR7 is considered crucial for migration and invasion and has been associated with the risk of miscarriage. However, the functional role of CCR7 in RSA is not fully understood. Our study found that CCR7 mRNA and protein abundance were significantly decreased in the villous from RSA patients compared with healthy controls. Knockdown of CCR7 caused a significant reduction of migration and invasion in JAR and JEG-3 cells. Meanwhile, CCR7 functioned as a positive upstream factor of the AKT pathway contributing to the expression of GATA2, promoting trophoblast migration, and invasion via MMP2. Notably, a decreased abundance of CCR7 was positively correlated with the phosphorylation of AKT and with an abundance of GATA2 and MMP2 in human villous specimens of RSA compared with the control group. CCL19, a ligand of CCR7, could promote trophoblast migration and invasion by activating the deregulation of the CCR7-mediated pathway in RSA. We are convinced that CCR7 and its downstream factors may be possible mechanisms for the pathogenesis of RSA.

Decreased brain and muscle ARNT-like protein 1 expression mediated the contribution of hyperandrogenism to insulin resistance in polycystic ovary syndrome.

BACKGROUND: The interface between environmental risk factors and genetic factors could contribute to the pathogenesis of hyperandrogenism and insulin resistance in polycystic ovary syndrome (PCOS); however, the underlying complex mechanism remains to be elucidated. METHODS: We used dehydroepiandrosterone (DHEA)-induced PCOS-like rat model to measure circadian clock genes and insulin resistance-related genes. Additionally, we performed in vitro experiments in mature adipocytes to verify the molecular mechanisms. RESULTS: DHEA-induced PCOS-like rats exhibited insulin resistance and arrhythmic expression of circadian clock genes in the liver and adipose tissues, particularly showing decreased brain and muscle ARNT-like protein 1 (BMAL1) expression. In addition, hyperandrogenism gave rise to negative regulation of BMAL1 expression to nicotinamide phosphoribosyltransferase and sirtuin 1, which further inhibited downstream glucose transporter type 4, leading to insulin resistance in mature adipocytes, which was consistent with our previous results in HepG2 cells. CONCLUSIONS: Decreased BMAL1 expression in the liver and adipose played a potentially novel role in the contribution of hyperandrogenism to insulin resistance, which might be a possible mechanism accounting for the pathogenesis of PCOS.

A candidate pathogenic gene, zinc finger gene 217 (ZNF217), may contribute to polycystic ovary syndrome through prostaglandin E2.

INTRODUCTION: Polycystic ovary syndrome is a complex endocrine condition with chronic inflammation. Prostaglandin E2 (PGE2) is a proinflammatory factor with an increased expression in the serum of women with polycystic ovary syndrome. Zinc finger gene 217 (ZNF217) is known as a candidate gene for polycystic ovary syndrome. We aimed to investigate the relation between ZNF217 and PGE2 in polycystic ovary syndrome. MATERIAL AND METHODS: We used a rat model of dehydroepiandrosterone-induced polycystic ovary syndrome and human granulosa cells both of women with polycystic ovary syndrome and of women without the syndrome to measure ZNF217 and other target gene expressions. In addition, we performed in vitro experiments with KGN human granulosa-like tumor cells to verify the molecular mechanisms. RESULTS: ZNF217 was decreased in the granulosa cells both of dehydroepiandrosterone-treated rats and of women with polycystic ovary syndrome. Cyclooxygenase 2, a key enzyme of PGE2 synthesis, was highly expressed in the granulosa cells of rats and women with the syndrome, and PGE2 concentration was increased in the follicular fluid. Furthermore, decreased ZNF217 expression was supposed to inhibit estradiol synthesis, which further promoted cyclooxygenase 2 and PGE2 synthesis. At the same time, PGE2 had an inhibitory effect on ZNF217 expression in a dose-dependent manner in KGN cells. CONCLUSIONS: Decreased ZNF217 expression in granulosa cells of women with polycystic ovary syndrome induced inflammation via PGE2, and PGE2 inhibited ZNF217 expression to establish a feedback loop. This mechanism might account for the pathogenesis of polycystic ovary syndrome.

Kisspeptin-10 inhibits OHSS by suppressing VEGF secretion.

The aim of the present study was to elucidate the effects of kisspeptin-10 (Kp-10) on ovarian hyperstimulation syndrome (OHSS) and its related mechanism in OHSS rat models, human umbilical vein endothelial cells (HUVECs) and human luteinized granulosa cells. OHSS is a systemic disorder with high vascular permeability (VP) and ovarian enlargement. KISS1R (KISS1 receptor) is the specific receptor of kisspeptin. The kisspeptin/KISS1R system inhibits the expression of vascular endothelial growth factor (VEGF), which is the main regulator of VP. In our study, decreased expression of Kiss1r was observed in both ovaries and lung tissue of OHSS rats. Injection of exogenous Kp-10 inhibited the increase of VP and VEGF while promoting the expression of Kiss1r in both the ovarian and lung tissue of OHSS rats. Using HUVECs, we revealed that a high level of 17-ß estradiol (E2), a feature of OHSS, suppressed the expression of KISS1R and increased VEGF and nitric oxide (NO) through estrogen receptors (ESR2). Furthermore, KISS1R mRNA also decreased in the luteinized human granulosa cells of high-risk OHSS patients, and was consistent with the results in rat models and HUVECs. In conclusion, Kp-10 prevents the increased VP of OHSS by the activation of KISS1R and the inhibition of VEGF.

Kisspeptin: a new marker for human pre-ovulation.

Kisspeptin is a polypeptide that plays an important role in reproductive endocrine regulation. The aim of present study was to investigate the dynamic trend of kisspeptin levels during the menstrual cycle and to elucidate the relationship between kisspeptin ovulation. First, 15 female volunteers with regular menstrual cycles were recruited to detect the change in serum and urine kisspeptin levels over one menstrual cycle within each individual. Subsequently, 114 serum samples and 79 urine samples from 114 individuals were randomly collected at the outpatient department to better ascertain the results. Kisspeptin levels showed a distinctive stage-specific pattern during the normal menstrual cycle in both serum and urine. It was low during the first 5 days, while the first surge appeared on the 11th day (the diameter of the dominant follicle was approximately 1.2 cm). Later, a second smaller surge appeared around the 14th day and the same changes were identified in serum and urine. Furthermore, serum kisspeptin levels were positively related to 17-ß estradiol (E2) level increase. Thus, kisspeptin surge in serum and urine may be used as a marker for dominant follicle development and pre-ovulation. Moreover, kisspeptin may also play a vital role in female reproduction through regulating hormonal state.

Estradiol Decline Before hCG Administration in COH Has a Negative Effect on IVF Outcomes in Patients Without OC Pretreatment.

Purpose: Together with ultrasound measurement of follicle size, serum estradiol (E2) provides guidance for controlled ovarian hyperstimulation (COH). However, during the COH process, some patients experience decreased serum E2 level, especially before human chorionic gonadotropin (hCG) trigger. In order to elucidate the effect of E2 reduction as well as the role of oral contraceptive pretreatment, a retrospective study was performed in our center from 2013 to 2019. Patients and Methods: In total, 333 patients who experienced an E2 decrease prior to hCG administration were recruited as E2 decline group, while 333 patients with continuously E2 increase during COH were considered as control group. Based on pretreatment strategy, the two groups were further categorized into oral contraceptive (OC) and non-OC sub-groups, and IVF and clinical outcomes were compared between paired groups. Results: Number of dominant follicles on hCG day and normally fertilized zygotes were significantly decreased in E2 decline group, and the significantly reduced live birth rate in E2 decline group indicated the close relationship between E2 decline and clinical outcomes. To analyse further, we found that in patients without OC pretreatment, the pregnancy rate and live birth rate of E2 decline group (n = 141) were significantly lower than control group (n = 136) (56.3% versus 68.0%, 50.8% versus 63.5%, respectively). However, for patients with OC pretreatment, no difference was detected between two groups, suggesting a potential effect of OC pretreatment on clinical outcomes. Conclusion: E2 decline prior to hCG-triggering day adversely affects IVF and clinical outcomes in patients without OC pretreatment, especially fertilization rate and live birth rate.

Single-cell RNA sequencing and ATAC sequencing identify novel biomarkers for bicuspid aortic valve-associated thoracic aortic aneurysm.

Introduction: Bicuspid aortic valve (BAV) is the most prevalent congenital cardiovascular defect and known to cause thoracic aortic aneurysms (TAAs). To improve our understanding of BAV pathogenesis, we characterized the cellular composition of BAV tissues and identified molecular changes in each cell population. Methods: Tissue samples from two patients with BAV and two heart transplant donors were analyzed using single-cell RNA sequencing, assay for transposase-accessible chromatin using sequencing, and weighted gene coexpression network analysis for differential gene analysis. TAA-related changes were evaluated by comparing the proportion of each cell type and gene expression profiles between TAA and control tissues. Further, by combining our single-cell RNA sequencing data with publicly available data from genome-wide association studies, we determined critical genes for BAV. Results: We found 20 cell subpopulations in TAA tissues, including multiple subtypes of smooth muscle cells, fibroblasts, macrophages, and T lymphocytes. This result suggested that these cells play multiple functional roles in BAV development. Several differentially expressed genes, including CD9, FHL1y, HSP90AA1, GAS6, PALLD, and ACTA2, were identified. Discussion: We believe that this comprehensive assessment of the cellular composition of TAA tissues and the insights into altered gene expression patterns can facilitate identification of novel diagnostic biomarkers and therapeutic targets for BAV-associated TAA.

SNHG 12 and hsa-miR-140-5P may play an important role in the ceRNA network related to hypertrophic cardiomyopathy.

Background: Competing endogenous RNA (ceRNA) networks play important roles in the mechanism and development of a variety of diseases. This study aimed to construct a ceRNA network of hypertrophic cardiomyopathy (HCM). Methods: We searched the Gene Expression Omnibus (GEO) database and then analyzed the RNAs of 353 samples to explore differentially expressed lncRNAs (DELs), microRNAs (miRNAs; DEMs), and messenger RNAs (DEmRNAs) during the progression of HCM. Weighted gene co-expression network analysis (WGCNA), Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and transcription factor (TF) prediction of miRNAs were also performed, and the GO terms, KEGG pathway terms, protein-protein interaction (PPI) network, and Pearson correlation network of the DEGs were visualized with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database and through Pearson analysis. In addition, a ceRNA network related to HCM was constructed on the basis of the DELs, DEMs, and DEs. Finally, the function of the ceRNA network was explored via GO and KEGG enrichment analyses. Results: Through our analysis, 93 DELs (77 upregulated and 16 downregulated), 163 DEMs (91 upregulated and 72 downregulated), and 432 DEGs (238 upregulated and 194 downregulated) were screened. The functional enrichment analysis results for miRNAs showed that the miRNAs were mainly related to the VEGFR signaling network and the INFr pathway and were mainly regulated by TFs such as SOX1, TEAD1, and POU2F1. Gene set enrichment analysis (GSEA), GO analysis, and KEGG enrichment analysis showed that the DEGs were enriched in the Hedgehog signaling pathway, IL-17 signaling pathway, and TNF signaling pathway. In addition, a ceRNA network including 8 lncRNAs (e.g., LINC00324, SNHG12, and ALMS1-IT1), 7 miRNAs (e.g., hsa-miR-217, hsa-miR-184, and hsa-miR-140-5p), and 52 mRNAs (e.g., IGFBP5, TMED5, and MAGT1) was constructed. The results revealed that SNHG12, hsa-miR-140-5p, hsa-miR-217, TFRC, HDAC4, TJP1, IGFBP5, and CREB5 may form an important network involved in the pathology of HCM. Conclusions: The novel ceRNA network that we have demonstrated will provide new research points about molecular mechanisms of HCM.

Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling.

Knowledge gaps that limit the development of therapies for polycystic ovary syndrome (PCOS) concern various environmental factors that impact clinical characteristics. Circadian dysrhythmia contributes to glycometabolic and reproductive hallmarks of PCOS. Here, we illustrated the amelioration of Limosilactobacillus reuteri (L. reuteri) on biorhythm disorder-ignited dyslipidemia of PCOS via a microbiota-metabolite-liver axis. A rat model of long-term (8 weeks) darkness treatment was used to mimic circadian dysrhythmia-induced PCOS. Hepatic transcriptomics certified by in vitro experiments demonstrated that increased hepatic galanin receptor 1 (GALR1) due to darkness exposure functioned as a critical upstream factor in the phosphoinositide 3-kinase (PI3K)/protein kinase B pathway to suppress nuclear receptors subfamily 1, group D, member 1 (NR1D1) and promoted sterol regulatory element binding protein 1 (SREBP1), inducing lipid accumulation in the liver. Further investigations figured out a restructured microbiome-metabolome network following L. reuteri administration to protect darkness rats against dyslipidemia. Notably, L. reuteri intervention resulted in the decrease of Clostridium sensu stricto 1 and Ruminococcaceae UCG-010 as well as gut microbiota-derived metabolite capric acid, which could further inhibit GALR1-NR1D1-SREBP1 pathway in the liver. In addition, GALR antagonist M40 reproduced similar ameliorative effects as L. reuteri to protect against dyslipidemia. While exogenous treatment of capric acid restrained the protective effects of L. reuteri in circadian disruption-induced PCOS through inhibiting GALR1-dependent hepatic lipid metabolism. These findings purport that L. reuteri could serve for circadian disruption-associated dyslipidemia. Manipulation of L. reuteri-capric acid-GALR1 axis paves way for clinical therapeutic strategies to prevent biorhythm disorder-ignited dyslipidemia in PCOS women.

Complement component 3 haplotypes influence serum complement activity and milk production traits in Chinese Holstein cattle.

Complement component 3 (C3) is the key molecule of the three pathways of complement activation (alternative, classical, and lectin pathways), which are involved in phagocytosis, inflammation, and immunoregulation processes to destroy infectious microorganisms. In this study, three novel single-nucleotide polymorphisms (SNPs) (g.-1293C>G located in the 5'-flanking region, g.56T>C in exon I, and g.7017C>T in exon XII) of the C3 gene were detected using created restriction site polymerase chain reaction, restriction fragment length polymorphism, and DNA sequencing in 952 cattle from three Chinese breeds. The genotypes and haplotypes were analyzed to investigate the polymorphisms and their possible implications, with particular investigative focus on their associations with serum C3 level, complement hemolytic activity (CH50 and ACH50), and milk production traits. The g.56T>C SNP in exon I affected the serum ACH50 (P<0.01) and the milk somatic cell score (SCS) (P<0.05), and the g.7017C>T SNP in exon XII significantly affected the serum ACH50 values (P<0.01). Moreover, statistical analyses revealed that individuals with genotypic combination CCC/GCC showed significantly lower SCS and the lowest C3 concentration in serum compared with cows with CCC/GTT (P = 0.0007) and CTT/CTT (P = 0.0021); the individuals with CCC/CCT had significantly higher ACH50 values than those with CCC/CTC (P = 0.0008) and CTC/GTC (P = 0.001); cows with CCT/CTT had higher values of CH50 and 305-day milk yield (P>0.05). The C3 expression levels were significantly increased in lung and mammary tissues (P<0.05), while significantly decreased in heart, spleen, liver, and kidney tissues in mastitis cows compared with those in healthy animals (P<0.01), respectively. Bacterial counts of serum antibacterial activities were also completed to verify the effect of SNPs on resistance to mastitis pathogens. Genetically resistant cows (CCC/GCC) had serum with noticeably higher antibacterial activity against S. aureus and E. coli in vitro than the genetically susceptible CCC/GTT cows (P<0.05). Results from this study imply that the C3 gene plays a role in resistance to bacterial infection and that it can be used as a molecular marker for complement activity and traits related to milk production.

Long-term environmental exposure of darkness induces hyperandrogenism in PCOS via melatonin receptor 1A and aromatase reduction.

Polycystic ovary syndrome (PCOS) is a common and complex disorder impairing female fertility, yet its etiology remains elusive. It is reported that circadian rhythm disruption might play a crucial role in PCOS pathologic progression. Here, in this research, we investigated the effect of environmental long-term circadian rhythm dysfunction and clarified its pathogenic mechanism in the development of PCOS, which might provide the targeted clinical strategies to patients with PCOS. Female SD rats were used to construct a circadian rhythm misalignment model with constant darkness (12/12-h dark/dark cycle), and the control group was kept under normal circadian rhythm exposure (12/12-h light/dark cycle) for 8 weeks. We measured their reproductive, endocrinal, and metabolic profiles at different zeitgeber times (ZTs). Different rescue methods, including melatonin receptor agonist and normal circadian rhythm restoration, and in vitro experiments on the KGN cell line were performed. We found that long-term darkness caused PCOS-like reproductive abnormalities, including estrous cycle disorder, polycystic ovaries, LH elevation, hyperandrogenism, and glucose intolerance. In addition, the expression of melatonin receptor 1A (Mtnr1a) in ovarian granulosa cells significantly decreased in the darkness group. Normal light/dark cycle and melatonin receptor agonist application relieved hyperandrogenism of darkness-treated rats. In vitro experiments demonstrated that decreased MTNR1A inhibited androgen receptor (AR) and CYP19A1 expression, and AR acted as an essential downstream factor of MTNR1A in modulating aromatase abundance. Overall, our finding demonstrates the significant influence of circadian rhythms on PCOS occurrence, suggests that MTNR1A and AR play vital roles in pathological progression of hyperandrogenism, and broadens current treatment strategies for PCOS in clinical practice.

Data mining analysis of the prognostic impact of N6-methyladenosine regulators in patients with endometrial adenocarcinoma.

We reanalyzed the expression of 16 acknowledged N6-methyladenosine (m6A) RNA regulators in 406 endometrial adenocarcinoma patients and 19 controls using The Cancer Genome Atlas (TCGA) dataset, and further verified our results using Gene Expression Omnibus (GEO) dataset and real-time quantitative polymerase chain reaction. Thirteen m6A RNA methylation regulators were differentially expressed between patients with endometrial adenocarcinoma and controls. FTO, RBM15, and YTHDF1, were identified as independent prognostic markers and closely associated with International Federation of Gynecology and Obstetrics grade in endometrial cancer patients. GEO dataset also verified the differential expression of FTO and RBM15 between patients with endometrial adenocarcinoma and hyperplasia. Functional enrichment and ingenuity pathway analysis network suggested that FTO and RBM15 contributed to the survival of patients with endometrial adenocarcinoma via the regulation of connective tissue development, catabolic process, RNA stability, oxidative demethylation, temperature homeostasis, and energy metabolism through IGF1, IRS1, RBM24, LARP1, and CBFA2T3. The decreased FTO expression and increased RBM15 expression in endometrial adenocarcinoma from our validation cohort was consistent with in silico analysis using TCGA and GEO datasets. In conclusion, m6A methylation regulators, especially FTO, RBM15, and YTHDF1, are critical in the progression and prognosis of endometrial adenocarcinoma.

Serum Sex Hormone Binding Globulin Concentration as a Predictor of Ovarian Response During Controlled Ovarian Hyperstimulation.

Purpose: Serum concentrations of sex hormone binding globulin (SHBG), a glycated homodimeric plasma transport protein, correlate positively with the total number of follicles in women with infertility. However, the relationship between serum SHBG concentrations and the ovarian response during controlled ovarian hyperstimulation (COH) and whether this relationship differs between women with and without polycystic ovary syndrome (PCOS) remains unclear. Methods: The study cohort included 120 participants (60 non-PCOS and 60 PCOS) undergoing in vitro fertilization. Serum samples were collected from each participant every 2-3 days during the COH cycle. The concentrations of serum SHBG and other sex hormones were determined to investigate the relationship between serum SHBG concentrations and the ovarian response in women with and without PCOS. Results: We found that the serum SHBG concentration was positively correlated with the ovarian response in non-PCOS patients but not in PCOS patients. Conclusion: The serum SHBG concentration may be clinically useful as a predictor of the ovarian response during COH in patients without PCOS.

In Silico, In Vitro, and In Vivo Analysis Identifies Endometrial Circadian Clock Genes in Recurrent Implantation Failure.

CONTEXT: Previous work has demonstrated the role of the circadian clock in ovarian steroid hormone synthesis and attributed embryo implantation failure associated with arrhythmic circadian clock genes to insufficient ovarian-derived progesterone synthesis. Research on expression of core circadian clock genes in the endometrium itself and possible roles in compromised endometrial receptivity and recurrent implantation failure (RIF) are limited. OBJECTIVE: We aimed to assess the core circadian clock gene profiling in human endometrium across the menstrual cycle and the possible gene interaction networks in the endometrial receptivity of window of implantation (WOI) as well as RIF. METHODS: The study was initially an in silico study, with confirmatory lab-based data from primary human endometrial stromal cells (hESCs) as well as endometrial biopsies obtained from 60 women undergoing gynecological surgery in a clinical research center. The study included 30 RIF women and 30 age-matched and body mass index-matched controls. RESULTS: Initial data mining and bioinformatics analysis of human endometrial microarray datasets across the menstrual cycle and between RIF women versus controls demonstrated the varied expression of core circadian clock genes across menstrual cycle, including the key role of PER2 in WOI and RIF. A PER2-centered network was investigated in the regulation of endometrial receptivity. We also confirmed the evidently increased mRNA expression of SHTN1, RXFP1, KLF5, and STEAP4 in the endometrium of RIF women, displaying the same trend as PER2 did, without any changes in MT1E and FKBP5. Treatment of PER2 siRNA in hESCs verified the positive regulation of PER2 to SHTN1, KLF5, and STEAP4. CONCLUSION: Aberrant expression of endometrial PER2 might contribute to impaired endometrial receptivity and development of RIF via regulating SHTN1, KLF5, and STEAP4.

Adenomyosis: Mechanisms and Pathogenesis.

Adenomyosis is a common disorder of the uterus, and is associated with an enlarged uterus, heavy menstrual bleeding (HMB), pelvic pain, and infertility. It is characterized by endometrial epithelial cells and stromal fibroblasts abnormally found in the myometrium where they elicit hyperplasia and hypertrophy of surrounding smooth muscle cells. While both the mechanistic processes and the pathogenesis of adenomyosis are uncertain, several theories have been put forward addressing how this disease develops. These include intrinsic or induced (1) microtrauma of the endometrial-myometrial interface; (2) enhanced invasion of endometrium into myometrium; (3) metaplasia of stem cells in myometrium; (4) infiltration of endometrial cells in retrograde menstrual effluent into the uterine wall from the serosal side; (5) induction of adenomyotic lesions by aberrant local steroid and pituitary hormones; and (6) abnormal uterine development in response to genetic and epigenetic modifications. Dysmenorrhea, HMB, and infertility are likely results of inflammation, neurogenesis, angiogenesis, and contractile abnormalities in the endometrial and myometrial components. Elucidating mechanisms underlying the pathogenesis of adenomyosis raise possibilities to develop targeted therapies to ameliorate symptoms beyond the current agents that are largely ineffective. Herein, we address these possible etiologies and data that support underlying mechanisms.

Altered circadian clock as a novel therapeutic target for constant darkness-induced insulin resistance and hyperandrogenism of polycystic ovary syndrome.

The mechanisms underlying metabolic and reproductive dysfunction caused by arrhythmic circadian clock and their involvement in polycystic ovary syndrome (PCOS) are not understood. Here, we addressed this issue using rats with constant light or darkness exposure for 8 weeks and human leukocytes and serum of PCOS and non-PCOS patients. Additionally, we utilized HepG2 cells and KGN cells to verify the molecular mechanisms. The arrhythmic expressions of circadian clock genes due to constant darkness induced the metabolic and reproductive hallmarks of PCOS in rats. After exposure to constant darkness, decreased brain and muscle ARNT-like protein 1 (BMAL1) promoted insulin resistance via glucose transporter 4 (GLUT4), and decreased period (PER) 1 and PER2 promoted androgen excess via insulin-like growth factor-binding protein 4 (IGFBP4) and sex hormone binding globulin (SHBG) in the liver. Hyperinsulinemia and hyperandrogenism shared a bidirectional link promoting aberrant expression of circadian genes and inducing apoptosis of ovarian granulosa cells. Notably, the altered expressions of circadian clock genes in darkness-treated rats matched those of PCOS patients. Furthermore, melatonin treatment relieved the hyperinsulinemia and hyperandrogenism of darkness-treated rats via BMAL1, PER1, and PER2. Restoring normal light/dark exposure for 2 weeks reversed these conditions via BMAL1. In conclusion, our findings elucidated the critical function of circadian clock genes, especially BMAL1, PER1, and PER2 in PCOS, which might aid the development of feasible preventive and therapeutic strategies for PCOS in women with biorhythm disorder.