CircFOXO3 mediates hypoxia-induced autophagy of endometrial stromal cells in endometriosis.

Endometriosis is a benign gynecological disease that shares some common features of malignancy. Autophagy plays vital roles in endometriosis and influences endometrial cell metastasis, and hypoxia was identified as the initiator of this pathological process through hypoxia inducible factor 1 alpha (HIF-1α). A newly discovered circular RNA FOXO3 (circFOXO3) is critical in cell autophagy, migration, and invasion of various diseases and is reported to be related to hypoxia, although its role in endometriosis remains to be elucidated up to now. In this study, a lower circFOXO3 expression in ectopic endometrium was investigated. Furthermore, we verified that circFOXO3 could regulate autophagy by downregulating the level of p53 protein to mediate the migration and invasion of human endometrial stromal cells (T HESCs). Additionally, the effects of HIF-1α on circFOXO3 and autophagy were examined in T HESCs. Notably, overexpression of HIF-1α could induce autophagy and inhibit circFOXO3 expression, whereas overexpressing of circFOXO3 under hypoxia significantly inhibited hypoxia-induced autophagy. Mechanistically, the direct combination between HIF-1α and HIF-1α-binding site on adenosine deaminase 1 acting on RNA (ADAR1) promoter increased the level of ADAR1 protein, which bind directly with circFOXO3 pre-mRNA to block the cyclization of circFOXO3. All these results support that hypoxia-mediated ADAR1 elevation inhibited the expression of circFOXO3, and then autophagy was induced upon loss of circFOXO3 via inhibition of p53 degradation, participating in the development of endometriosis.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

STUDY QUESTION: Is pronuclear transfer (PNT) capable of restoring embryo developmental arrest caused by cytoplasmic inferiority of in vitro-grown (IVG) mouse oocytes? SUMMARY ANSWER: PNT to in vivo matured cytoplasm significantly improved embryo development of IVG mouse oocytes, leading to living, fertile offspring. WHAT IS KNOWN ALREADY: In vitro follicle culture has been considered as a fertility preservation option for cancer patients. Studies describing the culture of human follicles remain scarce, owing to low availability of tissue. Mouse models have extensively been used to study and optimize follicle culture. Although important achievements have been accomplished, including the production of healthy offspring in mice, IVG oocytes are of inferior quality when compared to in vivo-grown oocytes, likely because of cytoplasmic incompetence. STUDY DESIGN SIZE DURATION: The study was carried out from September 2020 to February 2022. In total, 120 15-day-old B6D2 mice were used to perform secondary follicle culture and assess the quality of IVG oocytes. In vivo-grown control oocytes were obtained from 85 8- to 12-week-old B6D2 mice, following ovarian stimulation. For sperm collection, four B6D2 males between 10 and 14 weeks old were used. For embryo transfer, 14 8- to 12-week-old CD1 females served as surrogate mothers and 10 CD1 vasectomized males 10-24 weeks old were used to generate pseudo-pregnant females. Finally, for mating, four B6D2 female mice aged 8-10 weeks and two B6D2 male mice aged 10 weeks old were used to confirm the fertility of nuclear transfer (NT)-derived pups. PARTICIPANTS/MATERIALS SETTING METHODS: Secondary follicles from 15-day-old B6D2 mice were isolated from the ovaries and cultured for 9 days, before a maturation stimulus was given. Following 16-18 h of maturation, oocytes were collected and evaluated on maturation rate, oocyte diameter, activation rate, spindle morphology, calcium-releasing ability, and mitochondrial membrane potential. For every experiment, in vivo-grown oocytes were used as a control for comparison. When cytoplasmic immaturity and poor embryo development were confirmed in IVG oocytes, PNT was performed. For this, the pronuclei from IVG oocytes, created following parthenogenetic activation and IVF, were transferred to the cytoplasm of fertilized, in vivo-grown oocytes. Genetic analysis and embryo transfer of the generated embryos were implemented to confirm the safety of the technique. MAIN RESULTS AND THE ROLE OF CHANCE: Following 9 days of follicle culture, 703 oocytes were collected, of which 76% showed maturation to the metaphase II stage. Oocyte diameters were significantly lower in IVG oocytes, measuring 67.4 µm versus 73.1 µm in controls (P < 0.001). Spindle morphology did not differ significantly between IVG and control oocytes, but calcium-releasing ability was compromised in the IVG group. An average calcium release of 1.62 arbitrary units was observed in IVG oocytes, significantly lower than 5.74 in control oocytes (P < 0.001). Finally, mitochondrial membrane potential was inferior in IVG compared to the control group, reaching an average value of 0.95 versus 2.27 (P < 0.001). Developmental potential of IVG oocytes was assessed following parthenogenetic activation with strontium chloride (SrCl2). Only 59.4% of IVG oocytes cleaved to two cells and 36.3% reached the blastocyst stage, significantly lower than 89.5% and 88.2% in control oocytes, respectively (P < 0.001 and 0.001). Both PNT and spindle transfer (ST) were explored in pilot experiments with parthenogenetically activated oocytes, as a means to overcome poor embryo development. After the added value of NT was confirmed, we continued with the generation of biparental embryos by PNT. For this purpose, IVG and control oocytes first underwent IVF. Only 15.5% of IVG oocytes were normally fertilized, in contrast to 45.5% in controls (P < 0.001), with resulting failure of blastocyst formation in the IVG group (0 versus 86.2%, P < 0.001). When the pronuclei of IVG zygotes were transferred to the cytoplasm of control zygotes, the blastocyst rate was restored to 86.9%, a similar level as the control. Genetic analysis of PNT embryos revealed a normal chromosomal profile, to a rate of 80%. Finally, the generation of living, fertile offspring from PNT was possible following embryo transfer to surrogate mothers. LARGE-SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Genetic profiles of analysed embryos from PNT originate from groups that are too small to draw concrete conclusions, whilst ST, which would be the preferred NT approach, could not be used for the generation of biparental embryos owing to technical limitations. Even though promising, the use of PNT should be considered as experimental. Furthermore, results were acquired in a mouse model, so validation of the technique in human IVG oocytes needs to be performed to evaluate the clinical relevance of the technology. The genetic profiles from IVG oocytes, which would be the ultimate characterization for chromosomal abnormalities, were not analysed owing to limitations in the reliable analysis of single cells. WIDER IMPLICATIONS OF THE FINDINGS: PNT has the ability to overcome the poor cytoplasmic quality of IVG mouse oocytes. Considering the low maturation efficiency of human IVG oocytes and potential detrimental effects following long-term in vitro culture, NT could be applied to rescue embryo development and could lead to an increased availability of good quality embryos for transfer. STUDY FUNDING/COMPETING INTERESTS: A.C. is a holder of FWO (Fonds voor Wetenschappelijk Onderzoek) grants (1S80220N and 1S80222N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) 2018000504 (GOA030-18 BOF) funding. B.H. has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest.

Low dose of liraglutide combined with metformin leads to a significant weight loss in Chinese Han women with polycystic ovary syndrome: a retrospective study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder with complex pathophysiological mechanism. It is reported that even a modest weight loss of 5-10% substantially may improve the reproductive and metabolic profile. This study aims to assess the efficacy of the low dose of liraglutide (0.6 mg QD) combined with metformin (0.85 mg BID) in weight loss in Chinese Han women with PCOS. METHODS: We included clinical data of 102 obese/overweight (≥18 years, body mass index ≥28 kg/m2 or ≥24 kg/m2) women who were diagnosed with PCOS from October 2016 to March 2018 in Wuhan Union Hospital initially. They were treated with dinae-35, low dose of liraglutide (0.6 mg QD) and metformin (0.85 mg BID) for 12 weeks. The demographic and clinical data were retrieved retrospectively, and weight loss was the main outcome measure. Student's paired t-test and Wilcoxon rank sum test were used to compare the differences before and after therapy, p < 0.05 was considered statistically significant. RESULTS: Participants(n = 102)had lost a mean of 7.20 ± 3.42 kg of body weight (95%CI: 6.55-7.86, p < 0.001), and the mean reduction of BMI was 2.87 ± 1.36 kg/m2 (95%CI: 0.02-0.27, p < 0.001). A total of 88.24% of participants lost more than 5% of their body weight. CONCLUSION: The combination of low dose of liraglutide and metformin was associated with significant reduction of body weight in Chinese Han women with PCOS. Additionally, a larger randomized double-blind multicenter controlled clinical trial is needed to confirm that. TRIAL REGISTRATION: The study was registered on http://www.chictr.org.cn as ChiCTR1900024384.

Upregulation of the long noncoding RNA UBOX5 antisense RNA 1 (UBOX5-AS1) under hypoxic conditions promotes epithelial-mesenchymal transition in endometriosis.

BACKGROUND: Endometriosis is a debilitating gynecological condition that manifests many common malignant features, including migration and invasion. Hypoxia is a hallmark of endometriosis, characterized by endometrial cell metastasis via epithelial-mesenchymal transition (EMT). The long noncoding RNA (lncRNA) UBOX antisense RNA 1 (UBOX5-AS1) has been shown to be upregulated in ovarian endometriosis. However, the molecular mechanisms and biological functions of lncRNA UBOX5-AS1 in hypoxia-induced endometriosis EMT remain to be explored. METHODS: Normal, eutopic, and ectopic endometrium from ovarian endometriosis tissues were collected, and the expressions of hypoxia inducible factor (HIF)-1α, lncRNA UBOX5-AS1, E-cadherin, and vimentin were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting analysis. Primary human endometrial epithelial cells and human endometrial epithelial adenocarcinoma Ishikawa cell lines were cultured under hypoxic conditions, and western blotting analysis and immunocytochemistry were performed to investigate hypoxia-induced EMT. Moreover, HIF-1α and lncRNA UBOX5-AS1 were overexpressed and knocked down in endometrial epithelial cells to explore the role and mechanisms of lncRNA UBOX5-AS1 in hypoxia-triggered EMT. The migration and invasion potential of human endometrial epithelial cells was detected by Transwell migration/invasion assays. RESULTS: In ovarian endometriosis, the expression of hypoxia-inducible factor-1α (HIF-1α) and lncRNA UBOX5-AS1 were significantly increased, and this was accompanied by EMT. Furthermore, endometrial epithelial cells cultured under hypoxic conditions exhibited elevated lncRNA UBOX5-AS1 expression, as well as migration, invasion, and an EMT-like phenotype. This data indicated that HIF-1α signaling was crucial for hypoxia-induced lncRNA UBOX5-AS1 upregulation and the EMT process. Moreover, downregulation of lncRNA UBOX5-AS1 inhibited the hypoxia-induced EMT and attenuated cell migration and invasion. CONCLUSIONS: The present research demonstrated that hypoxia upregulated the expression of lncRNA UBOX5-AS1 via HIF-1α-dependent signaling. The increased expression of lncRNA UBOX5-AS1 plays a vital role in mediating the hypoxia-regulated EMT and invasiveness of endometriosis, suggesting that lncRNA UBOX5-AS1 may be an important potential therapeutic target for endometriosis.

Estrogen-decreased hsa_circ_0001649 promotes stromal cell invasion in endometriosis.

Endometriosis (EMs) is an estrogen (E2)-dependent inflammatory disorder. Although EMs is considered a benign disease, it presents with malignant characteristics, such as migration and invasion. An increasing number of studies have shown that aberrantly expressed circular RNAs (circRNAs) play an essential role in disease development and progression. However, the mechanisms by which circRNAs exert their pathological effects in EMs remain unclear. Hsa_circ_0001649, a novel cancer-associated circRNA, has been previously reported to be downregulated in several cancer types and related to cell migration and invasion. In the present study, real-time PCR (qRT-PCR) was carried out to measure hsa_circ_0001649 levels in human tissues, human primary endometrial stromal cells (ESCs) and a human endometrial stromal cell line (ThESCs). Matrix metalloproteinase 9 (MMP9) levels in ESCs and ThESCs were assessed by qRT-PCR and Western blotting, and the migration and invasion capacities of ThESCs were evaluated by transwell assay. As a result, hsa_circ_0001649 expression was significantly decreased in ectopic and eutopic endometrial samples compared with that in normal endometrial samples. E2 decreased hsa_circ_0001649 expression but increased MMP9 expression in ESCs and ThESCs. Furthermore, ThESCs were more invasive under E2 stimulation. However, these effects disappeared when ICI or hsa_circ_0001649 transfection was used. Collectively, our findings reveal that decreased hsa_circ_0001649 expression plays a role in E2-increased MMP9 expression through E2 receptors (ERs), which have critical functions in EMs.

E2 -mediated EMT by activation of ß-catenin/Snail signalling during the development of ovarian endometriosis.

Endometriosis is an oestrogen-dependent disease, and epithelial-mesenchymal transition (EMT) is involved in the process of endometriosis. Whether oestrogen could induce EMT in endometriosis remains largely unknown. Here, we reported that up-regulated expression of EMT markers in ovarian chocolate cyst is accompanied by high expression 17ß-hydroxysteroid dehydrogenase 1 (17ß-HSD1), and exposure of primary human endometrial epithelial cells to oestradiol conditions could promote EMT occurrence and activate both ß-catenin and Snail signalling. Furthermore, we found nuclear ß-catenin and Snail expression was closely linked in ovarian endometriosis, and ß-catenin knockdown abrogated oestrogen-induced Snail mediated EMT in vitro. This is due to that ß-catenin/ TCF-3 could bind to Snail promoter and activate its transcription. These results suggested that ß-catenin signalling functions as the Snail activator and plays a critical role in oestradiol-induced EMT in endometriosis.

Estradiol promotes EMT in endometriosis via MALAT1/miR200s sponge function.

Endometriosis is an estrogen-dependent benign gynecological disease that shares some common features of malignancy. Epithelial-mesenchymal transition (EMT) has been recognized as a core mechanism of endometriosis. MALAT1 is widely known as EMT promoter, while miR200 family members (miR200s) are considered as EMT inhibitors. Previous studies have reported that MALAT1 upregulation and miR200s downregulation are observed in endometriosis. MiR200c has been regarded as the strongest member of miR200s to interact with MALAT1. However, whether MALAT1/miR200c regulates EMT remains largely unclear. In this study, the roles of miR200s and MALAT1 in ectopic endometrium were investigated. Additionally, the effects of E2 on EMT and MALAT1/miR200s were examined in both EECs and Ishikawa cells. Notably, E2 could upregulate MALAT1 and downregulate miR200s expression levels and induce EMT in EECs and Ishikawa cells. PHTPP, an ERß antagonist, could reverse the effect of E2. Overexpression of miR200c and knockdown of MALAT1 significantly inhibited E2-mediated EMT, suggesting that both miR200c and MALAT1 are involved in the E2-induced EMT process in endometriosis. In addition, a reciprocal inhibition was found between miR200s and MALAT1. Therefore, the role of MALAT1/miR200c in EMT is influenced by the presence of estrogen during endometriosis development.

Association of characteristics of HBV quasispecies with hepatitis B surface antigen seroconversion after pegylated interferon-α-2a treatment in child patients.

BACKGROUND: The correlation between hepatitis B surface antigen (HBsAg) seroconversion and the characteristics of HBV quasispecies (QS) before and during pegylated interferon-α-2a (PEG-IFN-α-2a) treatment in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) children has not yet been reported. METHODS: 35 patients, including 18 HBsAg seroconverters (SS) and 17 non-seroconverters (SN), were enrolled. Serum samples were collected before treatment and at weeks 12 and 24 of treatment. Sequences within the basal core promoter/pre-core (BCP/PC) and S/reverse transcriptase (S/RT) region were analysed by next-generation sequencing. RESULTS: There was no significant difference in the baseline diversity of HBV QS (Shannon entropy [Sn]; Hamming distance [HD]) in either region between the two groups. The baseline mutations A1762T/G1764A, C1913A, and T2003A/G or C2004T were correlated with non-response to therapy (P=0.025, P=0.036, P=0.032, respectively). After 24 weeks of therapy, HBV diversity within the BCP/PC region in the SS group notably declined (Sn: P=0.002; HD: P=0.011), while that of the SN group was nearly unchanged. As for the S/RT region, 24 weeks of treatment made no significant difference on QS diversity in either group. CONCLUSIONS: Our data demonstrated that the baseline viral mutations and dynamic changes in HBV QS diversity within the BCP/PC region were closely related to HBsAg seroconversion in HBeAg-positive CHB children treated with PEG-IFN-α-2a.

Autophagy contributes to hypoxia-induced epithelial to mesenchymal transition of endometrial epithelial cells in endometriosis.

Endometriosis is a benign gynecologic disorder, and presents with malignant characteristics, such as migration and invasion. Hypoxia has been implicated in triggering epithelial-mesenchymal transition (EMT). Hypoxia is also known to induce autophagy. However, the relationship between autophagy and EMT under hypoxia conditions in endometriosis remains unknown. In the present study, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), microtubule associated protein light chain 3 (LC3), and mesenchymal cell marker vimentin was significantly higher in ectopic endometrium from patients with endometriosis, along with decreased expression of epithelial cell marker E-cadherin. After hypoxia treatment, endometrial epithelial cells exhibited enhanced migration and invasion abilities, as well as promoted autophagy and the EMT phenotype. Our analyses also show that HIF-1α was responsible for induction of autophagy. Moreover, inhibition of autophagy by chemical or genetic approaches suppressed hypoxia triggered EMT and reduced cell migration and invasion. Collectively, our findings identify that autophagy is critical for the migration and invasion of endometrial cells through the induction of EMT and indicate that inhibition of autophagy may be a novel useful strategy in the treatment of endometriosis.

Hypoxia-inducible factor-1α promotes endometrial stromal cells migration and invasion by upregulating autophagy in endometriosis.

Endometriosis is a benign gynecological disease that shares some characteristics with malignancy like migration and invasion. It has been reported that both hypoxia-inducible factor-1α (HIF-1α) and autophagy were upregulated in ectopic endometrium of patients with ovarian endometriosis. However, the crosstalk between HIF-1α and autophagy in the pathogenesis of endometriosis remains to be clarified. Accordingly, we investigated whether autophagy was regulated by HIF-1α, as well as whether the effect of HIF-1α on cell migration and invasion is mediated through autophagy upregulation. Here, we found that ectopic endometrium from patients with endometriosis highly expressed HIF-1α and autophagy-related protein LC3. In cultured human endometrial stromal cells (HESCs), autophagy was induced by hypoxia in a time-dependent manner and autophagy activation was dependent on HIF-1α. In addition, migration and invasion ability of HESCs were enhanced by hypoxia treatment, whereas knockdown of HIF-1α attenuated this effect. Furthermore, inhibiting autophagy with specific inhibitors and Beclin1 siRNA attenuated hypoxia triggered migration and invasion of HESCs. Taken together, these results suggest that HIF-1α promotes HESCs invasion and metastasis by upregulating autophagy. Thus, autophagy may be involved in the pathogenesis of endometriosis and inhibition of autophagy might be a novel therapeutic approach to the treatment of endometriosis.

Estrogen stabilizes hypoxia-inducible factor 1α through G protein-coupled estrogen receptor 1 in eutopic endometrium of endometriosis.

OBJECTIVE: To investigate whether G protein-coupled estrogen receptor (GPER, also known as GPR30 and GPER1) stabilizes hypoxia-inducible factor 1α (HIF-1α) in eutopic endometrium (EuEM) of endometriosis. DESIGN: Immunohistochemical analysis and experimental in vitro study. SETTING: University hospital. PATIENT(S): Patients with or without endometriosis. INTERVENTION(S): The EuEM and normal control endometrium (CoEM) were obtained by curettage. Primary cultured endometrial stromal cells (ESCs) were treated with 17ß-E2, G1, or G15. MAIN OUTCOME MEASURE(S): The EuEM and CoEM were collected for immunohistochemistry. Western blot, polymerase chain reaction, ELISA, and dual luciferase experiments were used to detect expression of GPER, HIF-1α, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 9 (MMP9) in ESCs. Estradiol and G1 were used as agonists of GPER, G15 as an antagonist. Migration of ESCs and endothelial tube formation of human umbilical vein endothelial cells cultured in medium collected from ESCs were measured. RESULT(S): Protein levels of GPER and HIF-1α were higher in EuEM than in CoEM. Protein levels of HIF-1α but not HIF-1α mRNA levels increased concurrently with GPER after E2 and G1 treatment. Furthermore, expression and activity of VEGF and MMP9 increased under E2 and G1 stimulation. However, these effects disappeared when GPER was blocked. CONCLUSION(S): G protein-coupled estrogen receptor stabilizes HIF-1α and thus promotes HIF-1α-induced VEGF and MMP9 in ESCs, which play critical roles in endometriosis.

[Stable expression of HBV C gene mutants in immortalized human B-cell lines].

OBJECTIVE: To provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus (HBV) genome carrying the hot-spot mutations V60, G87, and L97. METHODS: V60, G87, and L97 mutation points were introduced into HBV p3.8 II plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 II and p3.8 II-V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay. RESULTS: DNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV-transfected cell lysate or culture supernatant. CONCLUSION: Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.