Single-cell sequencing provides insights into the landscape of ovary in PCOS and alterations induced by CUMS.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder related to psychological distress. However, the mechanism underlying increased prevalence of depression in PCOS remained unclear. This study aimed to explore the unique transcriptional landscape of ovary and offered a platform to explore the mechanism of PCOS, as well as the influences caused by depression. The PCOS rat model was established by letrozole whereas PCOS rat model with depression was established by letrozole combined with chronic unpredicted mild stress (CUMS). Then single-cell RNA sequencing (scRNA-Seq) was applied to analyze the transcriptional features of rat ovaries. Granulosa cells (GCs) and fibroblasts (Fibros) accounted for the top two clusters of total 12 cell types. There were nine clusters in GCs, related to inflammatory response, endoplasmic reticulum (ER) stress, and steroidogenesis. The expression of differentially expressed genes (DEG) Hes1 was higher in PCOS and PCOS + CUMS groups, exhibiting enhanced expression by pseudotime and positively related to inflammation. Pseudotemporal analysis revealed that inflammation contributed to the different GCs distributions. Moreover, analysis of DEGs and gene ontology (GO) function enrichment revealed CUMS aggravated inflammation in PCOS GCs possibly via interferon signaling pathway. In theca cells (TCs), nine clusters were observed and some of them were relevant to inflammation, ER stress, and lipid metabolism. DEGs Ass1, Insl3, and Ifi27 were positively related to Cyp17a1, and Ces1d might contribute to the different trajectory of TCs. Subsequent scRNA-seq revealed a signature profile of endothelial cells (ECs) and Fibros, which suggest that inflammation-induced damage of ECs and Fibro, further exacerbated by CUMS. Finally, analysis of T cells and mononuclear phagocytes (MPs) revealed the existence of immune dysfunction, among which interferon signaling played a critical role. These findings provided more knowledge for a better understanding PCOS from the view of inflammation and identified new biomarkers and targets for the treatment of PCOS with psychological diseases.NEW & NOTEWORTHY In this study, we mapped the landscape of polycystic ovary syndrome (PCOS) ovary with rat model induced by letrozole and provided a novel insight into the molecular mechanism of PCOS accompanied by chronic unpredicted mild stress (CUMS) at single-cell transcriptomic level. These observations highlight the importance of inflammation in the pathogenesis of PCOS, which might also be the bridge between PCOS and psychological diseases.

Protocatechuic acid alleviates polycystic ovary syndrome symptoms in mice by PI3K signaling in granulosa cells to relieve ROS pressure and apoptosis.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complicated gynecological endocrine disease that occurs in women of childbearing age. Protocatechuic acid is a phenol-rich compound derived from herbs and owns vital functions in numerous diseases. Howbeit, protocatechuic acid's impact on PCOS is unknown. METHODS: A combination of in vivo and in vitro models was examined in this study. C57BL/6 mice were injected subcutaneously daily with dehydroepiandrosterone to establish a PCOS mouse model, and protocatechuic acid was intraperitoneally injected into PCOS mice. Granulosa cells of PCOS ovaries were also isolated. The function of protocatechuic acid was appraised using enzyme-linked immunosorbent assay, hematoxylin-eosin staining, reactive oxygen species (ROS) and LC3 levels analysis, flow cytometry, quantitative real-time PCR, and western blot. Meanwhile, the mechanism of protocatechuic acid was assessed with a series of molecular experiments. RESULTS: Protocatechuic acid owned no apparent toxic effect on mice. Functionally, protocatechuic acid owned a function of mitigating PCOS in vivo. Meanwhile, protocatechuic acid repressed ROS, autophagy, and apoptosis of PCOS ovarian granulosa cells in vitro. Mechanistically, rescue assays elucidated that the protective function of protocatechuic acid against PCOS was interrelated to the activation of the PI3K/AKT/mTOR axis. CONCLUSION: Protocatechuic acid alleviated PCOS symptoms in mice through PI3K signaling in granulosa cells to reduce ROS levels and apoptosis.

Diagnostic values of MRI indexes for polycystic ovary syndrome.

BACKGROUND: Magnetic resonance imaging (MRI) is a useful non-invasive modality for observation of ovarian morphologic characteristics. Few studies have focused on the value of MRI-derived indexes in reproductive-aged women with polycystic ovary syndrome (PCOS). PURPOSE: To assess the diagnostic value of MRI in women with PCOS. MATERIAL AND METHODS: This prospective case-control study included 85 women with PCOS and 50 controls who underwent pelvic MRI during 2017-2019. Ovarian volume (OV), follicle count (FC; counts of follicles sizing 2-3, 4-6, 7-9, 2-9 mm, respectively), follicular peripheral distribution, absence of a dominant follicle and stromal to total area ratio (S:A) were determined with MRI. The diagnostic value (sensitivity, specificity, area under the receiver operating characteristic curve [AUC]) of OV, FC2-9, and follicular peripheral distribution for PCOS were assessed. RESULTS: The AUCs were 0.94 for OV, 0.96 for FC2-9, and 0.78 for follicular peripheral distribution. The optimal threshold to detect PCOS was 8.5 mL for OV (sensitivity 78%; specificity 96%) and 26 for FC2-9 (sensitivity 85%; specificity 98%). Sensitivity and specificity were 73% and 82% for follicular peripheral distribution, respectively. Reproducibility was perfect for OV (ICC = 0.96) and absence of a dominant follicle (k = 0.85), substantial for FC2-9 (ICC = 0.79) and S:A (ICC = 0.69), and moderate for follicular peripheral distribution (k = 0.56). CONCLUSION: Detected by MRI, OV >8.5 mL or FC2-9 >26 are accurate for diagnosing PCOS.

[Regulation of vascular endothelial cell activator expression by tanshinone â ¡_A based on PI3K/Akt signaling pathway].

The present study investigated the regulatory effect of tanshinone Ⅱ_A(TAⅡ_A) on activator expression in human umbilical vein endothelial cells(HUVECs) and the effect on the phosphoinositide 3-kinase(PI3 K)/protein kinase B(Akt) signaling pathway in patients with antiphospholipid syndrome(APS). HUVECs cultured in vitro were divided into a medium group, a blank control group, an APS model group, an APS+LY5 group, an APS+LY10 group, an APS+LY20 group, an APS+TAⅡ_A5 group, an APS+TAⅡ_A10 group, an APS+TAⅡ_A20 group, and an APS+TAⅡ_A10+LY10 group. The effects of LY294002 and TAⅡ_A at different concentrations on the secretion of interleukin-6(IL-6), interleukin-8(IL-8), and monocyte chemoattractant protein-1(MCP-1) by HUVECs were investigated. The effects on the mRNA expression of annexin A2(ANXA2), PI3 K, Akt, and E-cadherin(E-cad) were detected by quantitative polymerase chain reaction(qPCR), and Western blot was used to determine the effects on the protein expression of ANXA2, p-PI3 K/PI3 K, p-Akt/Akt, and E-cad. The results revealed that compared with the APS model group, the APS+TAⅡ_A10 group showed statistically reduced IL-6 and MCP-1 and increased IL-8 in a concentration-dependent manner with the increase in TAⅡ_A dose, while the APS+TAⅡ_A10 group showed increased mRNA and protein expression of ANXA2, PI3 K, Akt, and E-cad(P<0.05 or P<0.01) in a concentration-dependent manner with the increase in TAⅡ_A dose. The findings indicated that the serum of APS patients could lead to the decreased mRNA and protein expression levels of ANXA2, PI3 K, Akt, and E-cad in HUVECs, increased secretion of IL-6 and MCP-1, and reduced secretion of IL-8, and activate vascular endothelial cells. In contrast, once the PI3 K/Akt signaling pathway was blocked, the mRNA and protein expression of ANXA2 and E-cad significantly decreased, IL-6 and MCP-1 secretion significantly increased, and IL-8 secretion was significantly reduced. It suggests that TAⅡ_A regulates the activation of vascular endothelial cells in APS patients by activating the PI3 K/Akt signaling pathway.

Transplantation of collagen sponge-based three-dimensional neural stem cells cultured in a RCCS facilitates locomotor functional recovery in spinal cord injury animals.

Numerous studies have indicated that microgravity induces various changes in the cellular functions of neural stem cells (NSCs), and the use of microgravity to culture tissue engineered seed cells for the treatment of nervous system diseases has drawn increasing attention. The goal of this study was to verify the efficacy of collagen sponge-based 3-dimensional (3D) NSCs cultured in a rotary cell culture system (RCCS) in treating spinal cord injury (SCI). The Basso-Beattie-Bresnahan score, inclined plane test, and electrophysiology results all indicated that 3D cultured NSCs cultured in a RCCS had better therapeutic effects than those cultured in a traditional cell culture environment, suggesting that the microgravity provided by the RCCS could enhance the therapeutic effect of 3D cultured NSCs. Our study indicates the feasibility of combining the RCCS with collagen sponge-based 3D cell culture for producing tissue engineered seed cells for the treatment of SCI. This novel and effective method shows promise for application in cell-based therapy for SCI in the future.

Upregulation of Apol8 by Epothilone D facilitates the neuronal relay of transplanted NSCs in spinal cord injury.

BACKGROUND: Microtubule-stabilizing agents have been demonstrated to modulate axonal sprouting during neuronal disease. One such agent, Epothilone D, has been used to treat spinal cord injury (SCI) by promoting axonal sprouting at the lesion site after SCI. However, the role of Epothilone D in the differentiation of neural stem cells (NSCs) in SCI repair is unknown. In the present study, we mainly explored the effects and mechanisms of Epothilone D on the neuronal differentiation of NSCs and revealed a potential new SCI treatment. METHODS: In vitro differentiation assays, western blotting, and quantitative real-time polymerase chain reaction were used to detect the effects of Epothilone D on NSC differentiation. Retrograde tracing using a pseudotyped rabies virus was then used to detect neuronal circuit construction. RNA sequencing (RNA-Seq) was valuable for exploring the target gene involved in the neuronal differentiation stimulated by Epothilone D. In addition, lentivirus-induced overexpression and RNA interference technology were applied to demonstrate the function of the target gene. Last, an Apol8-NSC-linear ordered collagen scaffold (LOCS) graft was prepared to treat a mouse model of SCI, and functional and electrophysiological evaluations were performed. RESULTS: We first revealed that Epothilone D promoted the neuronal differentiation of cultured NSCs and facilitated neuronal relay formation in the injured site after SCI. Furthermore, the RNA-Seq results demonstrated that Apol8 was upregulated during Epothilone D-induced neuronal relay formation. Lentivirus-mediated Apol8 overexpression in NSCs (Apol8-NSCs) promoted NSC differentiation toward neurons, and an Apol8 interference assay showed that Apol8 had a role in promoting neuronal differentiation under the induction of Epothilone D. Last, Apol8-NSC transplantation with LOCS promoted the neuronal differentiation of transplanted NSCs in the lesion site as well as synapse formation, thus improving the motor function of mice with complete spinal cord transection. CONCLUSIONS: Epothilone D can promote the neuronal differentiation of NSCs by upregulating Apol8, which may provide a promising therapeutic target for SCI repair.

Predicting necrosis in adnexal torsion in women of reproductive age using magnetic resonance imaging.

PURPOSE: To identify the diagnostic performance of magnetic resonance (MR) imaging for patients with adnexal torsion and to develop a predictive model for necrosis related to torsion. METHODS: The institutional ethics committee approved this retrospective study. A total of 56 women with a preoperative pelvic MR scan and a surgical and pathologic diagnosis of adnexal torsion were enrolled from five institutions. Three radiologists reviewed the MR images independently. The kappa value of interrater agreement was assessed. Differences between patients treated with conservative surgery and adnexectomy were evaluated by univariate and multivariate logistic regression analyses. Receiver operating characteristic (ROC) curve analysis was used to assess the ability of the model to predict ovarian necrosis. RESULTS: Fifty-six patients were divided into the conservative surgery group (24/56, 42.9%) or the adnexectomy group (32/56, 57.1%) depending on the surgical outcomes. The radiographic features related to torsion were interpreted by three raters retrospectively with substantial interrater agreement (kappa > 0.60). Older reproductive age and pedicle hemorrhagic infarction were significantly associated with adnexectomy (p < 0.05). At multivariate analysis, pedicle hemorrhagic infarction (odds ratio = 10.476 [95% confidence interval 1.103, 99.504; p = 0.041]) was associated with adnexectomy. Using the predictive model (older reproductive age and pedicle hemorrhagic infarction), a receiver operating characteristic curve was generated with an area under the curve (AUC = 0.870 ± 0.049). CONCLUSION: The presence of pedicle hemorrhagic infarction and older reproductive age can predict necrosis of adnexal torsion and may be used to guide the optimal treatment strategy. KEY POINTS: • Pedicle hemorrhagic infarction and older reproductive age are predictors of necrosis in adnexal torsion in patients of reproductive age (AUC = 0.870 ± 0.049). • Cystic wall thickening, enlarged vascular pedicle, tubal thickening, and uterine deviation are associated with a high risk for adnexal torsion, occurring in more than half of the cases in this study. • MR findings are useful for the definitive diagnosis of adnexal torsion and for the prediction of adnexal necrosis.

Repair of lumbar vertebral bone defects by bone particles combined with hUC-MSCs in weaned rabbit.

Aim: The major symptom of many closed spinal dysraphism patients is that the laminas or arches of vertebra are not fused well. To date, the bone repair of spina bifida for young children is a significant challenge in clinical practice. Materials & methods: Bovine bone collagen particle (BBCP) scaffolds combined with human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were implanted in the defect area. X-ray analysis was performed after 3 months. Tissues were harvested for gross observation, and histological and immunohistochemical staining. Results: The BBCP supported hUC-MSCs adhesion and growth. Implanted BBCP combined with hUC-MSCs also promoted bone regeneration in the vertebral lamina and arch defect area. Conclusion: This method represents a new strategy for vertebral lamina and arch reconstruction in children.

The Flavanone, Naringenin, Modifies Antioxidant and Steroidogenic Enzyme Activity in a Rat Model of Letrozole-Induced Polycystic Ovary Syndrome.

BACKGROUND Worldwide, polycystic ovary syndrome (PCOS) is a prevalent endocrine and metabolic disorder that affects women of reproductive age. Naringenin is a natural flavanone, derived from grapefruit. The aims of this study were to investigate the effects of naringenin on the antioxidant and steroidogenic enzyme activity in a rat model of letrozole-induced PCOS. MATERIAL AND METHODS The induction of PCOS was undertaken by giving 28 female Sprague-Dawley rats a dose of letrozole (1 mg/kg) daily for 21 days. There were four treatment groups: Group I (n=7) received 1% of carboxymethyl cellulose (CMC); Group II (n=7) received 1% CMC plus naringenin 20 mg/kg; Group III (n=7) received letrozole only; Group IV (n=7) received letrozole plus naringenin 20 mg/kg. Estradiol, testosterone, and steroidogenic enzyme activities, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activity were determined in the four treatment groups, and histology was performed on the rat ovarian tissue. Serum glucose levels were measured with a glucometer. RESULTS Naringenin treatment in a rat model of PCOS significantly increased the levels of the reactive oxygen species (ROS) scavenging enzymes CAT, SOD, and GPX (p<0.05), and prevented weight increase. Naringenin treatment resulted in a significant reduction in serum glucose levels (p<0.05), normalized estradiol and testosterone levels steroidogenic enzyme activity, and maintained the normal anatomy of the ovaries. CONCLUSIONS Naringenin treatment, in a rat model of PCOS, demonstrated antioxidant and steroidogenic enzyme activity.

Substrate-independent immunomodulatory characteristics of mesenchymal stem cells in three-dimensional culture.

Mesenchymal stem cells (MSCs) play important roles in tissue regeneration, and multi-lineage differentiation and immunomodulation are two major characteristics of MSCs that are utilized in stem cell therapy. MSCs in vivo have a markedly different three-dimensional (3D) niche compared to the traditional two-dimensional (2D) culture in vitro. A 3D scaffold is predicted to provide an artificial 3D environment similar to the in vivo environment. Significant changes in MSC differentiation are shown to be occurred when under 3D culture. However, the immunomodulatory characteristics of MSCs under 3D culture remain unknown. In this study, 3D culture systems were constructed using different substrates to evaluate the common immunomodulatory characteristics of MSCs. Compared to the MSCs under 2D culture, the MSCs under 3D culture, which had higher stemness and maintained cell phenotype, showed altered immunophenotypic pattern. Gene expression profile analysis at mRNA and protein level detected by gene chip and protein chip, respectively, further revealed the difference between 3D cultured MSCs and 2D cultured MSCs, which was mainly concentrated in the immunoregulation related aspects. Moreover, the immunoregulatory role of 3D culture was confirmed by our immunosuppressive experiments. These findings demonstrated that the immunomodulatory capacities of MSCs were enhanced by the 3D geometry of substrates.

Bone marrow-derived mesenchymal stem cells in three-dimensional culture promote neuronal regeneration by neurotrophic protection and immunomodulation.

Accumulating evidence has revealed three-dimensional (3D) culture could better mimic the stem cell niche in vivo in comparison with conventional two-dimensional (2D) culture. In this study, we found that bone marrow derived mesenchymal stem cells (BMSCs) cultured in 3D collagen scaffold (3D BMSCs) exhibited distinctive features including significantly enhancing neurotrophic factor secretions and reducing macrophage activations challenged by lipopolysaccharide (LPS) in vitro. To further evaluate 3D BMSCs' potential benefits to the regeneration of spinal cord injury (SCI), the 3D and 2D BMSCs were respectively implanted in rat hemisected SCI. Compared with 2D cohort, 3D BMSCs transplantation significantly reduced the expressions of inflammatory cytokines such as TNF-α, IL-1ß, and IL-6 at 5 days after transplantation, markedly enhanced axonal regeneration, and promoted motor functional recovery during 8 weeks of observation. When Nocodazole was used to depolymerize the cytoskeleton of 3D BMSCs, the changed expressions of neurotrophic factors and inflammatory cytokines were blunted, at least partially. Thus synergistic effects of neuronal protection and immunomodulation of 3D BMSCs may lead to a better functional recovery of SCI and the underlying mechanism may involve the alteration of their cellular morphology because of 3D culture. This study contributes to a better understanding of the cellular characteristics of 3D BMSCs and provides a novel strategy to promote the repair of the injured spinal cord. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1759-1769, 2016.

[Effect of electro-acupuncture on clinical outcomes and ovarian hyperstimulation syndrome in in vitro fertilization and embryo transplantation].

OBJECTIVE: To observe the effect of electro-acupuncture (EA) on clinical outcomes and the occurrence of ovarian hyperstimulation syndrome (OHSS) in in vitro fertilization and embryo transplantation. METHODS: Totally 109 patients who routinely received in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) at Reproductive Center were assigned to the control group (56 cases) and the EA group (53 cases) according to even and odd-numbered date. Patients in the control group received controlled ovarian hyperstimulation (COH) referring to GnRH-a long protocol. On the basis of COH, those in the EA group received EA from the day of Gn injection to the day of embryo transfer. Estradiol (E2), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and angiotensin (AT) II were measured in all patients on the day of hCG injection, the day of ovum pick up (OPU), and the day of embryo transfer (ET), respectively. The oocyte retrieval rate, good quality embryo rate, clinical pregnancy rate, the abortion rate, and the occurrence of OHSS were compared between the two groups. RESULTS: Compared with the control group, serum E2 levels on the day of OPU and the day of ET were significantly lower in the EA group (P < 0.05). On the day of OPU levels of VEGF and IL-6 also significantly decreased (P < 0.05). Serum levels of VEGF and IL-6 reached the highest line on the day of hCG in the two groups, and then showed a decreasing trend. Compared with the control group at the same time point, serum levels of VEGF and IL-6 obviously decreased on on the day of OPU, hCG, and ET (P < 0.05). The occurrence of OHSS and the canceling rate of transplant cycle were significantly lower in the EA group than in the control group (P < 0.05). CONCLUSIONS: EA, as an adjunctive therapy, could reduce the occurrence of OHSS in IVF. Besides, it did not decrease good embryo rates and pregnancy rates in IVF-ET, which might be associated with lowering local vascular permeability of ovaries.