Identification of molecular subtypes and immune infiltration in endometriosis: a novel bioinformatics analysis and In vitro validation.

Introduction: Endometriosis is a worldwide gynacological diseases, affecting in 6-10% of women of reproductive age. The aim of this study was to investigate the gene network and potential signatures of immune infiltration in endometriosis. Methods: The expression profiles of GSE51981, GSE6364, and GSE7305 were obtained from the Gene Expression Omnibus (GEO) database. Core modules and central genes related to immune characteristics were identified using a weighted gene coexpression network analysis. Bioinformatics analysis was performed to identify central genes in immune infiltration. Protein-protein interaction (PPI) network was used to identify the hub genes. We then constructed subtypes of endometriosis samples and calculated their correlation with hub genes. qRTPCR and Western blotting were used to verify our findings. Results: We identified 10 candidate hub genes (GZMB, PRF1, KIR2DL1, KIR2DL3, KIR3DL1, KIR2DL4, FGB, IGFBP1, RBP4, and PROK1) that were significantly correlated with immune infiltration. Our study established a detailed immune network and systematically elucidated the molecular mechanism underlying endometriosis from the aspect of immune infiltration. Discussion: Our study provides comprehensive insights into the immunology involved in endometriosis and might contribute to the development of immunotherapy for endometriosis. Furthermore, our study sheds light on the underlying molecular mechanism of endometriosis and might help improve the diagnosis and treatment of this condition.

Exposure to Di-(2-Ethylhexyl) phthalate drives ovarian dysfunction by inducing granulosa cell pyroptosis via the SLC39A5/NF-κB/NLRP3 axis.

Endocrine-disrupting chemicals (EDCs) have been reported to affect populations by disrupting the human endocrine system. Di-(2-ethylhexyl) phthalate (DEHP) is an EDC that is present in various consumer products. Exposure to DEHP could contribute to reproductive system dysfunction, with subsequent adverse female reproductive outcomes. Granulosa cells (GCs) play essential roles in ovarian function and fertility. To further reveal the underlying mechanism by which DEHP impairs female fertility and affects the normal function of GCs, in vivo and in vitro experiments were performed. Transcript sequencing was used to identify genes that were differentially expressed in GCs after DEHP treatment. SLC39A5 was shown to be overexpressed in the DEHP group compared to the normal control group. DEHP treatment and overexpression of SLC39A5 activated NF-κB-related factors, followed by an increase in the transcript expression level of NLRP3. NLRP3 inflammasomes play crucial roles in pyroptosis by acting as sensors. Pyroptosis is a type of inflammation-related cell death associated with various diseases, including ovarian cancer and polycystic ovary syndrome. Activation of NF-κB contributed to the upregulation of pyroptosis in GCs, while pyroptosis factors were downregulated after the inhibition of NF-κB with JSH-23. The same phenomenon was also observed in a mouse model in which DEHP-treated mice had higher expression levels of NF-κB and pyroptosis markers in GCs. Moreover, this phenomenon could be partially reversed by the NF-κB inhibitor JSH-23. DEHP treatment also disrupted the normal expression of ovarian function-related genes and inhibited the proliferation of GCs. Reproductive system impairment was observed in mice exposed to DEHP. DEHP-treated mice had a lower body weight, smaller reproductive organs, fewer healthy follicles, and diminished ovarian reserve. Thus, DEHP contributes to ovarian dysfunction by inducing pyroptosis via the SLC39A5/NF-κB/NLRP3 axis in GCs.

Identification and Validation of Autophagy-Related Genes in Primary Ovarian Insufficiency by Gene Expression Profile and Bioinformatic Analysis.

Background: To investigate the relationship between primary ovarian insufficiency and autophagy, we detected and got the expression profile of human granulosa cell line SVOG, which was with or without LPS induced. The expression profile was analyzed with the focus on the autophagy genes, among which hub genes were identified. Results: Totally, 6 genes were selected as candidate hub genes which might correlate with the process of primary ovarian insufficiency. The expression of hub genes was then validated by quantitative real-time PCR and two of them had significant expression change. Bioinformatics analysis was performed to observe the features of hub genes, including hub gene-RBP/TF/miRNA/drug network construction, functional analysis, and protein-protein interaction network. Pearson's correlation analysis was also performed to identify the correlation between hub genes and autophagy genes, among which there were four autophagy genes significantly correlated with hub genes, including ATG4B, ATG3, ATG13, and ULK1. Conclusion: The results indicated that autophagy might play an essential role in the process and underlying molecular mechanism of primary ovarian insufficiency, which was revealed for the first time and may help to provide a molecular foundation for the development of diagnostic and therapeutic approaches for primary ovarian insufficiency.

Downregulating HK2 inhibits proliferation of endometrial stromal cells through a noncanonical pathway involving phosphorylation of signal transducer and activator of transcription 1 in endometriosis.

BACKGROUND: Endometriosis is a benign gynecologic disease that causes chronic pelvic pain, dysmenorrhea and infertility and shares several characteristics with malignant tumors, afflicting women of reproductive age. Hexokinase 2 plays an essential role as the first rate-limiting enzyme in the metabolic glycolysis pathway, and its abnormal elevation in tumors is associated with tumor genesis and metastasis. However, the expression and role of hexokinase 2 in endometriosis remain unclear. METHODS: We sequenced the primary endometrial stromal cells from patients with endometrioma and utilized immunohistochemistry, quantitative real-time PCR, and western blot to determine the expression of hexokinase 2. Then wound healing assays, cell invasion assays, and cell proliferation assays were performed to explore the functions of hexokinase 2 in endometrial stromal cells. Furthermore, mice models of endometriosis were used to observe the effects of hexokinase 2 inhibitors in vivo. Lastly, glycolysis metabolism detection and transcriptome sequencing were carried out in hexokinase 2-knockdown endometrial stromal cells to analyze the mechanism of hexokinase 2 affecting cell function. RESULTS: Endometrial stromal cells of endometrioma displayed active glycolysis metabolism and elevated expression of hexokinase 2. Downregulating hexokinase 2 reduced the migration, invasion, and proliferation capacity of endometrial stromal cells. Knockdown of hexokinase 2 induced upregulation of signal transducer and activator of transcription 1 and their phosphorylation to attenuate the proliferation of endometrial stromal cells. CONCLUSIONS: Hexokinase 2 is associated with the migration, invasion, and proliferation of endometrial stromal cells, which might provide new insights into the pathogenesis and treatment of endometriosis. SUMMARY SENTENCE: HK2 is upregulated in ovarian endometrioma and knockdown of HK2 induced upregulation of signal transducer and activator of transcription 1 (STAT1) and their phosphorylation to attenuate the proliferation of endometrial stromal cells.

Establishment and Mechanism Study of a Primary Ovarian Insufficiency Mouse Model Using Lipopolysaccharide.

This study is aimed at establishing a lipopolysaccharide- (LPS-) induced primary ovarian insufficiency (POI) mouse model and investigating the underlying mechanism. C57BL/6N female mice were intraperitoneally injected with low-dose LPS (0.5 mg/kg) once daily for 14 days, high-dose LPS (2.5 mg/kg) twice weekly for 2 weeks, or cyclophosphamide (CTX; 150 mg/kg) once weekly for 2 weeks. Ovarian function was assessed by measuring the length of estrous cycle, the number of primordial follicles, and the levels of serum hormones. Expression and production of interleukin 1ß (IL-1ß) were determined to evaluate ovarian inflammation. Histopathological examination was performed to examine ovarian fibrosis. TUNEL assay was carried out to evaluate granulosa cell apoptosis. Western blotting was performed to measure the levels of inflammation-, fibrosis-, and apoptosis-related proteins in the mouse ovaries. Like CTX, both low- and high-dose LPS significantly impaired ovarian functions in mice, as evidenced by extended lengths of estrous cycles, reduced counts of primordial follicles, and alterations in the levels of serum hormones. Also, LPS promoted granulosa cell apoptosis and ovarian fibrosis in mice. However, LPS but not CTX promoted IL-1ß expression and production in mice. Moreover, LPS but not CTX enhanced TLR, p-p65, p65, and MyD88 expression in mouse ovaries, suggesting that LPS differs from CTX in triggering ovarian inflammation. In general, continuous low-dose LPS stimulation was less potent than high-dose LPS to affect the ovarian functions. In conclusion, LPS may induce ovarian inflammation, fibrosis, and granulosa cell apoptosis and can be used to establish a POI model in mice.

Do endometrial lesions require removal? A retrospective study.

BACKGROUND: This study aimed to evaluate the management of asymptomatic intrauterine lesions detected by ultrasonography. METHODS: Patients who underwent diagnostic hysteroscopy for asymptomatic lesions, including pre- and post-menopausal endometrial polyps, post-menopausal endometrial thickening (ET ≥5 mm) and reduplicative endometrial heterogeneity detected by transvaginal ultrasonography (TVUS), were recruited for this study. RESULTS: In the 792 recruited patients, the symptom-free focal masses within the uterine cavity detected by TVUS included 558 patients with pre- or post-menopausal endometrial polyps and 234 patients with postmenopausal endometrial thickening. No pre-menopausal patient presented with carcinoma. The polyp diameter (PD) was not identified as an independent risk factor for malignancy in this study. A significant difference (P = 0.036, < 0.05) in both benign and malignant endometrial lesions was observed between two groups of post-menopausal women stratified using an endometrial thickness cut-off of ≥11 mm. The TVUS was highly sensitive (94%) for pre-menopausal polyps. This technique had a specificity and positive predictive value of 84.4 and 92.7%, respectively, for postmenopausal polyps. The TVUS was clearly valuable for ruling out polyps, as indicated by a negative likelihood ratio (LR-) of 0.087. Among postmenopausal women with endometrial thickening, the area under the receiver operating characteristic curve was 0.828 (P < 0.001). An ET cut-off value of 12.5 mm yielded a sensitivity of 72.7% and specificity of 86%. CONCLUSION: We recommend follow-up alone for women with asymptomatic uterine polyps, particularly those who are pre-menopausal. Additionally, gynaecologists should consider risk factors such as age, obesity, polycystic ovarian syndrome, and diabetes. Prospective long-term follow-up studies should be conducted after hysteroscopic polypectomy to evaluate the recurrence rate of endometrial lesions.

Two steps modification for improvement of cyclobenzaprine transdermal delivery: releasing from patch and penetrating through skin.

The aim of this study was to improve the transdermal delivery of cyclobenzaprine (CBZ) from drug-in-adhesive patch which showed less side effects and better compliance. CBZ base was prepared and then characterized using differential scanning calorimetry (DSC). The interaction between CBZ and pressure-sensitive adhesive (PSA) was determined by Fourier Transform Infrared Spectroscopy (FT-IR). The influences of PSAs, penetration enhancers, patch thickness and drug content on the transdermal delivery of CBZ were studied thoroughly in vitro. Both CBZ releasing from patch and penetrating through the skin showed very great effect on the transdermal delivery of CBZ. The percentage of drug released from patch was increased with the decreasing of patch thickness, and so did the permeation percentage. The stratum corneum (SC) contributed approximately 57% resistance of total skin permeation resistance, and Span 20 increased the transdermal permeation by approximately 1.59-fold. The pharmacokinetic parameters were obtained through in vivo experiments of the optimized formulation using rabbit. Furthermore, the in vitro skin permeation results of CBZ patch correlated well with the in vivo absorption results in rabbit.

Probing the role of chemical enhancers in facilitating drug release from patches: Mechanistic insights based on FT-IR spectroscopy, molecular modeling and thermal analysis.

In patches, a drug must release from patches prior to its percutaneous absorption. Chemical enhancers have been used for several decades, but their roles in drug release from patches are poorly understood. In this work, the roles of chemical enhancers in bisoprolol tartrate (BSP-T) release from patches were probed in vitro and in vivo. More importantly, an innovative mechanism insight of chemical enhancers in drug release process was provided at molecular level. FT-IR spectroscopy and molecular modeling were employed to investigate the influence of chemical enhancers on drug-adhesive interaction. The results showed chemical enhancers like Span 80, which had a strong ability forming hydrogen bonds, could decrease drug-adhesive interaction leading to the release of drug from adhesive of patches. Thermal analysis was conducted to research the influence of chemical enhancers on the thermodynamic properties of patch system. It showed that chemical enhancers promoted the formation of free volume of adhesive, which facilitated drug release process. By contrast, the influence on the thermodynamic properties of BSP-T was less effective in influencing BSP-T release process. In conclusion, chemical enhancers played an important role in facilitating BSP-T release from the adhesive DURO-TAK® 87-2287 of patches by decreasing drug-adhesive interaction and promoting the formation of free volume of adhesive. This work may be an important step in understanding the important roles of chemical enhancers in drug release process.