Validation of mitochondrial biomarkers and immune dynamics in polycystic ovary syndrome.

PROBLEM: Polycystic ovary syndrome (PCOS), a prevalent endocrine-metabolic disorder, presents considerable therapeutic challenges due to its complex and elusive pathophysiology. METHOD OF STUDY: We employed three machine learning algorithms to identify potential biomarkers within a training dataset, comprising GSE138518, GSE155489, and GSE193123. The diagnostic accuracy of these biomarkers was rigorously evaluated using a validation dataset using area under the curve (AUC) metrics. Further validation in clinical samples was conducted using PCR and immunofluorescence techniques. Additionally, we investigate the complex interplay among immune cells in PCOS using CIBERSORT to uncover the relationships between the identified biomarkers and various immune cell types. RESULTS: Our analysis identified ACSS2, LPIN1, and NR4A1 as key mitochondria-related biomarkers associated with PCOS. A notable difference was observed in the immune microenvironment between PCOS patients and healthy controls. In particular, LPIN1 exhibited a positive correlation with resting mast cells, whereas NR4A1 demonstrated a negative correlation with monocytes in PCOS patients. CONCLUSION: ACSS2, LPIN1, and NR4A1 emerge as PCOS-related diagnostic biomarkers and potential intervention targets, opening new avenues for the diagnosis and management of PCOS.

Ginsenosides in endometrium-related diseases: Emerging roles and mechanisms.

Ginsenosides, agents extracted from an important herb (ginseng), are expected to provide new therapies for endometrium-related diseases. Based on the molecular types of ginsenosides, we reviewed the main pharmacological effects of ginsenosides against endometrium-related diseases (e.g., endometrial cancers, endometriosis, and endometritis). The mechanism of action of ginsenosides involves inducing apoptosis of endometrium-related cells, promoting autophagy of endometrium-related cells, regulating epithelial-mesenchymal transition (EMT) in endometrium-related cells, and activating the immune system to kill cells associated with endometrial diseases. We hope to provide a theoretical foundation for the treatment of endometrium-related diseases by ginsenosides.

RANKL up-regulated by progesterone aggravates lipopolysaccharide-induced acute lung injury during pregnancy.

Acute lung injury (ALI) is a common acute respiratory disease with high morbidity and mortality rate in pregnant women. Receptor activator of NF-κB ligand (TNFSF11, also known as RANKL) exerts either pro-inflammatory or anti-inflammatory effects on the immune response. LPS administration reduced the survival time (n = 10, p < 0.01), increased wet/dry ratio (n = 10, p < 0.001) and lung injury score (n = 10, p < 0.001), the elevated proportions of plasmacytoid dendritic cells (pDCs) (n = 10, p < 0.0001), tissue-resident DCs (resDCs) (n = 10, p < 0.0001), macrophages (n = 10, p < 0.0001), and neutrophils (n = 10, p < 0.0001), and the expressions of costimulatory molecules and inflammation cytokines (n = 10, p < 0.05) in lungs of pregnant mice, compared with non-pregnant mice. In vitro, progesterone up-regulated the expression of RANKL (n > 6, p < 0.05) on pulmonary fibroblasts. The results of cytokine arrays showed that the cytokines associated with inflammatory response and leukocyte differentiation were decreased in pulmonary fibroblasts after treatment with anti-RANKL neutralizing antibody, compared with control pulmonary fibroblasts. More notably, we found that Tnfsf11-/- pregnant mice had longer survival durations (n = 10, p < 0.01), lower lung injury scores (n = 10, p < 0.05), and lower immune cell infiltration (n = 10, p < 0.05). These data imply that the RANKL/RANK axis plays an essential role in LPS-induced ALI during pregnancy possibly through a variety of pathways.

Single-cell transcriptome profiling of the human endometrium of patients with recurrent implantation failure.

Introduction: Despite great advances in assisted reproductive technology (ART), recurrent implantation failure (RIF) cannot be effectively avoided. Notably, cellular characteristics and communication that regulate endometrial receptivity and differentiation, and its disorders in RIF at window of implantation (WOI) remain rudimentary. Objectives: In this study, we profiled the endometrial cells present at the WOI timing in RIF patients and healthy controls using single-cell RNA sequencing (scRNA-seq) and provided a detailed molecular and cellular map of a healthy and RIF endometrium at the WOI. Method: In the current study, the endometrium from RIF patient (n = 6; age range, 32 - 35 years) and control (Ctrl) (n = 3; age range, 29 - 35 years) groups were studied at a single-cell resolution. single-cell RNA-seq and analysis were performed on the endometrium of patients with RIF and Ctrl. Immunofluorescence, flow cytometry assays, and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to verify cellular identity and function. Results: We profiled the transcriptomes of 60222 primary human endometrial cells isolated from control and RIF patients at a single-cell resolution. We discovered dramatic differential expression of endometrial receptivity-related genes in four major endometrial fibroblast-like cells from RIF patients compared to the control endometrium. We observed that CD49a+CXCR4+NK cells were diminished in proportion with RIF. The decrease in subset of CD63highPGRhigh endometrial epithelial cells with high levels of progesterone receptor, autophagy and exosomes should contribute to the decrease in subset of NK cells. Additionally, we characterized aberrant molecular and cellular characteristics and endometrial cell-cell communication disorders in RIF patients. Conclusion: Our study provides deeper insights into endometrial microenvironment disorder of RIF that are potentially applicable to improving the etiological diagnosis and therapeutics of unexplained RIF.

Excess Heme Promotes the Migration and Infiltration of Macrophages in Endometrial Hyperplasia Complicated with Abnormal Uterine Bleeding.

In patients, endometrial hyperplasia (EH) is often accompanied by abnormal uterine bleeding (AUB), which is prone to release large amounts of heme. However, the role of excess heme in the migration and infiltration of immune cells in EH complicated by AUB remains unknown. In this study, 45 patients with AUB were divided into three groups: a proliferative phase group (n = 15), a secretory phase group (n = 15) and EH (n = 15). We observed that immune cell subpopulations were significantly different among the three groups, as demonstrated by flow cytometry analysis. Of note, there was a higher infiltration of total immune cells and macrophages in the endometrium of patients with EH. Heme up-regulated the expression of heme oxygenase-1 (HO-1) and nuclear factor erythroid-2-related factor 2 (Nrf2) in endometrial epithelial cells (EECs) in vitro, as well as chemokine (e.g., CCL2, CCL3, CCL5, CXCL8) levels. Additionally, stimulation with heme led to the increased recruitment of THP-1 cells in an indirect EEC-THP-1 co-culture unit. These data suggest that sustained and excessive heme in patients with AUB may recruit macrophages by increasing the levels of several chemokines, contributing to the accumulation and infiltration of macrophages in the endometrium of EH patients, and the key molecules of heme metabolism, HO-1 and Nrf2, are also involved in this regulatory process.

A defective lysophosphatidic acid-autophagy axis increases miscarriage risk by restricting decidual macrophage residence.

Massive infiltrated and enriched decidual macrophages (dMφ) have been widely regarded as important regulators of maternal-fetal immune tolerance and trophoblast invasion, contributing to normal pregnancy. However, the characteristics of metabolic profile and the underlying mechanism of dMφ residence remain largely unknown. Here, we observe that dMφ display an active glycerophospholipid metabolism. The activation of ENPP2-lysophosphatidic acid (LPA) facilitates the adhesion and retention, and M2 differentiation of dMφ during normal pregnancy. Mechanistically, this process is mediated through activation of the LPA receptors (LPAR1 and PPARG/PPARγ)-DDIT4-macroautophagy/autophagy axis, and further upregulation of multiple adhesion factors (e.g., cadherins and selectins) in a CLDN7 (claudin 7)-dependent manner. Additionally, poor trophoblast invasion and placenta development, and a high ratio of embryo loss are observed in Enpp2±, lpar1-/- or PPARG-blocked pregnant mice. Patients with unexplained spontaneous abortion display insufficient autophagy and cell residence of dMφ. In therapeutic studies, supplementation with LPA or the autophagy inducer rapamycin significantly promotes dMφ autophagy and cell residence, and improves embryo resorption in Enpp2± and spontaneous abortion mouse models, which should be dependent on the activation of DDIT4-autophagy-CLDN7-adhesion molecules axis. This observation reveals that inactivation of ENPP2-LPA metabolism and insufficient autophagy of dMφ result in resident obstacle of dMφ and further increase the risk of spontaneous abortion, and provides potential therapeutic strategies to prevent spontaneous abortion.Abbreviations: ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; Atg5: autophagy related 5; ATG13: autophagy related 13; BECN1: beclin 1; CDH1/E-cadherin: cadherin 1; CDH5/VE-cadherin: cadherin 5; CFSE: carboxyfluorescein succinimidyl ester; CLDN7: claudin 7; CSF1/M-CSF: colony stimulating factor 1; CSF2/GM-CSF: colony stimulating factor 2; Ctrl: control; CXCL10/IP-10: chemokine (C-X-C) ligand 10; DDIT4: DNA damage inducible transcript 4; dMφ: decidual macrophage; DSC: decidual stromal cells; ENPP2/ATX: ectonucleotide pyrophosphatase/phosphodiesterase 2; Enpp2±: Enpp2 heterozygous knockout mouse; ENPP2i/PF-8380: ENPP2 inhibitor; EPCAM: epithelial cell adhesion molecule; ESC: endometrial stromal cells; FGF2/b-FGF: fibroblast growth factor 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPCPD1: glycerophosphocholine phosphodiesterase 1; HE: heterozygote; HIF1A: hypoxia inducible factor 1 subunit alpha; HNF4A: hepatocyte nuclear factor 4 alpha; HO: homozygote; ICAM2: intercellular adhesion molecule 2; IL: interleukin; ITGAV/CD51: integrin subunit alpha V; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11b: integrin subunit alpha X; ITGB3/CD61: integrin subunit beta 3; KLRB1/NK1.1: killer cell lectin like receptor B1; KRT7/cytokeratin 7: keratin 7; LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; lpar1-/-: lpar1 homozygous knockout mouse; LPAR1i/AM966: LPAR1 inhibitor; LY6C: lymphocyte antigen 6 complex, locus C1; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; Lyz2: lysozyme 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MARVELD2: MARVEL domain containing 2; 3-MA: 3-methyladenine; MBOAT2: membrane bound O-acyltransferase domain containing 2; MGLL: monoglyceride lipase; MRC1/CD206: mannose receptor C-type 1; MTOR: mechanistic target of rapamycin kinase; NP: normal pregnancy; PDGF: platelet derived growth factor; PLA1A: phospholipase A1 member A; PLA2G4A: phospholipase A2 group IVA; PLPP1: phospholipid phosphatase 1; pMo: peripheral blood monocytes; p-MTOR: phosphorylated MTOR; PPAR: peroxisome proliferator activated receptor; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGi/GW9662: PPARG inhibitor; PTPRC/CD45: protein tyrosine phosphatase receptor type, C; Rapa: rapamycin; RHEB: Ras homolog, mTORC1 binding; SA: spontaneous abortion; SELE: selectin E; SELL: selectin L; siCLDN7: CLDN7-silenced; STAT: signal transducer and activator of transcription; SQSTM1: sequestosome 1; TJP1: tight junction protein 1; VCAM1: vascular cell adhesion molecule 1; WT: wild type.

Protopanaxadiol improves endometriosis associated infertility and miscarriage in sex hormones receptors-dependent and independent manners.

Background: Patients with endometriosis (EMs) have high risks of infertility and spontaneous abortion. How to remodel the fertility of patients with EMs has always been the hot spot and difficulty in the field of reproductive medicine. As an aglycone of ginsenosides, protopanaxadiol (PPD) possesses pleiotropic biological functions and has high medicinal values. We aimed to investigate the effect and potential mechanism of PPD in the treatment of EMs-associated infertility and spontaneous abortion. Methods: The EMs mice models were constructed by allotransplantation. The pregnancy rates, embryo implantation numbers and embryo resorption rates of control and EMs were counted. RNA sequencing, qRT-PCR, enzyme linked immunosorbent assay (ELISA) and FCM analysis were performed to screen and confirm the expression of endometrial receptivity/decidualization-related molecules, inflammation cytokines and NK cell function-related molecules in vitro and/or in vivo. The SWISS Target Prediction, STRING and Cytoscape were carried out to predict the potential cellular sensory proteins, the protein-protein interaction (PPI) network between sensory proteins and fertility-related molecules, respectively. Micro-CT detection, liver and kidney function tests were used to evaluate the safety. Results: Here, we observe that PPD significantly up-regulates endometrial receptivity-related molecules (e.g., Lif, Igfbp1, Mmps, collagens) and restricts pelvic inflammatory response (low levels of IL-12 and IFN-γ) of macrophage, and further remodel and improve the fertility of EMs mice. Additionally, PPD increases the expression of decidualization-related genes and Collagens, and promotes the proliferation, residence, immune tolerance and anagogic functions of decidual NK cells (low levels of CD16 and NKp30, high levels of Ki67, VEGF, TGF-ß) in pregnant EMs mice, and further triggers decidualization, decidual NK cell-mediated maternal-fetal immune tolerance and angiogenesis, preventing pregnant EMs mice from miscarriage. Mechanically, these effects should be dependent on ESRs, PGR and other sensory proteins (e.g., AR). Compared with GnRHa (the clinic first-line drug for EMs), PPD does not lead to the decline of serum estrogen and bone loss. Conclusion: These data suggest that PPD prevents EMs-associated infertility and miscarriage in sex hormones receptors-dependent and independent manners possibly, and provides a potential therapeutic strategy with high efficiency and low side effects to remodels the fertility of patients with EMs.

CD45RO-CD8+ T cell-derived exosomes restrict estrogen-driven endometrial cancer development via the ERß/miR-765/PLP2/Notch axis.

Rationale: Estrogen-dependent cancers (e.g., breast, endometrial, and ovarian cancers) are among the leading causes of morbidity and mortality in women worldwide. Recently, exosomes released by tumor-infiltrating CD8+ T cells have been under the spotlight in the field of cancer immunotherapy. Our study aims at elucidating the underlying mechanisms of the crosstalk between estrogen signaling and CD8+ T cells, and possible intervention values in uterine corpus endometrial cancer (UCEC). Methods: Micro RNA-seq was conducted to screen differentially expressed micro RNA in UCEC. Bioinformatic analysis was processed to predict the target of miR-765. RNA silencing or overexpressing and pharmacologic inhibitors were used to assess the functions of ERß/miR-765/PLP2/Notch axis in UCEC cell proliferation and invasion in vivo and in vitro. In vivo imaging was performed to evaluate the metastasis of tumor in mice. Combined fluorescent in situ hybridization for miR-765 and immunofluorescent labeling for CD8 was carried out to prove the co-localization between miR-765 and CD8+ T cells. Exosomes derived from CD45RO-CD8+ T cells were isolated to detect the regulatory effects on UCEC. Results: miR-765 is characterized as the most downregulated miRNA in UCEC, and there is a negative correlation between miR-765 and Proteolipid protein 2 (PLP2) in UCEC lesion. Estrogen significantly down-regulates miR-765 level, and facilitates the development of UCEC by estrogen receptor (ER) ß. Mechanistically, this process is mediated through the miRNAs (e.g., miR-3584-5p, miR-7-5p, miR-150-5p, and miR-124-3p) cluster-controlled regulation of the PLP2, which further regulates Ki-67 and multiple epithelial-mesenchymal transition (EMT)-related molecules (e.g, E-cadherin and Vimentin) in a Notch signaling pathway-dependent manner. Interestingly, the selective ER degrader Fulvestrant alleviates estrogen-mediated miR-765/PLP2 expression regulation and UCEC development in ERß-dependent and -independent manners. Additionally, CD45RO-CD8+ T cell-derived exosomes release more miR-765 than that from CD45RO+CD8+ T cells. In therapeutic studies, these exosomes limit estrogen-driven disease development via regulation of the miR-765/PLP2 axis. Conclusions: This observation reveals novel molecular mechanisms underlying estrogen signaling and CD8+ T cell-released exosomes in UCEC development, and provides a potential therapeutic strategy for UCEC patients with aberrant ERß/miR-765/PLP2/Notch signaling axis.

Ovarian hormones-autophagy-immunity axis in menstruation and endometriosis.

Menstruation occurs in few species and involves a cyclic process of proliferation, breakdown and regeneration under the control of ovarian hormones. Knowledge of normal endometrial physiology, as it pertains to the regulation of menstruation, is essential to understand disorders of menstruation. Accumulating evidence indicates that autophagy in the endometrium, under the regulation of ovarian hormones, can result in the infiltration of immune cells, which plays an indispensable role in the endometrium shedding, tissue repair and prevention of infections during menstruation. In addition, abnormal autophagy levels, together with resulting dysregulated immune system function, are associated with the pathogenesis and progression of endometriosis. Considering its potential value of autophagy as a target for the treatment of menstrual-related and endometrium-related disorders, we review the activity and function of autophagy during menstrual cycles. The role of the estrogen/progesterone-autophagy-immunity axis in endometriosis are also discussed.

Changes in subsets of immunocytes in endometrial hyperplasia.

PROBLEM: Endometrial hyperplasia (EH) is characterized by an endometrial gland-to-stroma ratio >1 and is one of the most common gynecological diseases in the world. The role of immunocyte subsets in the development of EH remains unknown. METHODS: Patients who underwent dilatation and curettage due to abnormal uterine bleeding were recruited in the present study. Alterations in the numbers of different types of immune cell subsets in the endometrium of patients were analyzed by flow cytometry. RESULTS: The present study included 48 patients who were divided into three groups, based on the pathological results: (a) proliferative period (PP, n = 12); (b) simple EH (SEH, n = 30); and (c) complex EH (CEH, n = 6). The results showed that immune cell subpopulations were significantly different between these three groups. Compared with the PP group, the proportion of CD45+ cells and neutrophils and the subtypes of T cells and macrophages were significantly increased in the SEH patients. Compared with the PP and SEH groups, subsets of immunocytes in the CEH group were significantly decreased, including the population of CD45+ cells and the subtypes of T cells and natural killer cells; in contrast, the proportion of macrophages was significantly increased. There were no significant differences between the other cell subsets in each group. CONCLUSION: The changes in immune cell subsets may be closely associated with the progression of EH. Although the specific role of different immune cell subsets in the development of the diseases requires further study, the changes in the proportions of immune cell subsets should not be ignored.

Estrogen restricts the apoptosis of endometrial stromal cells by promoting TSLP secretion.

Endometriosis (EMS) is a female hormone­ dependent disease with controversial reports of its etiology and pathogenesis. Apoptosis is particularly important in the human endometrium due to the dynamic cycles of proliferation and shedding. Estrogen possessed antiapoptotic effects on endometrial stromal cells (ESCs), which appears to be exacerbated in women with EMS; however, the underlying mechanism of the antiapoptotic effects of estrogen on ESC remains unknown. The present study aimed to determine whether estrogen regulates the apoptosis of ESCs via thymic stromal lymphopoietin (TSLP) and the associated mechanism. An ELISA was conducted to detect TSLP content in the ESC culture medium treated with estrogen. Subsequently, the early apoptotic rate and expression of B­cell lymphoma (Bcl­2) of ESCs were analyzed by flow cytometry in the presence of recombinant human TSLP, anti­human TSLP neutralizing antibody or estrogen. In the present study, it was reported that ESCs exhibited basal TSLP secretion in the absence of estrogen as reported in previous studies, and that estrogen promoted TSLP secretion of ESCs in a dose­dependent manner. The results demonstrated that estrogen suppressed the apoptosis of ESCs associated with the promotion of Bcl­2 expression, which may be partly reversed by inhibiting TSLP. Therefore, the findings of the present study revealed a novel mechanism of estrogen­dependent apoptotic suppression of ESCs associated with TSLP secretion and Bcl­2 regulation. Endogenous and estrogen­induced endometrial TSLP may promote the initiation and development of EMS via the inhibition of apoptosis.

[Exploration of serum endothelin-1 as a marker of early radiation lung injury].

AIM: To explore the possibility of endothelin-1(ET-1) as a serological marker of early diagnosis and progression of radiation induced lung injury. METHODS: One hundred and ninety female rats were randomly divided into control group (group C) and experimental groups, namely, radiation group (group R), fluvastatin treatment group (group Flu), retinoic acid treatment group (group Ra) and dexemethasone treatment group (group Dex). The chests of rats in experimental groups were exposed to radiation by linear accelerator after anesthesia. The radiation dose for each rat was 15Gy, 2Gy per minute, and radiation distance was 1 meter. The next day after radiation, fluvastatin (20 mg. kg(-1). d(-1) ) was administered orally in group Flu, retinoic acid (20 mg. kg(-1). d(-1)) in group Ra and dexemethasone (3.33 mg. kg(-1). d(-1)) in group Dex. The rats in group C and group R were medicated with the equal volume of normal saline. On the 5th, 15th, 30th, and 60th day after radiation, five rats were randomly chosen from each group respectively. The sera were harvested by decapitation or cardiopuncture and at the same time, lung tissues were cut off. The levels of serum ET-1 and LN were detected by radioimmunological assay(RIA). The pathologic changes of lung tissue were observed under light microscope. RESULTS: Compared to the control group, serum ET-1 level began to increase on the 5th day after exposure to radiation and reached the peak on the 60th day in group R. The levels of laminin and hyaluronic acid began to rise on the 30th day and the 60th day respectively. The elevation of serum ET-1 level in group R was obviously earlier than that in other groups and correlated to extent of lung injury. CONCLUSION: The serum ET-1 can be used as a marker of early diagnosis and dynamic changes of radiation lung injury.