Microbial signatures and continuum in endometrial cancer and benign patients.

BACKGROUND: Endometrial cancer is a multifactorial disease with inflammatory, metabolic and potentially microbial cues involved in disease pathogenesis. The endometrial cancer microbiome has been poorly characterised so far and studies have often overestimated bacterial biomass due to lack of integration of appropriate contamination controls. There is also a scarcity of evidence on the functionality of microbial microenvironments in endometrial cancer. This work addresses that knowledge gap by interrogating the genuine, contamination-free microbial signatures in the female genital tract and rectum of women with endometrial cancer and the mechanistic role of microbiome on carcinogenic processes. RESULTS: Here we sampled different regions of the reproductive tract (vagina, cervix, endometrium, fallopian tubes and ovaries) and rectum of 61 patients (37 endometrial cancer; 24 benign controls). We performed 16S rRNA gene sequencing of the V1-V2 hypervariable regions and qPCR of the 16S rRNA gene to qualitatively and quantitatively assess microbial communities and used 3D benign and endometrial cancer organoids to evaluate the effect of microbial products of L. crispatus, which was found depleted in endometrial cancer patients following primary analysis, on endometrial cell proliferation and inflammation. We found that the upper genital tract of a subset of women with and without endometrial cancer harbour microbiota quantitatively and compositionally distinguishable from background contaminants. Endometrial cancer was associated with reduced cervicovaginal and rectal bacterial load together with depletion of Lactobacillus species relative abundance, including L. crispatus, increased bacterial diversity and enrichment of Porphyromonas, Prevotella, Peptoniphilus and Anaerococcus in the lower genital tract and endometrium. Treatment of benign and malignant endometrial organoids with L. crispatus conditioned media exerted an anti-proliferative effect at high concentrations but had minimal impact on cytokine and chemokine profiles. CONCLUSIONS: Our findings provide evidence that the upper female reproductive tract of some women contains detectable levels of bacteria, the composition of which is associated with endometrial cancer. Whether this is a cause or consequence of cancer pathophysiology and what is the functional significance of this finding remain to be elucidated to guide future screening tools and microbiome-based therapeutics. Video Abstract.

Eff ect of platelet-rich autoplasma on endometrial thickness and receptor sensitivity to estrogen and progesterone.

AIM: The effect of platelet-rich autoplasma on endometrial thickness and receptor sensitivity to estrogen and progesterone. MATERIALS AND METHODS: This prospective clinical study included 200 patients. The participants in the study were divided into two groups. The first control group received hormone replacement therapy (HRT). The second study group received an intrauterine infusion of platelet-rich autoplasma (PRP group). On the 19th day of the menstrual cycle, an ultrasound examination was performed to assess endometrial thickness, as well as an immunohistochemical analysis to determine receptor sensitivity to estrogen and progesterone. RESULTS: In the course of the study, we found that the use of platelet-rich autoplasma increased the thickness of the endometrium by 0.85 mm; the average thickness of the endometrium in the group who received PRP therapy was 8.25 (8.25-8.61)  mm; and in the group of patients who only received HRT, it was 7.40 (7.34-7.65)  mm. The sensitivity of receptors to estrogen in the experimental group increased by 3.5, in the experimental group it was 75.00 (71.43-74.22), and in the control group it was 71.50 (67.05-70.85). The sensitivity of receptors to progesterone also increased by 9.0, in the experimental group it was 95.0 (91.4-93.8), and in the control group it was 86.0 (83.47-86.27). CONCLUSION: Due to the action of platelet factors, PRP therapy has a positive effect on the endometrium, increasing its thickness and improving its receptivity. Therefore, it can be concluded that this method can find great practical application to improve the outcomes of assisted reproductive technology programs.

Embryo Quality Conditions the Secretory Profile of Tolerogenic Dendritic Cell DC-10 During the Peri-Implantation Period.

PROBLEM: The decidualization process conditions monocytes to the immunosuppressive and tolerogenic dendritic cell (DC)-10 profile, a DC subset with high IL-10 production. Since the implantation process implies an embryo-endometrium-immune crosstalk, here we focused on the ability of embryonic soluble factors to modify decidual DC conditioning accordingly with its quality. METHOD OF STUDY: Human endometrial stromal cell line (HESC) decidualized with medroxyprogesterone and dibutyryl-cAMP (Dec) was stimulated with human embryo-conditioned media (ECM), classified as normal (ND) or impaired developed (ID) for 48 h (n = 18/group). Monocytes isolated from six healthy women were differentiated to DCs with rhGM-CSF+rhIL-4 in the presence/absence of conditioned media (CM) from decidualized cells stimulated with ECM or nontreated. RESULTS: We found that decidualized cells stimulated with ECM sustain a myeloid regulatory cell profile on monocyte-derived culture with increased frequency of CD1a-CD14+ and CD83+CD86low cells. ND-Dec sustained the higher expression of the DC-10 markers, HLA-G and IL-10 whereas ID-Dec diminished IL-10 production (ID-Dec: 135 ± 37.4 vs. Dec: 223.3 ± 49.9 pg/mL, p < 0.05). The treatment with ECM-Dec sustained a higher IL-10 production and prevented the increase of CD83/CD86 after LPS challenge regardless of embryo quality. Notably, TNF-α production increased in ID-Dec cultures (ID-Dec: 475.1 ± 134.7 vs. Dec: 347.5 ± 98 pg/mL, p < 0.05). CONCLUSIONS: Although remaining in a tolerogenic profile compatible with DC-10, DCs can differentially respond to decidual secreted factors based on embryo quality, changing their secretome. These results suggest that in the presence of arrested embryo, DCs could differentially shape the immunological microenvironment, contributing to arrested embryo clearance during the menstrual phase.

DKK1 loss promotes endometrial fibrosis via autophagy and exosome-mediated macrophage-to-myofibroblast transition.

INTRODUCTION: Intrauterine adhesions (IUA) manifest as endometrial fibrosis, often causing infertility or recurrent miscarriage; however, their pathogenesis remains unclear. OBJECTIVES: This study assessed the role of Dickkopf WNT signaling pathway inhibitor 1 (DKK1) and autophagy in endometrial fibrosis, using clinical samples as well as in vitro and in vivo experiments. METHODS: Immunohistochemistry, immunofluorescence and western blot were used to determine the localization and expression of DKK1 in endometrium; DKK1 silencing and DKK1 overexpression were used to detect the biological effects of DKK1 silencing or expression in endometrial cells; DKK1 gene knockout mice were used to observe the phenotypes caused by DKK1 gene knockout. RESULTS: In patients with IUA, DKK1 and autophagy markers were down-regulated; also, α-SMA and macrophage localization were increased in the endometrium. DKK1 conditional knockout (CKO) mice showed a fibrotic phenotype with decreased autophagy and increased localization of α-SMA and macrophages in the endometrium. In vitro studies showed that DKK1 knockout (KO) suppressed the autophagic flux of endometrial stromal cells. In contrast, ectopic expression of DKK1 showed the opposite phenotype. Mechanistically, we discovered that DKK1 regulates autophagic flux through Wnt/ß-catenin and PI3K/AKT/mTOR pathways. Further studies showed that DKK1 KO promoted the secretion of interleukin (IL)-8 in exosomes, thereby promoting macrophage proliferation and metastasis. Also, in DKK1 CKO mice, treatment with autophagy activator rapamycin partially restored the endometrial fibrosis phenotype. CONCLUSION: Our findings indicated that DKK1 was a potential diagnostic marker or therapeutic target for IUA.

Significant increased isolation of Escherichia coli in Iranian women with endometriosis: a case control - study.

BACKGROUND: The role of bacterial contamination in the development and progression of endometriosis lesions is currently a hot topic for gynecologists. In this study, we decided to compare the endometrial cultures of women affected by endometriosis with those of non-endometriotic women, focusing on specific microbial pathogens. MATERIAL AND METHOD: In this cross-sectional case-control study, 30 women with endometriosis in stages 4 of the disease whose endometriosis was confirmed based on clinical, ultrasound, and histopathological findings, and 30 women without endometriosis who were candidates for surgery due to benign uterine diseases with regular menstrual cycle, underwent endometrial biopsy with Novak Kort in sterile conditions before starting their operation, and the results of their endometrial culture were analyzed and compared. RESULTS: Results of the study indicate that there were no significant differences in terms of age, BMI, smoking, education level, place of residency, use of the intrauterine device, or vaginal douche, and age of menarche between the case and control groups. The only demographic difference observed was in parity, where the control group had a significantly higher parity than the case group (P = 0.001). Out of the 60 cultures, only 15 samples were positive in the endometriosis group, and E. coli was the most prevalent species, with 10 (33.3%) samples testing positive for it. Klebsiella spp. and Enterobacteria spp. were also detected in 3 (10.0%) and 2 (6.7%) samples, respectively. The comparison between the two groups showed that only E. coli had a significant association with the presence of endometriosis (P = 0.001). There was no significant relationship between the location of endometriosis in the pelvic cavity and culture results. It was observed that parity among the E. coli negative group was significantly higher compared to the E. coli positive group (P < 0.001). CONCLUSION: Based on The high occurrence of E. coli in women with endometriosis, along with its potential involvement in the progression and/or recurrence of this condition, the researchers propose that treating women with endometriosis and recurrent IVF failure, as well as those with endometriosis recurrence after surgical treatment, with suitable antibiotics and repeated culture until the culture becomes negative, could be beneficial.

[Quercetin Alleviates H2O2-Induced Oxidative Stress Damage to Human Endometrial Stromal Cells by Inhibiting the p38 MAPK/NOX4 Signaling Pathway]. / æ§²çš®ç´ é€šè¿‡æŠ‘åˆ¶p38 MAPK/NOX4信号通路减轻H2O2è¯±å¯¼çš„äººå­å®«å† è†œåŸºè´¨ç»†èƒžæ°§åŒ–åº”æ¿€æŸä¼¤.

Objective: This study aims to systematically evaluate the protective role of quercetin (QCT), a naturally occurring flavonoid, against oxidative damage in human endometrial stromal cells (HESCs) induced by hydrogen peroxide (H2O2). Oxidative stress, such as that induced by H2O2, is known to contribute significantly to cellular damage and has been implicated in various reproductive health issues. The study is focused on investigating how QCT interacts with specific molecular pathways to mitigate this damage. Special attention was given to the p38 MAPK/NOX4 signaling pathway, which is crucial to the regulation of oxidative stress responses in cellular systems. By elucidating these mechanisms, the study seeks to confirm the potential of QCT not only as a protective agent against oxidative stress but also as a therapeutic agent that could be integrated in treatments of conditions characterized by heightened oxidative stress in endometrial cells. Methods: I n vitro cultures of HESCs were treated with QCT at different concentrations (0, 10, 20, and 40 µmol/L) for 24 h to verify the non-toxic effects of QCT on normal endometrial cells. Subsequently, 250 µmol/L H2O2 was used to incubate the cells for 12 h to establish an H2O2-induced HESCs injury model. HESCs were pretreated with QCT for 24 h, which was followed by stimulation with H2O2. Then, CCK-8 assay was performed to examine the cell viability and to screen for the effective intervention concentration. HESCs were divided into 3 groups, the control group, the H2O2 model group, and the H2O2+QCT group. Intracellular levels of reactive oxygen species (ROS) were precisely quantified using the DCFH-DA fluorescence assay, a method known for its accuracy in detecting and quantifying oxidative changes within the cell. The mitochondrial membrane potential was determined by JC-1 staining. Annexin Ⅴ/PI double staining and flow cytometry were performed to determine the effect of QCT on H2O2-induced apoptosis of HESCs. Furthermore, to delve deeper into the cellular mechanisms underlying the observed effects, Western blot analysis was conducted to measure the expression levels of the critical proteins involved in oxidative stress response, including NADPH oxidase 4 (NOX4), p38 mitogen-activated protein kinase (p38 MAPK), and phosphorylated p38 MAPK (p-p38 MAPK). This analysis helps increase understanding of the specific intracellular signaling pathways affected by QCT treatment, giving special attention to its potential for modulation of the p38 MAPK/NOX4 pathway, which plays a significant role in cellular defense mechanisms against oxidative stress. Results: In this study, we started off by assessing the toxicity of QCT on normal endometrial cells. Our findings revealed that QCT at various concentrations (0, 10, 20, and 40 µmol/L) did not exhibit any cytotoxic effects, which laid the foundation for further investigation into its protective roles. In the H2O2-induced HESCs injury model, a significant reduction in cell viability was observed, which was linked to the generation of ROS and the resultant oxidative damage. However, pretreatment with QCT (10 µmol/L and 20 µmol/L) significantly enhanced cell viability after 24 h (P<0.05), with the 20 µmol/L concentration showing the most substantial effect. This suggests that QCT can effectively reverse the cellular damage caused by H2O2. Furthermore, the apoptosis assays demonstrated a significant increase in the apoptosis rates in the H2O2 model group compared to those in the control group (P<0.01). However, co-treatment with QCT significantly reversed this trend (P<0.05), indicating QCT's potential protective role in mitigating cell apoptosis. ROS assays showed that, compared to that in the control group, the average fluorescence intensity of ROS in the H2O2 model group significantly increased (P<0.01). QCT treatment significantly reduced the ROS fluorescence intensity in the H2O2+QCT group compared to the that in the H2O2 model group, suggesting an effective alleviation of oxidative damage (P<0.05). JC-1 staining for mitochondrial membrane potential changes revealed that compared to that in the control, the proportion of cells with decreased mitochondrial membrane potential significantly increased in the H2O2 model group (P<0.01). However, this proportion was significantly reduced in the QCT-treated group compared to that of the H2O2 model group (P<0.05). Finally, Western blot analysis indicated that the expression levels of NOX4 and p-p38 MAPK proteins were elevated in the H2O2 model group compared to those of the control group (P<0.05). Following QCT treatment, these protein levels significantly decreased compared to those of the H2O2 model group (P<0.05). These results suggest that QCT may exert its protective effects against oxidative stress by modulating the p38 MAPK/NOX4 signaling pathway. Conclusion: QCT has demonstrated significant protective effects against H2O2-induced oxidative damage in HESCs. This protection is primarily achieved through the effective reduction of ROS accumulation and the inhibition of critical signaling pathways involved in the oxidative stress response, notably the p38 MAPK/NOX4 pathway. The results of this study reveal that QCT's ability to modulate these pathways plays a key role in alleviating cellular damage associated with oxidative stress conditions. This indicates not only its potential as a protective agent against cellular oxidative stress, but also highlights its potential for therapeutic applications in treating conditions characterized by increased oxidative stress in the endometrium, thereby offering the prospect of enhancing reproductive health. Future studies should explore the long-term effects of QCT and its clinical efficacy in vivo, thereby providing a clear path toward its integration into therapeutic protocols.

[Notch1/Akt/Foxo1 Pathway Regulated by Kisspeptin Is Involved in Endometrial Decidualization in Patients With Recurrent Spontaneous Abortion]. / kisspeptin调控Notch1/Akt/Foxo1é€šè·¯å‚ä¸Žå¤å‘æ€§æµäº§æ‚£è€ å­å®«å† è†œèœ•è†œåŒ–.

Objective: Kisspeptin, a protein encoded by the KISS1 gene, functions as an essential factor in suppressing tumor growth. The intricate orchestration of cellular processes such as proliferation and differentiation is governed by the Notch1/Akt/Foxo1 signaling pathway, which assumes a central role in maintaining cellular homeostasis. In the specific context of this investigation, the focal point lies in a meticulous exploration of the intricate mechanisms underlying the regulatory effect of kisspeptin on the process of endometrial decidualization. This investigation delves into the interplay between kisspeptin and the Notch1/Akt/Foxo1 signaling pathway, aiming to elucidate its significance in the pathophysiology of recurrent spontaneous abortion (RSA). Methods: We enrolled a cohort comprising 45 individuals diagnosed with RSA, who were admitted to the outpatient clinic of the Reproductive Center at the Second Affiliated Hospital of Soochow University between June 2020 and December 2020. On the other hand, an additional group of 50 women undergoing elective abortion at the outpatient clinic of the Family Planning Department during the same timeframe was also included. To comprehensively assess the molecular landscape, Western blot and RT-qPCR were performed to analyze the expression levels of kisspeptin (and its gene KISS1), IGFBP1 (an established marker of decidualization), Notch1, Akt, and Foxo1 within the decidua. Human endometrial stromal cells (hESC) were given targeted interventions, including treatment with siRNA to disrupt KISS1 or exposure to kisspeptin10 (the bioactive fragment of kisspeptin), and were subsequently designated as the siKP group or the KP10 group, respectively. A control group comprised hESC was transfected with blank siRNA, and cell proliferation was meticulously evaluated with CCK8 assay. Following in vitro induction for decidualization across the three experimental groups, immunofluorescence assay was performed to identify differences in Notch1 expression and decidualization morphology between the siKP and the KP10 groups. Furthermore, RT-qPCR and Western blot were performed to gauge the expression levels of IGFBP1, Notch1, Akt, and Foxo1 across the three cell groups. Subsequently, decidualization was induced in hESC by adding inhibitors targeting Notch1, Akt, and Foxo1. The expression profiles of the aforementioned proteins and genes in the four groups were then examined, with hESC induced for decidualization without adding inhibitors serving as the normal control group. To establish murine models of normal pregnancy (NP) and RSA, CBA/J×BALB/c and CBA/J×DBA/2 mice were used. The mice were respectively labeled as the NP model and RSA model. The experimental groups received intraperitoneal injections of kisspeptin10 and kisspeptin234 (acting as a blocker) and were designated as RSA-KP10 and NP-KP234 groups. On the other hand, the control groups received intraperitoneal injections of normal saline (NS) and were referred to as RSA-NS and NP-NS groups. Each group comprised 6 mice, and uterine tissues from embryos at 9.5 days of gestation were meticulously collected for observation of embryo absorption and examination of the expression of the aforementioned proteins and genes. Results: The analysis revealed that the expression levels of kisspeptin, IGFBP1, Notch1, Akt, and Foxo1 were significantly lower in patients diagnosed with RSA compared to those in women with NP (P<0.01 for kisspeptin and P<0.05 for IGFBP1, Notch1, Akt, and Foxo1). After the introduction of kisspeptin10 to hESC, there was an observed enhancement in decidualization capability. Subsequently, the expression levels of Notch1, Akt, and Foxo1 showed an increase, but they decreased after interference with KISS1. Through immunofluorescence analysis, it was observed that proliferative hESC displayed a slender morphology, but they transitioned to a rounder and larger morphology post-decidualization. Concurrently, the expression of Notch1 increased, suggesting enhanced decidualization upon the administration of kisspeptin10, but the expression decreased after interference with KISS1. Further experimentation involved treating hESC with inhibitors specific to Notch1, Akt, and Foxo1 separately, revealing a regulatory sequence of Notch1/Akt/Foxo1 (P<0.05). In comparison to the NS group, NP mice administered with kisspeptin234 exhibited increased fetal absorption rates (P<0.001) and decreased expression of IGFBP1, Notch1, Akt, and Foxo1 (P<0.05). Conversely, RSA mice administered with kisspeptin10 demonstrated decreased fetal absorption rates (P<0.001) and increased expression levels of the aforementioned molecules (P<0.05). Conclusion: It is suggested that kisspeptin might exert its regulatory influence on the process of decidualization through the modulation of the Notch1/Akt/Foxo1 signaling cascade. A down-regulation of the expression levels of kisspeptin could result in suboptimal decidualization, which in turn might contribute to the development or progression of RSA.

[Effect of Intrauterine Perfusion of Granulocyte Colony-Stimulating Factor on Endometrium and Blood Flow Parameters in Patients With Thin Endometrium: A Prospective Controlled Clinical Trial]. / å®«è ”çŒæ³¨ç²’ç»†èƒžé›†è½åˆºæ¿€å› å­æ”¹å–„è–„åž‹å­å®«å† è†œæ‚£è€ å† è†œåŠè¡€æµå‚æ•°çš„å‰çž»æ€§ä¸´åºŠå¯¹ç §è¯•éªŒ.

Objective: To investigate the effects of intrauterine perfusion with granulocyte colony-stimulating factor (G-CSF) on the endometrial thickness, volume, and blood flow parameters of patients with thin endometrium and their clinical outcomes. Methods: We designed a prospective non-randomized synchronous controlled trial and recruited patients with thin endometrium who underwent frozen-thawed embryo transfer (FET) at Mianyang Central Hospital between September 1, 2021 and September 1, 2023. They were divided into two groups, an experimental group of patients who received the experimental treatment of intrauterine perfusion with G-CSF and a control group of patients who did not receive the experimental treatment. The general data and the clinical outcomes of the two groups were analyzed and compared. The endometrial thickness, volume and blood flow parameters of patients in the experimental group before and after intrauterine perfusion with G-CSF were analyzed. Results: The clinical data of 83 patients were included in the study. The experimental group included 51 cases, while the control group included 31 cases. There were no significant differences in the baseline data between the two groups. The clinical pregnancy rate of the experimental group (56.86%) was higher than that of the control group (50.00%) and the rate of spontaneous abortion in the experimental group (27.59%) was lower than that in the control group (37.50%), but the differences were not statistically significant (P>0.05). In the experimental group, the postperfusion endometrial thickness ([0.67±0.1] cm) was greater than the preperfusion endometrial thickness ([0.59±0.09] cm), the postperfusion ([1.84±0.81] cm3) was greater than the preperfusion endometrial volume ([1.54±0.69] cm3), and the postperfusion vascularization flow index (VFI) (1.97±2.82) was greater than the preperfusion VFI (0.99±1.04), with all the differences being statistically significant (P<0.05). Conclusion: Intrauterine perfusion with G-CSF can enhance the endometrial thickness, volume, and some blood flow parameters in patients with thin endometrium.

Proangiogenic properties of complement protein C1q can contribute to endometriosis.

Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM.

Inflammatory Changes after Medical Suppression of Suspected Endometriosis for Implantation Failure: Preliminary Results.

Unexplained euploid embryo transfer failure (UEETF) is a frustrating and unanswered conundrum accounting for 30 to 50% of failures in in vitro fertilization using preimplantation genetic testing for aneuploidy (PGT-A). Endometriosis is thought by many to account for most of such losses and menstrual suppression or surgery prior to the next transfer has been reported to be beneficial. In this study, we performed endometrial biopsy in a subset of women with UEETF, testing for the oncogene BCL6 and the histone deacetylase SIRT1. We compared 205 PGT-A cycles outcomes and provide those results following treatment with GnRH agonist versus controls (no treatment). Based on these and previous promising results, we next performed a pilot randomized controlled trial comparing the orally active GnRH antagonist, elagolix, to oral contraceptive pill (OCP) suppression for 2 months before the next euploid embryo transfer, and monitored inflammation and miRNA expression in blood, before and after treatment. These studies support a role for endometriosis in UEETF and suggest that medical suppression of suspected disease with GnRH antagonist prior to the next transfer could improve success rates and address underlying inflammatory and epigenetic changes associated with UEETF.

The Molecular Link between Obesity and the Endometrial Environment: A Starting Point for Female Infertility.

Female infertility constitutes a growing health problem in developing countries and could be associated with several possible causes including reproductive disorders, congenital malformations, infections and hormonal dysfunction. Nonetheless, a series of additional factors can also negatively impact female fertility and are represented by chronic exposure to environmental pollutants, stress, unhealthy lifestyle choices such as cigarette smoking and, among others, obesity. Excess weight is associated with several chronic diseases, and growing evidence demonstrates that it can compromise reproductive physiology due to its influence on endometrial gene expression and receptivity. Thus, the current review of the literature mainly focused on how obesity can impair uterine receptivity, mostly from a molecular point of view throughout the window of implantation (WOI) period at an endometrial level. It was also highlighted that an obesity-related increase in adipose tissue may lead to a modulation in the expression of multiple pathways, which could cause a hostile endometrial environment with a consequent negative impact on the uterine receptivity and the establishment of pregnancy. Thanks to the use of the endometrial receptivity assay (ERA), a specific microarray that studies the expression of a series of genes, it is now possible to evaluate the endometrial status of patients with infertility problems in a more detailed manner. Moreover, female fertility and endometrial receptivity could be affected by endometriosis, a chronic benign gynecological disease, whose cause-and-effect relationship to obesity is still uncertain. Therefore, further investigations would be required to better elucidate these mechanisms that govern embryo implantation and could be potentially useful for the generation of new strategies to overcome implantation failure and improve the pregnancy rates in obese women.

The Extremely-Low-Frequency Electromagnetic Field Affects Apoptosis and Oxidative-Stress-Related Genes and Proteins in the Porcine Endometrium-An In Vitro Study.

Nowadays, the extremely-low-frequency electromagnetic field (ELF-EMF) is recognized as environmental pollution. The data indicate that the ELF-EMF may affect factors related to epigenetic regulation and alter important biological processes in the uterus. The impact of the ELF-EMF on apoptosis and oxidative-stress-related genes has not been documented in porcine endometrium. This raises the question of whether the exposure to the ELF-EMF can induce apoptosis and/or oxidative stress in the endometrium of pigs during the peri-implantation period. Porcine endometrial slices (100 ± 5 mg) collected (n = 5) during the peri-implantation period were treated in vitro with ELF-EMF at a frequency of 50 Hz and flux density of 8 × 104 mG for 2 h. To determine the effect of ELF-EMF on apoptosis and oxidative stress in the endometrium, CASP3, CASP7, CIDEB, GADD45G, NOS1, NOS2, NOS3, and TP53I3 mRNA transcript were analyzed using real-time PCR, and protein abundance of CASP3, CASP7 using Western blot, and eNOS using ELISA were determined. Moreover, CASP3/7 and NOS activity was analyzed using flow cytometry and colorimetry, respectively. The decreased CASP7 and increased NOS3 mRNA transcript and protein abundance in ELF-EMF-treated endometrium were observed. Moreover, CIDEB, GADD45G, and TP53I3 mRNA transcript abundance was increased. Only p ≤ 0.05 was considered a statistically significant difference. The documented alterations indicate the potential of the ELF-EMF to affect apoptosis and generate oxidative stress in the endometrium. The insight into observed consequences documents for the first time the fact that the ELF-EMF may influence endometrial cell proliferation, angiogenesis, and/or tissue receptivity during peri-implantation.

CBLL1 promotes endometrial stromal cell senescence via inhibiting PTEN in recurrent spontaneous abortion.

Recurrent spontaneous abortion (RSA) is a common pregnancy-related disorder. Cbl proto-oncogene like 1 (CBLL1) is an E3 ubiquitin ligase, which has been reported to vary with the menstrual cycle in the endometrium. However, whether CBLL1 is involved in the occurrence and development of RSA remains unclear. This study aimed to investigate the effects of CBLL1 on RSA. We analyzed the expression of CBLL1 in the decidua of RSA patients, as well as its functional effects on cellular senescence, oxidative stress, and proliferation of human endometrial stromal cells (HESCs). RNA sequencing was employed to identify a key downstream target gene regulated by CBLL1. We found that CBLL1 was upregulated in the decidua of RSA patients. Additionally, overexpression of CBLL1 promoted HESC senescence, increased oxidative stress levels, and inhibited proliferation. Phosphatase and tensin homolog located on chromosome 10 (PTEN) was identified as one of the important downstream target genes of CBLL1. In vivo experiments demonstrated that CBLL1 overexpression in the endometrium caused higher embryo absorption rate in mice. Consequently, elevated CBLL1 expression is a potential cause of RSA, representing a novel therapeutic target for RSA.

Nuclear actin assembly is an integral part of decidualization in human endometrial stromal cells.

Decidualization of the human endometrium is critical for establishing pregnancy and is entailed by differentiation of endometrial stromal cells (ESCs) into decidual cells. During decidualization, the actin cytoskeleton is dynamically reorganized for the ESCs' morphological and functional changes. Although actin dynamically alters its polymerized state upon external stimuli not only in the cytoplasm, but also in the nucleus, nuclear actin dynamics during decidualization have not been elucidated. Here, we show that nuclear actin was specifically assembled during decidualization of human ESCs. This decidualization-specific formation of nuclear actin filaments was disassembled following the withdrawal of the decidualization stimulus, suggesting its reversible process. Mechanistically, RNA-seq analyses revealed that the forced disassembly of nuclear actin resulted in the suppression of decidualization, accompanied with the abnormal upregulation of cell proliferation genes, leading to incomplete cell cycle arrest. CCAAT/enhancer-binding protein beta (C/EBPß), an important regulator for decidualization, was responsible for downregulation of the nuclear actin exporter, thus accelerating nuclear actin accumulation and its assembly for decidualization. Taken together, we demonstrate that decidualization-specific nuclear actin assembly induces cell cycle arrest for establishing the decidualized state of ESCs. We propose that not only the cytoplasmic actin, but also nuclear actin dynamics profoundly affect decidualization process in humans for ensuring pregnancy.

Oxidative Stress and the Nrf2/PPARγ Axis in the Endometrium: Insights into Female Fertility.

Successful pregnancy depends on precise molecular regulation of uterine physiology, especially during the menstrual cycle. Deregulated oxidative stress (OS), often influenced by inflammatory changes but also by environmental factors, represents a constant threat to this delicate balance. Oxidative stress induces a reciprocally regulated nuclear factor erythroid 2-related factor 2/peroxisome proliferator-activated receptor-gamma (Nrf2/PPARγ) pathway. However, increased PPARγ activity appears to be a double-edged sword in endometrial physiology. Activated PPARγ attenuates inflammation and attenuates OS to restore redox homeostasis. However, it also interferes with physiological processes during the menstrual cycle, such as hormonal signaling and angiogenesis. This review provides an elucidation of the molecular mechanisms that support the interplay between PPARγ and OS. Additionally, it offers fresh perspectives on the Nrf2/PPARγ pathway concerning endometrial receptivity and its potential implications for infertility.

Comparative Assessment of miR-185-5p and miR-191-5p Expression: From Normal Endometrium to High-Grade Endometrial Cancer.

Endometrial cancer (EC) is a significant cause of cancer-related deaths in women. MicroRNAs (miRs) play a role in cancer development, acting as oncogenes or tumor suppressors. This study evaluated the diagnostic potential of hsa-miR-185-5p and hsa-miR-191-5p in EC and their correlation with clinical and histopathological features. A cross-sectional study analyzed formalin-fixed, paraffin-embedded tissue samples from 59 patients: 18 with EC, 21 with endometrial hyperplasia (EH), 17 with normal endometrium (NE), and 3 with endometrial polyps (EPs). Quantitative reverse transcription-polymerase chain reaction and TaqMan probes were used for miR expression analysis. The Shapiro-Wilk test was used to analyze the normal distribution of the data. Subsequently, parametric or non-parametric tests were used to evaluate the associations between the expression levels of each miR and clinical parameters. Both miRs were underexpressed in some precursor and malignant lesions compared to certain NE subtypes and benign lesions. Specifically, hsa-miR-185-5p showed underexpression in grade 3 EC compared to some NE and EH subtypes (FC: -57.9 to -8.5, p < 0.05), and hsa-miR-191-5p was underexpressed in EH and EC compared to secretory endometrium and EPs (FC: -4.2 to -32.8, p < 0.05). SETD1B, TJP1, and MSI1 were common predicted target genes. In conclusion, hsa-miR-185-5p and hsa-miR-191-5p are underexpressed in EC tissues, correlating with histopathological grades, highlighting their potential as diagnostic biomarkers and their role as tumor suppressors in EC.

The impact of clomiphene citrate on the endometrium in comparison to gonadotropins in intrauterine insemination cycles: is it thinner and does it matter?

Objective: To determine whether endometrial thickness (EMT) differs between i) clomiphene citrate (CC) and gonadotropin (Gn) utilizing patients as their own controls, and ii) patients who conceived with CC and those who did not. Furthermore, to investigate the association between late-follicular EMT and pregnancy outcomes, in CC and Gn cycles. Methods: Retrospective study. Three sets of analyses were conducted separately for the purpose of this study. In analysis 1, we included all cycles from women who initially underwent CC/IUI (CC1, n=1252), followed by Gn/IUI (Gn1, n=1307), to compare EMT differences between CC/IUI and Gn/IUI, utilizing women as their own controls. In analysis 2, we included all CC/IUI cycles (CC2, n=686) from women who eventually conceived with CC during the same study period, to evaluate EMT differences between patients who conceived with CC (CC2) and those who did not (CC1). In analysis 3, pregnancy outcomes among different EMT quartiles were evaluated in CC/IUI and Gn/IUI cycles, separately, to investigate the potential association between EMT and pregnancy outcomes. Results: In analysis 1, when CC1 was compared to Gn1 cycles, EMT was noted to be significantly thinner [Median (IQR): 6.8 (5.5-8.0) vs. 8.3 (7.0-10.0) mm, p<0.001]. Within-patient, CC1 compared to Gn1 EMT was on average 1.7mm thinner. Generalized linear mixed models, adjusted for confounders, revealed similar results (coefficient: 1.69, 95% CI: 1.52-1.85, CC1 as ref.). In analysis 2, CC1 was compared to CC2 EMT, the former being thinner both before [Median (IQR): 6.8 (5.5-8.0) vs. 7.2 (6.0-8.9) mm, p<0.001] and after adjustment (coefficient: 0.59, 95%CI: 0.34-0.85, CC1 as ref.). In analysis 3, clinical pregnancy rates (CPRs) and ongoing pregnancy rates (OPRs) improved as EMT quartiles increased (Q1 to Q4) among CC cycles (p<0.001, p<0.001, respectively), while no such trend was observed among Gn cycles (p=0.94, p=0.68, respectively). Generalized estimating equations models, adjusted for confounders, suggested that EMT was positively associated with CPR and OPR in CC cycles, but not in Gn cycles. Conclusions: Within-patient, CC generally resulted in thinner EMT compared to Gn. Thinner endometrium was associated with decreased OPR in CC cycles, while no such association was detected in Gn cycles.

Endometrial senescence is mediated by interleukin 17 receptor B signaling.

BACKGROUND: We previously identified Il17RB, a member of the IL17 superfamily, as a candidate marker gene for endometrial aging. While IL17RB has been linked to inflammation and malignancies in several organ systems, its function in the endometrium has not been investigated and is thus poorly understood. In the present study, we performed a functional analysis of this receptor with the aim of determining the effects of its age-associated overexpression on the uterine environment. METHODS: We analyzed IL17RB-related signaling pathways and downstream gene expression in an immortalized human endometrial glandular epithelial cell line ("hEM") forced to express the receptor via lentiviral transduction ("IL17RB-hEM"). We also prepared endometrial organoids from human endometrial tissue sourced from hysterectomy patients ("patient-derived EOs") and exposed them to cytokines that are upregulated by IL17RB expression to investigate changes in organoid-forming capacity and senescence markers. We analyzed RNA-seq data (GEO accession number GSE132886) from our previous study to identify the signaling pathways associated with altered IL17RB expression. We also analyzed the effects of the JNK pathway on organoid-forming capacity. RESULTS: Stimulation with interleukin 17B enhanced the NF-κB pathway in IL17RB-hEM, resulting in significantly elevated expression of the genes encoding the senescence associated secretory phenotype (SASP) factors IL6, IL8, and IL1ß. Of these cytokines, IL1ß inhibited endometrial organoid growth. Bioinformatics analysis showed that the JNK signaling pathway was associated with age-related variation in IL17RB expression. When IL17RB-positive cells were cultured in the presence of IL17B, their organoid-forming capacity was slightly but non-significantly lower than in unexposed IL17RB-positive cells, but when IL17B was paired with a JNK inhibitor (SP600125), it was restored to control levels. Further, IL1ß exposure significantly reduced organoid-forming capacity and increased p21 expression in endometrial organoids relative to non-exposure (control), but when IL1ß was paired with SP600125, both indicators were restored to levels comparable to the control condition. CONCLUSIONS: We have revealed an association between IL17RB, whose expression increases in the endometrial glandular epithelium with advancing age, and cellular senescence. Using human endometrial organoids as in vitro model, we found that IL1ß inhibits cell proliferation and leads to endometrial senescence via the JNK pathway.

Role of autophagy and ferroptosis in the development of endometriotic cysts (Review).

It is considered that the etiology of endometriosis is retrograde menstruation of endometrial tissue. Although shed endometrial cells are constantly exposed to a challenging environment with iron overload, oxidative stress and hypoxia, a few cells are able to survive and continue to proliferate and invade. Ferroptosis, an iron­dependent form of non­apoptotic cell death, is known to play a major role in the development and course of endometriosis. However, few papers have concentrated on the dynamic interaction between autophagy and ferroptosis throughout the progression of diseases. The present review summarized the current understanding of the mechanisms underlying autophagy and ferroptosis in endometriosis and discuss their role in disease development and progression. For the present narrative review electronic databases including PubMed and Google Scholar were searched for literature published up to the October 31, 2023. Autophagy and ferroptosis may be activated at early stages in endometriosis development. On the other hand, excessive activation of intrinsic pathways (e.g., estrogen and mechanistic target of rapamycin) may promote disease progression through autophagy inhibition. Furthermore, suppression of ferroptosis may cause further progression of endometriotic lesions. In conclusion, the autophagy and ferroptosis pathways may play a dual role in disease initiation and progression. The present review discussed the temporal transition of non­apoptotic cell death regulation during disease progression from retrograde endometrium to early lesions to established lesions.

In mares resistant to endometrial infection, periovulatory treatment with ecbolic drugs does not influence uterine clearance or luteal development.

We aimed to determine associations between experimentally impaired uterine clearance or treatment with ecbolic drugs on luteal development in estrous mares after insemination. In a crossover design, eight mares were treated with saline (CON), clenbuterol (CLEN), oxytocin (OXY) and carbetocin (CARB) from the day of first insemination until 2 days after ovulation. Between treatments, the mares rested for one cycle. Estrous mares were examined for the presence of free intrauterine fluid by transrectal ultrasound. Endometrial swabs for cytology and bacteriology were collected on days 1 and 14. Blood samples were collected daily before AI until day 14 after ovulation for determination of progesterone and PGF2α metabolites (PGFM). Differences between treatments were compared by a general linear model for repeated measures (SPSS 29). One mare was excluded because of a uterine infection in the control cycle. In all other mares, only minor amounts of free intrauterine fluid were present after insemination and decreased over time (P<0.05) with no treatment x time interaction. There was no effect of treatment on polymorphonucleated cells (PMN) in endometrial cytology after ovulation or PGFM secretion. Progesterone release from day 1-14 as well as pregnancy rate and conceptus size on day 14 was not influenced by treatment. In conclusion, treatment with clenbuterol does not impair uterine clearance in estrous mares resistant to endometritis. Repeated injection of the oxytocin analogue carbetocin during the early postovulatory period is not detrimental to corpus luteum function and can be recommended to enhance uterine clearance.