Endometrial Cultures Research Articles

P-279 Significant increased isolation of escherichia coli in Iranian women with endometriosis: a case control – study

Abstract Study question Considering the role of bacterial infection in causing endometriosis, we decided to compare the cultures of endometriosis-affected women a in this study Summary answer Considering the possible role of E. coli in the progression and/or recurrence of endometriosis and also infertility What is known already (5). Due to the finding of endotoxin of E. coli species in the peritoneal cavity and menstrual fluid of endometriosis patients, infection with this bacterium has recently been proposed as an effective factor in the occurrence and growth of endometriosis lesions. Study design, size, duration This prospective case-control study was carried out in the Women’s Department of Shiraz University of Medical Sciences in collaboration with the Bacteriology and Virology Department between 60 participants. Participants/materials, setting, methods 30 women with endometriosis in stages 3 and 4 of the disease, and 30 women without endometriosis who were candidates for surgery due to benign uterine diseases, underwent endometrial biopsy with Novak Kort in sterile conditions before starting their operation, and the results of their endometrial culture were analyzed and compared in terms of the presence of Enterobacter spp., Klebsiella spp., and E. coli species. Main results and the role of chance Based on the comparison between the two groups, only E. coli species 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. Parity among the E. coli negative group was significantly higher compared to the E. coli positive group (P < 0.001). Limitations, reasons for caution Lack of sample trail for repeat testing and antibiotic susceptibility testing, low taxonomical resolution, and costly laboratory setup and maintenance are other limitations of this technology Wider implications of the findings we suggest that in women who have endometriosis and recurrent IVF failure and also in cases of endometriosis recurrence after surgical treatment, treatment of couples with appropriate antibiotics and repeated culture along with subsequent antibiotic susceptibility testing until their culture becomes negative could be beneficial. Trial registration number IR.SUMS.MED.REC.1400.097

Correlation between hysteroscopic features and specific microbial species in women with chronic endometritis

Objective and rationaleChronic endometritis (CE) has recently been associated with unexplained infertility and recurrent miscarriages. The current gold standard for CE detection is histopathological examination. However, office hysteroscopy and endometrial cultures are also significant, due to the possible link between CE and various microorganisms. Bacterial colonization of the endometrium has been associated with reduced success rates of in vitro fertilisation embryo transfer. Few studies have tried to correlate CE hysteroscopy findings with pathogenic microorganisms. This prospective cohort study sought to establish whether hysteroscopic diagnostic lesions correlate with specific microbial species. MethodsThe study encompassed women undergoing diagnostic tests for a range of subfertility health issues. 189 women completed the standard office diagnostic hysteroscopy (DH). 181 had also endometrial samples taken for microbial culture investigation. Correlation analysis (χ2 and Fisher's exact test) between hysteroscopic findings suggestive of CE and endometrial cultures was carried out. Logistic regression models were also fitted to measure whether a positive endometrial culture could affect CE conditions. ResultsA significant association of E. coli was observed between the hysteroscopically characterized CE + group with focal hyperplasia, when compared to the non–CE group. Logistic regression analysis revealed that women positive for E. coli were 4.423 times more likely to have focal endometrial hyperplasia. No other significant correlations were identified between DH and positive endometrial cultures. ConclusionsThe presence of E. coli in the endometrium was significantly correlated with focal hyperplasia findings from hysteroscopy, emphasizing the importance of microbial cultures in the diagnosis and targeted treatment of CE in women with subfertility.

Chronic endometritis and reproduction: Correlation of pathology reports with endometrial culture results

Chronic endometritis and reproduction: Correlation of pathology reports with endometrial culture results

RNA in situ hybridization of Escherichia coli in equine endometrial biopsies.

Endometritis is one of the major causes of infertility in mares. Escherichia coli and β-haemolytic streptococci are among the bacterial species most frequently isolated from the equine uterus. Some bacteria such as β-hemolytic streptococci, can persist in dormant forms and cause prolonged, latent or recurrent infections. Dormant bacteria may be present despite negative bacterial cultures, and they are resistant to antimicrobial treatment due to their resting metabolic state. The purpose of this study was to study formalin-fixed paraffin-embedded equine endometrial biopsies for the presence and localization of E. coli-bacteria, with a chromogenic RNAscope®-method for detection of E. coli-related 16S ribosomal RNA. Hematoxylin-eosin-stained endometrial biopsies were evaluated to determine the level of inflammation and degeneration. During estrus, samples were taken for endometrial culture and cytology with a double-guarded uterine swab. The samples included eight samples with moderate to severe endometrial inflammation detected in endometrial histopathology, and growth of E. coli in bacterial culture, six samples with moderate to severe endometrial inflammation but negative bacterial culture, and five samples with no endometrial pathology (grade I endometrial biopsy, negative endometrial culture and cytology) serving as controls. Positive and negative control probes were included in the RNA in situ hybridization, and results were confirmed with a fluorescence detection method (fluorescence in situ hybridization). Only unspecific signals of limited size and frequency of occurrence were detected in all samples, with random localization in the endometrium. No samples contained rod-shaped signals corresponding to bacterial findings. In conclusion, there was no evidence of bacterial invasion in the endometrium regardless of the inflammatory status of the biopsy or previous bacterial culture results. According to these findings on a small number of samples, invasion of E. coli is not a common finding in the lamina propria of mares, but these bacteria may also evade detection due to localized foci of infections, or supra-epithelial localization under the cover of biofilm. These bacteria and biofilm covering the epithelium may also be lost during formalin-fixation and processing.

Use of endometrial swab and cytobrush for detection of inflammatory diagnostic markers of endometritis

Screening methods for endometritis are often performed through endometrial culture and cytology with a variety of techniques including endometrial swab, cytobrush, low-volume lavage, or biopsy. With growing research interest in equine reproductive immunology and low sensitivity of current screening tools for endometritis, one area that would be of immediate benefit to clinicians and clients is inflammatory diagnostic development. Previous research detected inflammatory markers in low-volume lavage fluid in mares with chronic and acute endometritis (Lection et al. AAEP Proceedings. 2020; 66: 150-151). Therefore, our objective was to investigate the utility of the endometrial swab and cytobrush to screen mares for endometritis. Mares (n=84) of reproductive age ranging 3-20 years of age had an endometrial swab (n=103) and/or followed by cytobrush (n=94) taken once in the same estrous cycle and placed in Amies media (n=172) or PBS (n=25). Aerobic culture was performed in 139 of those samples. An endometrial biopsy was taken on a subset of mares (n=46) and graded by a board-certified veterinary pathologist. The media in which the cytobrushes and swabs were stored was used in a bead-based multiplex assay (Luminex Corp. Austin, TX) to quantify concentration of the following equine specific biomarkers: IFN-α, IFN-γ, IL-1β, IL-4, IL-10, IL-17, sCD14, TNF-α, CCL2, CCL3, CCL5, and CCL11. A Shapiro-Wilk test was used to check normality, followed by a Wilcoxon testperformed in JMP Pro 16 (Cary, NC) to assess for significant differences in inflammatory marker concentrations between healthy mares and those with either cytology-diagnosed endometritis (≥1 neutrophil/hpf), positive bacterial culture, or poor biopsy scores (IIB or III on the Kenney-Doig scale). IFN-γ (P≤0.02) and IL-17 (P≤0.04) were significantly increased in mares with poor biopsy scores in both swab and cytobrush compared to mares with biopsy I or IIA. Mares with inflammation on cytology showed increased concentration of IL-1β (P≤0.05) and decreased levels of IFN-γ (P≤0.02) and CCL5 (P≤0.03) in swab. Mares with intrauterine fluid on ultrasound had increased IL-17 (P≤0.05) on cytobrush. Bacterial growth was detected in 62/139 samples, with 31 of those being gram-positive. sCD14 and TNF-α tended to be increased in cytobrush samples from mares with a positive culture, and with gram-positive bacteria compared to gram-negative, respectively (P=0.06). Cytokine levels significantly differed between cytobrush and swab for IFN-γ (P≤0.004) with increased levels for cytobrush, likely due to the higher amount of cellular exfoliation. Several inflammatory markers show promise as an ancillary screening test for equine endometritis. In conclusion, as equine reproductive immunology expands as a field, our understanding of patterns of inflammatory cytokines and chemokines can allow for new diagnostic techniques for endometritis in mares.

Interleukin-1 beta treatment alters prostaglandin-related mRNA expression and cytokine abundance in equine endometrial culture

Interleukin-1β (IL-1β) is a proinflammatory cytokine that modulates the immunoinflammatory response and induces prostaglandin synthesis. IL-1β has a variety of roles in the endometrium during inflammation, decidualization, pregnancy, and post-partum. It can cause changes in endometrial gene expression and may affect cytokine, chemokine, and prostaglandin-related pathways. The relationship between IL-1β, prostaglandins, and downstream inflammatory responses in the equine uterine environment is not fully known. Our objective was to determine the effect that IL-1β has on prostaglandin-related gene expression, as well as cytokine and chemokine abundance, in endometrial cell culture. Light breed mares (n=8) were examined daily by transrectal ultrasound until ovulation. An endometrial biopsy was collected at day 11 post ovulation, and used for primary endometrial cell culture. Cells were cultured in DMEM with 10% FBS. At 80% confluence, cells were either not supplemented or supplemented with: 100 nM Progesterone (Prog); 1 nM Estradiol-17β (Est); or 100 nM Prog and 1 nM Est (P+E). After 24 hours, cells were treated with recombinant equine IL-1β at either 10 ng/mL (Low) or 1000 ng/mL (High), or 0.2% DMSO as an untreated control. Twenty-four hours post-treatment, supernatant was collected, and a bead-based multiplex assay was used to quantify concentration of twelve cytokines and chemokines. Total RNA was extracted from the cells and mRNA expression was evaluatedfor 10 prostaglandin-related genes, using the ΔCT method with ACTB as the reference gene. Data analysis was performed using JMP Pro 16, using a restricted maximum likelihood model with treatment and supplementation as factors, and mare as a random factor. Treatment with IL-1β High resulted in increased levels of CCL2, CCL5, and CCL11 (p<0.001) in cell culture supernatant, as well as increased mRNA expression of PTGS2 (p<0.02), regardless of supplementation. Cells supplemented with Prog or P+E and treated with IL-1β High had increased expression of IL1R1, IL1RAP, PTGS1, and PTGES compared to non-supplemented cells (p<0.05). IL-1β High treatment increased LiF expression in cells supplemented with Prog or P+E compared to untreated cells (p<0.05). IL-1β likely acts to modulate inflammatory pathways in the endometrium, altering chemokine levels and activating expression of PTGS2, leading to changes in downstream prostaglandin synthesis and other related pathways. Progesterone appears to increase expression of several target genes when compared to non-supplemented cells. Progesterone has been shown to be involved in the suppression of several inflammatory pathways, and investigation into the ovarian steroid regulation of genes and proteins in the equine endometrium is warranted. Further studies on the effect of IL-1β on molecular pathways and prostaglandin regulation could lead to a greater understanding of the uterine environment in the pregnant and non-pregnant mare.

High serum AMH inhibits pathological growth of the low biomass endometrial microbiome.

Although host microbiome play a role in both hormonal status and fertility, this issue has not yet been clarified. Since the endometrium is a sterile tissue, it is accepted that microbiota does not grow under normal conditions. The aim of the study was to reveal the characteristics of endometrial microbiota according to serum AMH levels in women with implantation failure. Forty-five women aged 20-30 years with two or more implantation failures were included in the study. They were divided into 3 groups according to their serum AMH values: Group 1 -AMH <1.3 ng/ml; Group 2 - AMH between 1.3-2.6 ng/ml; Group 3 - AMH >2.6 ng/ml. Twenty-two healthy fertile women who were the same age as the infertile group and applied for cervical smear screening were accepted as the control group. Following the embryo transfer, the tip of the catheter was inserted into the transport medium under sterile conditions. Sowing was carried out by touching the tips of the catheter to the blood agar medium. After the evaluation of the petri dishes at the end of 48 hours of incubation, colonies were stained with Gram stain. Microorganisms in the colonies were identified with the Vitek-2 device according to their gram-staining characteristics and their antibiograms were made. A negative correlation was detected between low AMH values and the microbiome detection rates in endometrial cultures. In patients with low serum AMH levels, the chance of endometrial microbiota growth was higher in the endometrial culture medium. The most common bacteria were found to be MSSA, MRKNS and lactobacillus. Clinical pregnancy rates were found to be significantly higher in the group with high AMH levels. As AMH levels increased, positive flora detection rates decreased, while clinical pregnancy rates increased. Low serum AMH level increases the rate of positive endometrial microbiome in culture and decreases clinical pregnancy rates.

THE USE OF ENDOMETRIAL CULTURE FOR TARGETED TREATMENT OF ENDOMETRITIS IN PATIENTS EXPERIENCING INFERTILITY AND RECURRENT PREGNANCY LOSS

To evaluate whether endometrial culture in addition to endometrial biopsy reduces time to clear chronic endometritis (CE) and fewer endometrial biopsies in patients experiencing infertility and recurrent pregnancy loss (RPL).

P-297 Regulation and functions of PGRMC1 in the human endometrium and in endometriosis

Abstract Study question What is the role of PGRMC1 in the human endometrium (especially in tissue remodeling) and its potential contribution to the development of endometrial diseases? Summary answer In primary endometrial stromal cell culture, PGRMC1 downregulation modifies expression of genes implicated in extracellular matrix remodeling as well as in cell migration and invasion. What is known already It has been suggested that Progesterone Receptor Membrane Component-1 (PGRMC1) is involved in gynecological pathologies. In particular, Pgrmc1 conditional KO in the mouse female reproductive tract induced subfertility and spontaneous development of endometrial cysts. Moreover, siRNA-mediated downregulation of PGRMC1 expression influenced TNF-α-induced activity of matrix metalloproteinase-9 (MMP-9) in cytotrophoblast cells, suggesting that PGRMC1 is a regulator of MMPs. In addition to playing a major role in the physiological breakdown of the human endometrium at menstruation, MMPs are strongly suspected to favor the pathogenesis of endometriosis lesions. Study design, size, duration Not applicable Participants/materials, setting, methods We compared transcriptomes obtained by RNA-sequencing from distinct primary endometrial stromal cell (ESC) cultures transfected with one of two specific siRNA targeting PGRMC1 mRNA or with a control siRNA. Expression changes for selected genes were confirmed by RT-qPCR. Migration and invasion capacities of the cells were also studied using Transwell assay. Finally, immunocytochemistry was performed on these primary cultures to localize the presence of PGRMC1 as well as the canonical progesterone receptor (PR). Main results and the role of chance RNA-sequencing data converged to show that the reduction of PGRMC1 expression by the specific siRNA significantly increased (up to 3-fold) the expression of several genes involved in the extracellular matrix (ECM) remodeling, as well as in the processes of migration and invasion. These changes were reproduced by RT-qPCR on other distinct primary endometrial stromal cultures isolated from other patients. In parallel, Transwell assays showed no significant changes in the migration and invasion capacities of the primary endometrial stromal cell cultures previously invalidated for PGRMC1 by transfection of the cells with a specific siRNA by comparison with a control siRNA. However, immunocytochemistry studies on these primary endometrial stromal cells showed that progesterone receptor (PR) labeling was restricted to cell nuclei, suggesting a constitutively activated PR in these cultures. Limitations, reasons for caution The absence of changes in migration and invasion properties of primary ESC cultures observed by Transwell assay could be due to the fact that the progesterone receptor seems to be constitutively activated in these cultures potentially causing a repressive effect on the migration and invasion capacity of the cells. Wider implications of the findings The modification of expression of genes implicated in ECM remodeling, and cell migration and invasion after PGRMC1 downregulation in primary ESC cultures is interesting in the context of endometriosis in which reduction of PGRMC1 expression could improve the migration and invasion capacities of lesions to implant into the host tissue. Trial registration number Not applicable

Uterine diagnostics: what is the best sample?

Uterine disease, particularly endometritis, is commonly encountered in equine reproduction and has major implications for mare fertility and breeding economics. A thorough reproductive examination, combined with selection of appropriate uterine diagnostics and interpretation of those findings, is essential in the work up of cases of mare infertility. Addition of endometrial cytology and alternative culture methods, such as the low-volume lavage and uterine biopsy, will increase the clinician's diagnostic sensitivity and specificity. The practitioner's approach to the investigation of uterine disease in the field and required techniques are described in this review article.

Variances in the Expression Profile of the EMT-Related Genes in Endometrial Cancer Lines In Vitro Study.

The aim of the study was to evaluate the variances in the expression pattern of mRNAs and miRNAs related to the EMT in the Ishikawa (histological grade 1; G1), EC-1A (histological grade 2; G2), and KLE (histological grade 3; G3) cell cultures under cisplatin treatment. Endometrial cancer cell lines were exposed to 75.22 mg (an average concentration of the drug used in patients with endometrial cancer) for 12.24 and 48 hours in comparison to the untreated cells (control). The molecular analysis included: extraction of total RNA, microarray analysis (mRNA and miRNA), RTqPCR, and the ELISA assay. Out of 226 mRNAs associated with the EMT, the number of mRNAs differentially expressed in endometrial cancer cell cultures treated with cisplatin compared to a control culture was as follows: Ishikawa line - 87 mRNAs; EC-1A - 84 mRNAs; KLE - 71 mRNAs (p<0.05). The greatest changes in the Ishikawa line treated with the drug compared to the control were noticed for mRNA STAT1 TGFβ1, SMAD3, FOXO8, whereas in EC-1A they were mRNA TGFβ1, BAMBI, SMAD4, and in KLE mRNA COL1A1, FOXO8, TGFβ1. The analysis also showed that miR-106a, miR-30d, miR-300 are common for all cell lines used in this experiment. Cisplatin changes the expression profile of genes associated with EMT in endometrial cancer cell lines. It seems that the expression pattern of TGFβ1 might be a promising, supplementary molecular marker of the effectiveness of cisplatin therapy. The analysis showed that miR-30d, miR-300, and miR-106a are involved in the regulation of the expression of EMT-related genes.

O-026 Advanced Glycation Endproducts: A new player in obesity related infertility

Abstract text Globally, 39% of the adult population is overweight or obese, with the prevalence of obesity following an upward trajectory over the recent decades (WHO). Up to 30% of women of reproductive age in Western countries are obese before conception, and obese women experience higher rates of infertility and pregnancy complications than lean women; however, the mechanisms underpinning obesity-related infertility are poorly understood. Advanced Glycation Endproducts (AGEs) are a proinflammatory modification of proteins exposed to sugars, formed through the Maillard reaction. AGEs are elevated four-fold in the uterine fluid of obese, infertile women, compared to lean. AGEs equimolar to those in the obese microenvironment negatively impact the functions of endometrial epithelial and stromal cells, and adhesion and invasion of trophoblast cells, reducing the potential for successful maternal-fetal interactions (Antoniotti et al., 2018). This research further investigated preimplantation embryo development and endometrial cell functions in the presence of AGEs equimolar to those in obese uterine fluid. Altered local environments in very early life can set offspring up for a lifetime of health or disease (DoHAD); thus, uterine AGEs may contribute to the prevalence of non-communicable disease in children of obese parents. Preimplantation mouse embryos were cultured in vitro with AGEs equimolar with uterine fluid concentrations from lean and obese women, and their development and implantation potential assessed. “Obese” AGEs did not impact the proportion of embryos reaching blastocyst stage by day 4, but significantly reduced the proportion of blastocysts hatching by day 5 (P &amp;lt; 0.01). AGEs equimolar with the obese uterine environment detrimentally impacted trophectoderm formation and function: reduced trophectoderm cell number (P &amp;lt; 0.01), reduced outgrowth on fibronectin (indicative of reduced implantation potential, (P &amp;lt; 0.01), but did not increase cell apoptosis (TUNEL assay). RAGE antagonism, but neither metformin nor antioxidants, improved trophectoderm cell number. Thus, obesity-associated AGEs link obesity and reduced fertility through poor placentation potential of embryos (Hutchison et al, 2020). Endometrial epithelial cell function was examined in the presence of lean and obese concentrations of AGEs. Obese AGEs significantly reduced the rate of proliferation (xCelligence real time cell analysis) of the endometrial epithelial cell line ECC-1 versus lean AGEs (P = 0.04). Antioxidants successfully restored the rate of proliferation in the presence of obese AGEs (P = 0.7 versus lean AGEs). Subsequently, human endometrial epithelial organoid culture was utilised as a more physiologically relevant experimental paradigm. When cultured as organoids, primary endometrial epithelial cells were functionally responsive to obesity-associated AGEs, expressing both RAGE and TLR4. The morphology of organoids in culture was not impacted by the presence of obese AGEs versus lean; however, the proliferation of epithelial cells retrieved from organoid culture was altered by obese AGEs versus lean. Obese AGEs also increased the secretion of proinflammatory CXCL16 versus vehicle control (P = 0.04) while increased secretion of other proinflammatory cytokines and chemokines including TNFa approached significance in the presence of obese AGEs. As the inflammatory milieu is altered in the uterine fluid of infertile women, elevated AGEs may promote an infertile endometrial inflammatory environment. AGEs link obesity and reduced fertility, being detrimental to preimplantation embryo development and endometrial cell function when present at concentrations equal to those in obese uterine fluid. Antioxidants and RAGE antagonism provide beneficial effects to cell function in the presence of obesity-associated AGEs. This research provides evidence supporting AGEs as a factor contributing to obesity related infertility, and as an emerging frontier for reproductive health. Clinically, reduction of uterine AGEs may improve fertility for obese couples wishing to conceive. Antoniotti et al (2018). Hum Rep. 33(4), 654-665. PMID: 29471449 Hutchison et al (2020). RBMO. 41(5), 757-766. PMID: 32972872

ENDOG Impacts on Tumor Cell Proliferation and Tumor Prognosis in the Context of PI3K/PTEN Pathway Status.

Simple SummaryThe PI3K/AKT pathway is involved in cell survival and proliferation. Molecular aberrations and/or hyperactivation of the PI3K-PTEN-AKT axis are frequent in distinct cancer types such as endometrial carcinoma, the most common type of cancer of the female genital tract, and chronic lymphocytic leukemia (CLL), a mature B-cell neoplasm depending on B-cell receptor (BCR) activity, which induces chronical activation of this pathway. The mitochondrial nuclease ENDOG was found to influence PI3K/AKT activity in somatic cells. Our aim was to assess the value of ENDOG gene silencing to block cancer cell proliferation and to evaluate the relevance of ENDOG as prognostic marker. In vivo, in vitro and in silico experiments show that Endog/ENDOG silencing blunts proliferation of tumor cells dependent on high p-AKT/low PTEN activity and that ENDOG has prognostic value in specific cancer types.EndoG influences mitochondrial DNA replication and is involved in somatic cell proliferation. Here, we investigated the effect of ENDOG/Endog expression on proliferation in different tumor models. Noteworthy, ENDOG deficiency reduced proliferation of endometrial tumor cells expressing low PTEN/high p-AKT levels, and Endog deletion blunted the growth of PTEN-deficient 3D endometrial cultures. Furthermore, ENDOG silencing reduced proliferation of follicular thyroid carcinoma and glioblastoma cell lines with high p-AKT expression. High ENDOG expression was associated with a short time to treatment in a cohort of patients with chronic lymphocytic leukemia (CLL), a B-cell lymphoid neoplasm with activation of PI3K/AKT. This clinical impact was observed in the less aggressive CLL subtype with mutated IGHV in which high ENDOG and low PTEN levels were associated with worse outcome. In summary, our results show that reducing ENDOG expression hinders growth of some tumors characterized by low PTEN activity and high p-AKT expression and that ENDOG has prognostic value for some cancer types.

Immunomodulatory Factors in Primary Endometrial Cell Cultures Isolated from Cancer and Noncancerous Human Tissue-Focus on RAGE and IDO1.

Background: Immune modulatory factors like indoleamine 2,3-dioxygenase 1 (IDO1) generating kynurenine (Kyn) and receptor for advanced glycation end-products (RAGE) contribute to endometrial and cancer microenvironment. Using adequate experimental models is needed to learn about the significance of these molecular factors in endometrial biology. In this paper we study IDO1 activity and RAGE expression in the in vitro cultured primary human endometrial cells derived from cancerous and noncancerous tissue. Methods: The generated primary cell cultures from cancer and noncancerous endometrial tissues were characterized using immunofluorescence and Western Blot for expression of endometrial and cancer markers. IDO1 activity was studied by Kyn quantification with High Performance Liquid Chromatography with Diode Array Detector. Results: The primary cultures of endometrial cells were obtained with 80% success rate and no major genetic aberrations. The cells retained in vitro expression of markers (mucin MUC1 and HER2) or immunomodulatory factors (RAGE and IDO1). Increased Kyn secretion was associated with cancer endometrial cell culture in contrast to the control one. Conclusions: Primary endometrial cells express immune modulatory factors RAGE and IDO1 in vitro associated with cancer phenotype of endometrium.

Intrauterine Blood Plasma Platelet-Therapy Mitigates Persistent Breeding-Induced Endometritis, Reduces Uterine Infections, and Improves Embryo Recovery in Mares.

Microorganisms, including pathogenic or opportunistic bacteria and fungi, may gain access to the uterus during breeding, and infectious endometritis plays a major role in equine subfertility. This study aimed to assess the post-breeding inflammatory response, endometrial culture, and embryo recovery of mares susceptible to persistent breeding-induced endometritis (PBIE) treated with plasma-rich (PRP) or -poor (PPP) plasma. Mares (n = 12) susceptible to PBIE had three cycles randomly assigned to receive intrauterine infusions of lactate ringer solution (LRS, control), or autologous PRP or PPP pre- (−48 and −24 h) and post-breeding (6 and 24 h). Mares were bred with fresh semen from one stallion. Intrauterine fluid accumulation (IUF) and endometrial neutrophils were assessed every 24 h up to 96 h post-breeding. Uterine cytokines (Ilβ, IL6, CXCL8, and IL10) were evaluated before (0 h), 6, and 24 h post-breeding, and endometrial culture three and nine days after breed. Embryo flushing was performed 8 days post-ovulation. Data were analyzed with mixed model, Tukey’s post-hoc test, and multivariate regression. PRP treatment reduced endometrial neutrophils, post-breeding IUF, and pro-inflammatory cytokines when compared to control-assigned cycles, but not significantly different than PPP. Controls had a significantly higher percentage of positive bacterial cultures (33%) in comparison to PRP-assigned cycles (0%), whereas cycles treated with PPP were not significantly different from the other groups (25%). The PRP-assigned cycles had significantly greater embryo recovery rates (83%) than the control (33%), though not significantly different than PPP (60%). Plasma infusion reduced the duration and intensity of the post-breeding inflammatory response and improved embryo recovery in mares susceptible to PBIE. Platelets incrementally downregulate PBIE and appear to have a dose-dependent antimicrobial property.

Antibiotic therapy versus no treatment for chronic endometritis: a case-control study

To demonstrate the infectious nature of chronic endometritis (CE) in an inductive way by comparing the results of germ-oriented antibiotic therapy vs. no treatment in women with CE. Retrospective, nonconcurrent case-control study. Tertiary hysteroscopic center in a university teaching hospital. Sixty-four consecutive women with CE who received antibiotic therapy (Group A) compared with a historical group of 64 patients with CE who refused antibiotic therapy (Group B). CE was diagnosed through hysteroscopy, histology, and immunohistochemistry for CD138. Patients in both groups were tested for CE twice to evaluate the cure rate after antibiotic therapy (Group A) or no treatment (Group B). For patients with persistent disease, antibiotic therapy was repeated up to 3 times. Antibiotics were chosen based on endometrial culture (with antibiogram). The primary outcome was to compare the cumulative cure rate of CE (defined as the percentage of patients without CE at the test of cure) between groups. Among Group A, 20 patients (31.25%) experienced CE resolution after 1 antibiotic cycle, an additional 20 patients (31.25%) after 2 antibiotic cycles, and 12 patients (19.35%) after 3 antibiotic cycles. In 12 cases (18.75%), CE was persistent after 3 cycles of antibiotics. The cure rate of CE in Group A after 1 cycle of antibiotics was significantly higher than that of Group B (32.25% vs. 6%). Similarly, the cumulative cure rate was considerably higher in Group A vs. Group B (81.3% vs. 6%). Notably, the number of positive cases decreased significantly with all techniques between the first and second evaluation, whereas at the third evaluation, there was a statistical decrease only with hysteroscopy and CD138+ cell count but not with histology. The cumulative number of cases of CE diagnosed at hysteroscopy was significantly higher than histology and immunohistochemistry. Our study demonstrated the superiority of antibiotic therapy compared with no treatment for CE cure. Accordingly, the infectious nature of CE is inferred.

A Natural Xenogeneic Endometrial Extracellular Matrix Hydrogel Toward Improving Current Human in vitro Models and Future in vivo Applications.

Decellularization techniques support the creation of biocompatible extracellular matrix hydrogels, providing tissue-specific environments for both in vitro cell culture and in vivo tissue regeneration. We obtained endometrium derived from porcine decellularized uteri to create endometrial extracellular matrix (EndoECM) hydrogels. After decellularization and detergent removal, we investigated the physicochemical features of the EndoECM, including gelation kinetics, ultrastructure, and proteomic profile. The matrisome showed conservation of structural and tissue-specific components with low amounts of immunoreactive molecules. EndoECM supported in vitro culture of human endometrial cells in two- and three-dimensional conditions and improved proliferation of endometrial stem cells with respect to collagen and Matrigel. Further, we developed a three-dimensional endometrium-like co-culture system of epithelial and stromal cells from different origins. Endometrial co-cultures remained viable and showed significant remodeling. Finally, EndoECM was injected subcutaneously in immunocompetent mice in a preliminary study to test a possible hypoimmunogenic reaction. Biomimetic endometrial milieus offer new strategies in reproductive techniques and endometrial repair and our findings demonstrate that EndoECM has potential for in vitro endometrial culture and as treatment for endometrial pathologies.

Steroid hormones and human choriogonadotropin influence the distribution of alpha6-integrin and desmoplakin 1 in gland-like endometrial epithelial spheroids

In human glandular endometrial epithelial cells, desmosomal and adherens junction proteins have been shown to extend from a subapically restricted lateral position to the entire lateral membrane during the implantation window of the menstrual cycle. Similarly, a menstrual cycle stage-dependent redistribution of the extracellular matrix adhesion protein α6-integrin has been reported. These changes are believed to be important for endometrial receptiveness and successful embryo implantation. To prove the hypothesis that steroid hormones and human choriogonadotropin can induce the redistribution of these adhesion molecules, we used the human endometrial cell line Ishikawa in a 3D culture system. Gland-like spheroids were grown in reconstituted basement membrane (Matrigel™). The lumen-bearing spheroids were treated for 2 or 4 days with ovarian steroids or human choriogonadotropin and then assessed by immunofluorescence microscopy. In addition, human endometrial biopsies were obtained from patients, who were in therapy for assisted reproductive technology, and were examined in parallel. Lateral redistribution of the desmosomal plaque protein desmoplakin 1 was observed in the spheroids treated either with progesterone, medroxyprogesterone acetate or human choriogonadotropin. Furthermore, the extracellular matrix adhesion protein α6-integrin showed an increased lateral membrane localization upon gestagen stimulation in the 3D culture system. The results of this study demonstrate that the 3D endometrial Ishikawa cell culture might be suited as an experimental model system to prove the effect of hormonal changes like those occurring during the window of implantation.

Relation between chronic endometritis and recurrent miscarriage.

IntroductionGrowing interest has focused on the relation between chronic endometritis (CE) or asymptomatic inflammation of the endometrium and recurrent miscarriage (RM).The aim of the study was to assess the relation between CE and RM and the accuracy of hysteroscopy in diagnosing CE.Material and methodsOne hundred and ten (110) women with unexplained RM were included in this study. Participants were subjected to diagnostic hysteroscopy for uterine cavity, and endometrium evaluation. The diagnosis of CE during hysteroscopic examination was based on CE-related hysteroscopic signs (micro-polyps, stromal edema, and/or hyperemia). At the end of hysteroscopy, an endometrial biopsy was taken from participants for culture, and immunohistochemical (IHC) staining. Collected data were analyzed to assess the relation between CE and RM and the accuracy of hysteroscopy in diagnosing CE.ResultsThe prevalence of CE in women with RM was 31.8% using CE-related hysteroscopic signs, while it was 38.2% using IHC staining and endometrial cultures (p = 0.4). CE-related hysteroscopic signs had 64.1% sensitivity, 85.9% specificity, 71.4% positive predictive value (PPV), 81.3% negative predictive value (NPV), and 78.2% overall accuracy in diagnosing CE. Most cases of CE (> 81%) were caused by Mycoplasma and common pathogens.ConclusionsThe prevalence of CE in women with RM was 31.8% using CE-related hysteroscopic signs, while it was 38.2% using IHC staining and endometrial cultures. CE-related hysteroscopic signs had 64.1% sensitivity, 85.9% specificity, 71.4% PPV, 81.3% NPV, and 78.2% overall accuracy in diagnosing CE. Most cases of CE (> 81%) were caused by Mycoplasma and common pathogens.

Opaque Polyploid Cells in Ishikawa Endometrial Cultures Are Capable of Forming Megamitochondria, Organelles Derived from the Adaptation of Fused Mitochondria Whose Capacity to Develop Gaseous Vacuoles Suggests CO&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; Retention and Hypoxic Metabolism

Opaque polyploid cells capable of forming megamitochondria are a constant feature in colonies of Ishikawa endometrial epithelia, accounting for approximately 5% - 10% of the cells. Opaque cells appear to communicate with other opaque cells via membrane extensions and with other cells in a colony by extra-cellular vesicles. Opaque cells form first as rectangular structures, somewhat larger than surrounding monolayer cells. The cells eventually round up, remaining in the colony for 20 or more hours before detaching. The most unusual characteristic of Ishikawa opaque cells is their capacity to form mitonucleons, megamitochondria that surround aggregated chromatin. This paper reviews evidence that adaptations resulting in megamitochondria include a loss of the capacity for oxidative phosphorylation leaving the adapted megamitochondria reliant on metabolism such as reductive carboxylation.