Eutopic Endometrium Of Women Research Articles

Исследование экспрессии генов в эутопическом эндометрии женщин с эндометриоидными кистами яичников

Исследование экспрессии генов в эутопическом эндометрии женщин с эндометриоидными кистами яичников

KRAS Activation and over-expression of SIRT1/BCL6 Contributes to the Pathogenesis of Endometriosis and Progesterone Resistance

Endometriosis is an inflammatory condition that is associated with progesterone resistance and cell proliferation, resulting in pain, infertility and pregnancy loss. We previously demonstrated phosphorylation of STAT3 in eutopic endometrium of infertile women with this disorder leading to over-expression of the oncogene BCL6 and stabilization of hypoxia-induced factor 1 alpha (HIF-1α). Here we report coordinated activation of KRAS and over-expression of Sirtuin 1 (SIRT1), a histone deacetylase and gene silencer, in the eutopic endometrium from women with endometriosis throughout the menstrual cycle. The mice with conditional activation of KRAS in the PGR positive cells reveal an increase of SIRT1 expression in the endometrium compared to control mice. The expression of progesterone receptor target genes including the Indian Hedgehog pathway genes are significantly down-regulated in the mutant mice. SIRT1 co-localizes with BCL6 in the nuclei of affected individuals and both proteins bind to and suppress the promoter of GLI1, a critical mediator of progesterone action in the Indian Hedgehog pathway, by ChIP analysis. In eutopic endometrium, GLI1 expression is reduced in women with endometriosis. Together, these data suggest that KRAS, SIRT1 and BCL6 are coordinately over-expressed in eutopic endometrium of women with endometriosis and likely participate in the pathogenesis of endometriosis.

Identification of global transcriptome abnormalities and potential biomarkers in eutopic endometria of women with endometriosis: A preliminary study.

The etiology and pathophysiology of endometriosis remain unclear. The aim of the current study was to identify a candidate pathogenic gene, as well as potential biomarkers of endometriosis using messenger RNA (mRNA) sequencing (mRNA-seq). Twenty-three eutopic endometria from women with endometriosis and 20 endometria from control subjects were investigated. Eight eutopic endometria and five normal endometria were selected for mRNA-seq. Differentially expressed genes (DEGs) were identified and functional analysis was conducted. Validation of certain DEGs was performed in the remaining cases and control subjects by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A total of 72 DEGs (66 upregulated and 6 downregulated) were identified in samples from women with endometriosis and compared with the control subjects. High DEGs included those involved in various functions, such as extracellular matrix (ECM) remodeling, angiogenesis, cell proliferation and differentiation. Enriched by these DEGs, 100 Gene Ontology terms were identified as significantly important, particularly ‘ECM’ and ‘endogenous stimulus’. Validation using RT-qPCR indicated that matrix metallopeptidase 11, dual specificity phosphatase 1, Fos proto-oncogeneand serpin family E member 1 were significantly upregulated and adenosine deaminase 2 was significantly downregulated in the eutopic endometrium of patients with endometriosis. The identified DEGs may be involved in the pathogenesis of endometriosis and may be potential biomarkers in the eutopic endometrium. The current study provides a comprehensive, but preliminary insight for elucidating the mechanisms of endometriosis, which require further in-depth studies for confirmation.

Perfil diferencial de transcritos em endométrio eutópico de mulheres inférteis com endometriose e controles durante a janela de implantação

ObjectivesMolecular alterations in the eutopic endometrium of women with endometriosis may be involved in the endometriosis‐related infertility. This study aimed to compare the differentially expressed genes (DEG) in eutopic endometrium of infertile women with endometriosis, infertile controls (IC; male and/or tubal factor) and fertile controls (FC) through RNA‐Seq. Material and methodsEndometrial biopsies were obtained from 17 patiens (6 infertile women with endometriosis, 6 IC and 5 FC) during the implantation window. The RNA was extracted and the RNA‐Seq was performed at a HISEQ 2500 Illumina Platform, high output, paired end. Standardization and differential expression were conducted in the statistical R environment using DESeq2 package. ResultsThe groups IC and FC were similar. No DEG has been identified comparing CF and endometriosis groups. Five DEGs (SCUBE1, CCL20, LGALS9C, TRIM 29 e WNT11) were identified in the advanced endometriosis (EIII/IV) group, and 1 (KANSL1‐AS1) in the initial endometriosis (EI/II) group compared to FC. Two DEGs (KANSL1‐AS1 and VGLL3) were identified by comparing EI/II and EIII/IV groups. ConclusionsThese data suggest that the eutopic endometrium of infertile women with endometriosis, especially those with advanced disease, may be molecularly different from those of fertile women during the implantation window.

Matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of matrix metalloproteinases (TIMP-1) and transforming growth factor-β2 (TGF-β2) expression in eutopic endometrium of women with peritoneal endometriosis

The prevalence of endometriosis among reproductive age women is 7-17%; however, these figures reach 20-50% in patients suffering from infertility. Matrix metalloproteinases (MMPs) activity is thought to be particularly essential in the early phases of endometriosis development. Any changes in the equilibrium between MMPs activity and their tissue inhibitors (TIMPs) could be potentially harmful, promoting endometriosis development. The aim of this study was to investigate whether the MMP-2, MMP-9, TIMP-1 or TGF-B2 expression in eutopic endometrium from women with early endometriosis differ when compared with healthy subjects. The results were referred to the serum progesterone levels. Endometrial biopsy was taken from 42 patients (18 in the study group, 22 in thecontrol group) at the time of hysteroscopy for routine histology and for RT-PCR procedures. Comparison of the quantity of gene products was performed with a programme for densitometry and compared to GADPH product, which was a reference value. The obtained results did not reveal any statistical difference in endometrial expression of MMP-2, MMp-9, TIMP-1, and TGF-β2 or serum progesterone level between women with endometriosis and without visible signs of this illness. Despite the lack of statistical differences, it was observed that both examined metalloproteinases expressed a tendency to higher gene expression in the eutopic endometrium of women with endometriosis. However, both TIMP-1 and TGF-β2 expressions had the same tendency - higher values in endometriosis patients.

Pregnancy outcome in women with endometriosis achieving pregnancy with IVF.

Are women with endometriosis who conceive with IVF at increased risk of preterm birth? Women with endometriosis who conceive with IVF do not face an increased risk of preterm birth. The eutopic endometrium of women with endometriosis has been repeatedly shown to present molecular and cellular alterations. On this basis, it has been hypothesized that pregnancy outcome may be altered in affected women. However, to date, available evidence from epidemiological studies is scanty and conflicting. Data tended to be partly consistent only for an increased risk of preterm birth and placenta previa. Retrospective matched case-control study of women achieving an IVF singleton pregnancy progressing beyond 12 weeks' gestation. Women achieving IVF singleton pregnancies that progressed beyond 12 weeks' gestation at two infertility units were reviewed. Cases were women with a history of surgery for endometriosis and/or with a sonographic diagnosis of the disease at the time of the IVF cycle. Controls were women without current or past evidence of endometriosis who were matched to cases by age (±6 months), type of cycle (fresh or frozen cycle) and study period. Male factor and unexplained infertility were the most common diagnoses in the control group. Two hundred and thirty-nine women with endometriosis and 239 controls were selected. The main outcome of the study was the rate of preterm birth (birth<37 weeks' gestation) regardless of the cause. Secondary analyses were performed for the most common obstetrical complications. The rate of preterm birth was similar in the two study groups (14% and 14%, respectively, p=0.89). The rate of live birth and the incidence of hypertensive disorders, gestational diabetes, small and large for gestational age newborns and neonatal problems also did not differ. In contrast, placenta previa was more common in women with endometriosis than controls (6% versus 1%, respectively; p=0.006): The adjusted odds ratio was 4.8 (95% confidence interval: 1.4-17.2). As for all observational studies, confounders cannot be totally excluded. Moreover, the retrospective study design exposes the findings to some inaccuracies. For example, the independent role of adenomyosis could not be reliably assessed because this diagnosis is complex and would necessitate a prospective recruitment. Second, the selection of controls may also be a matter of concern because some affected women may have been erroneously included in this group. Third, even if the sample size is significant, it is insufficient for robust subgroup analyses. Finally, it is mandatory to point out that our conclusions are valid for IVF pregnancies only, and specific data from properly designed studies are required to support any inference for natural pregnancies. The results of our study suggest that women with endometriosis conceiving with IVF can be reassured regarding the risk of preterm birth. The observed association with placenta previa requires further investigation and may open a new avenue of research. No external funding was used for this study. None of the authors have any conflict of interest to declare.

Morphological features eutopic endometrium in women with adenomyosis and chronic pelvic pain syndrome.

Background. Chronic pelvic pain syndrome is one of the most frequent pathologies that reduce quality of woman`s life. The main reason for its occurrence is gynecological diseases (extragenital endometriosis, adenomyosis, uterine leiomyoma). But role of morphological changes in the endometrium for the occurrence of these diseases is still unexplored. Objective. Assess the links between morphological and immunohistochemical (markers ER, PGR, Ki-67, VEGF, COX-2, NF) features and the level of pain on VAS. Methods. 10 samples were divided according to expression of investigated marker in glands (ER, PGR, Ki-67, VEGF, COX-2, NF) and in stroma (ER, PGR, Ki-67). Rating staining immunohistochemical markers was conducted in at least 10 fields of view. Statistical processing of the data included non­parametric tests. Results. We observed heterogeneous expression of ER, PGR, more importat role of high level expression PRG in stroma, increased proliferative activity in glands is focal in the most cases. Expression of VEGF is often higher, when the level of VAS is up. Conclusion. Formation of the CPP and adenomyosis is associated with hyperplastic processes of endometrium , increased proliferative activity , and severe expression of COX - 2 ( p &lt; 0.05 ).

MiR-196a overexpression activates the MEK/ERK signal and represses the progesterone receptor and decidualization in eutopic endometrium from women with endometriosis.

Do microRNAs (miRNAs) contribute to aberrant progesterone receptor (PGR) expression in the eutopic endometrium of women with endometriosis? SUMMARY ANSWER: miR-196a upregulates MEK/ERK signalling, mediating a downregulation of PGR expression in the eutopic endometrium of women with minimal or mild endometriosis. Implantation failure is strongly suggested as an underlying cause for the observed infertility in minimal or mild endometriosis. Progesterone resistance, which is mainly caused by aberrantly expressed progesterone receptor in the eutopic endometrium, is considered as a key factor of decreased endometrial receptivity; thus far, epigenetics, but not miRNA, has been shown to affect PGR expression in the endometrium. Microarray analysis was used to analyse the eutopic endometrium. The differential expression of miR-196a was validated. Bioinformatics analysis predicted that miR-196a targets the 3'-untranslated region (UTR) of the PGR. The relationship between the miR-196a level and PGR expression was studied and the role of the MEK/ERK signal pathway was investigated. Total RNA was extracted from eutopic endometrium samples in three infertile women with mild/minimal endometriosis and three disease-free control subjects. The miRNA and mRNA expression levels were analysed by microarray analysis. The miR-196a expression was validated by qRT-PCR [endometriosis (n=22) and control (n=20)], while functional analysis utilised in vitro transfection of endometrial stromal cells (ESCs), induction of decidualization of ESCs, and luciferase reporter assays in 293 T cell lines. A total of 66 dysregulated miRNAs and 357 dysregulated mRNAs were screened by microarray analysis. miR-196a and P-MEK/P-ERK were both found to be significantly upregulated in the eutopic endometrium in patients with mild/minimal endometriosis. PGR and PGR-B mRNA were inhibited by miR-196a overexpression and upregulated by miR-196a inhibition. Luciferase reporter failed to confirm the target regulation of miR-196a on PGR. Transfection of ESCs with a miR-196a mimic led to an increase in the P-MEK/P-ERK protein levels, decrease in the PGR protein levels, and atypical decidualization. Following miR-196a inhibition, the P-MEK/P-ERK protein was downregulated and the PGR protein was upregulated. Inhibition of P-MEK/P-ERK also increased PGR expression. Data are presented in Supplementary Tables SI and SII. This study focused on the role of miR-196a, and therefore does not involve other miRNAs; hence, it is possible that other miRNAs may also be responsible for progestin resistance in endometriosis. Our data revealed altered miRNA expression and activated MEK/ERK signalling in the eutopic endometrium in minimal or mild endometriosis. We showed that the miR-196a level is associated with reduced expression of PGR isoforms through MEK/ERK, suggesting that miR-196a and MEK/ERK are both potential biomarkers of endometriosis. These results provide a novel approach to target the mechanisms behind progesterone resistance in endometriosis. This research was supported by the National Natural Science Foundation of China (No. 81370693). The authors have no conflicts of interest.

Estradiol Reduces Connexin43 Gap Junctions in the Uterus during Adenomyosis in Cows.

Adenomyosis is defined as the presence of glandular foci external to the endometrium of the uterus, either in the myometrium or/and perimetrium, depending on the progress of this dysfunction. To date, we showed that steroids secretion and prolactin expression and proliferative processes are disturbed during uterine adenomyosis in cows. During endometriosis in eutopic endometrium in women, gap junctions are down regulated. The transmembrane gap junction protein, connexin (Cx43) is necessary for endometrial morphological, biochemical and angiogenic functions. The aim of this study is recognition of adenomyosis etiology by determination of the role of Cx43 in this process. Immunolocalization and comparison of Cx43 mRNA and protein expression in healthy (N=9) and adenomyotic uterine tissue (N=9), and Cx43 mRNA expression (real time PCR) in uterine stromal - myometrium co-culture under 24-hour stimulation with 17-beta estradiol (10-7M) isolated from healthy (N=5) and adenomyotic (N=5) cows were determined. Cx43 was localized in healthy and adenomyotic uteri. mRNA and protein expression was down-regulated in uterine tissue in adenomyotic compared with healthy cows (p<0.05). Estradiol stimulated Cx43 mRNA expression in myometrial cell culture and co-culture of stromal and myometrial cells in adenomyotic compared with healthy cows (p<0.05). In summary, down-regulation of Cx43 expression in the junction zone might play an important role in pathogenesis of adenomyosis. Estradiol modulates gap junctions during adenomyosis.

Endometrial BCL6 Overexpression in Eutopic Endometrium of Women With Endometriosis.

The objective of this study was to examine B-cell CLL/lymphoma 6 (BCL6) expression in human eutopic endometrium across the menstrual cycle in women with and without endometriosis and to establish a cutoff for future studies. This design was a series of case-control studies in tertiary University teaching hospitals. We examined BCL6 expression by messenger RNA and immunohistochemically in prospectively collected samples in both the proliferative (P) and the secretory phases. BCL6 is minimally increased in the mid-secretory phase of the menstrual cycle compared to the P phase in normal patients. BCL6 protein expression was significantly higher in the secretory phase of patients with endometriosis (n = 29) versus fertile controls without endometriosis at laparoscopy (n = 20; P < .0001). Normal fertile controls (n = 28) recruited for endometrial biopsy also had low levels of secretory phase BCL6 expression compared to women with unexplained infertility (UI; n = 119). A receiving-operator characteristic analysis of these data revealed an area under the curve of 94% (95% confidence interval 85%-100%; P < .0001) with an HSCORE cutoff of 1.4 to differentiate cases with and without endometriosis. Using this cutoff value, BCL6 was positive in 88% of cases with UI. Laparoscopic examination of a subset of 65 patients confirmed abnormalities in 98% of cases; 61 (93.8%) were found to have endometriosis, 3 (4.6%) with hydrosalpinx, and 1 (1.5%) with a normal pelvis. These data suggest that BCL6 is a promising candidate as a single diagnostic biomarker for detection of endometriosis in women with otherwise UI and may be associated with endometrial dysfunction, including progesterone resistance.

Dysregulated infertility-related genes in women with endometriosis

A number of mechanisms may be responsible for endometriosis-related infertility, such as inflammatory microenvironment, altered immune response, reduced/absent adhesion molecules and oxidative stress. Many genes play important roles in these processes and their expression levels may be altered in women with endometriosis. A prospective case-control study was conducted to assess expression of dysregulated genes in the eutopic endometrium of women with endometriosis compared to that of women without endometriosis. Eutopic endometrial biopsies from 29 women (N=16 with endometriosis; N=13 without endometriosis) were obtained by Pipelle curette between 1 January 2015 and 31 December 2015 in Akanksha IVF centre. Paralleled gene expression profiling using high density oligonucleotide microarrays was applied to investigate dysregulated genes in eutopic endometrium of women with endometriosis compared to women without endometriosis. Data analysis was conducted by GeneSpring version 4.0.4. T-test using P value >0.05 and a fold-change expression >1.5 was applied to assess statistical significance. Selected dysregulated genes were randomly chosen and validated by RT-PCR. This study has shown that in the eutopic endometrium from women with endometriosis infertility-related genes were significantly dysregulated. Particularly, genes involved in inflammation, immune response and oxidative stress were upregulated whereas genes involved in the expression of adhesion molecules were downregulated. Dysregulation of genes involved in infertility likely contributes to decreased pregnancy rates in women with endometriosis. Increased inflammation in the eutopic endometrium may contribute to increased cytokine production and altered immune environment leading to oxidative stress thereby impairing embryo implantation. Moreover, downregulation of adhesion molecules in the eutopic endometrium may affect the endometrial receptivity in women with endometriosis. An improved understanding of the underlying molecular infertility mechanisms may aid in identifying candidate genes for managing endometriosis-associated infertility.Tabled 1Table 1: Significantly dysregulated genes in eutopic endometrium of women with endometriosis (n=16)Gene SymbolGene nameFunctionFold ChangeCOL16A1Collagen, type XVI, alpha 1Adhesion-1.5ITGA6Integrin, alpha 6Adhesion-1.6GZMBGranzyme BInflammation3.05CXCL14Chemokine (C-X-C-motif) ligand 14Inflammation10.7ERO1LERO1-likeoxidative stress1.65HIST1H2ACHistone cluster 1, H2acoxidative stress2.29MAFFv-Mafmusculoaponeuroticfibrosarcoma oncogene homolog Foxidative stress2.26MAP3K5mitogen-activated protein kinase kinase kinase 5oxidative stress3.15CD68CD68 moleculeImmune response1.53CD58CD58 moleculeImmune response1.69TLR4Toll-like receptor 4Immune response1.75 Open table in a new tab

Low prevalence of endometrial biopsies containing nerve fibers in women with and without endometriosis

To test the hypothesis that nerve fiber density is higher in eutopic endometrium of women with endometriosis compared to controls without endometriosis in samples collected in a university fertility center.

Aberrant Endometrial DNA Methylome and Associated Gene Expression in Women with Endometriosis.

Endometriosis is an estrogen-dependent, progesterone-resistant disorder largely derived from retrograde transplantation of menstrual tissue/cells into the pelvis, eliciting an inflammatory response, pelvic pain, and infertility. Eutopic endometrium (within the uterus), giving rise to pelvic disease, displays cycle-dependent transcriptomic, proteomic, and signaling abnormalities, and although its DNA methylation profiles dynamically change across the cycle in healthy women, studies in endometriosis are limited. Herein, we investigated the DNA methylome and associated gene expression in three phases of the cycle in eutopic endometrium of women with severe endometriosis versus controls, matched for ethnicity, medications, smoking, and no recent contraceptive steroid use. Genome-wide DNA methylation and gene expression were coassessed in each sample. Cycle phase was determined by histology, serum hormone levels, and unsupervised principal component and hierarchical cluster analyses of microarray data. Altered endometrial DNA methylation in endometriosis was most prominent in the midsecretory phase (peak progesterone), with disruption of the normal pattern of cycle-dependent DNA methylation changes, including a bias toward methylation of CpG islands, suggesting wide-range abnormalities of the chromatin remodeling machinery in endometriosis. DNA methylation changes were associated with altered gene expression relevant to endometrial function/dysfunction, including cell proliferation, inflammation/immune response, angiogenesis, and steroid hormone response. The data provide insight into epigenetic reprogramming and steroid hormone actions in endometrium contributing to the pathogenesis and pathophysiology of endometriosis.

Comprehensive study of angiogenic factors in women with endometriosis compared to women without endometriosis

ObjectiveEndometriosis is a benign gynaecological disease, affecting women during their reproductive years. Angiogenesis represents a crucial step in the pathogenesis of endometriosis, because endometriotic lesions require neovascularization. In this study several angiogenesis-related genes have been studied in the context of endometriosis. Some of the analyzed angiogenic factors as well as their interactions were studied the first time regarding a possible association with endometriosis. Study designThis case-control study consisted of 205 biopsies of 114 patients comprising 61 endometriosis patients and 53 control patients. Among them in 29 cases paired samples were obtained. VEGFA, VEGFR2, HIF1A, HGF, NRP1, PDGFB, FGF18, TNFα, TGFB2, EPHB4, EPO and ANG mRNA expression was analyzed by qRT-PCR in ectopic tissue samples, in eutopic endometrium of women with and without endometriosis, and in unaffected peritoneum of women with and without endometriosis. ResultsVEGFR2, HIF1A, HGF, PDGFB, NRP1 and EPHB4 are overexpressed in ectopic lesions compared to eutopic tissues. VEGFR2, HGF, PDGFB, NRP1, and EPHB4 showed highest mRNA levels in peritoneal implants, in contrast HIF1A showed the highest expression in ovarian endometriomas. Correlation analyses of angiogenic factors in ectopic lesions revealed the strongest associations between VEGFR2, PDGFB, and EPHB4. We further showed a significant upregulation of VEGFR2, HIF1A and EPHB4 in eutopic endometrium of women with endometriosis compared to that of controls and a trend towards upregulation of HGF. Additionally, a significant downregulation for HIF1A, HGF and EPHB4 was observed in unaffected peritoneal tissues of women with endometriosis compared to controls. ConclusionWe identified new genes (EPHB4 and NRP1) that may contribute to angiogenesis in endometriosis beside known factors (VEGFA, VEGFR2, HIF1A, HGF, and PDGFB). Correlation studies revealed the putative importance of EBHB4 in association with endometriosis. Our analyses support preliminary reports that angiogenic factors seem to be differently expressed in peritoneal implants, ovarian endometriomas and deep infiltrating endometriosis. Our observation that angiogenic factors are differently expressed in the unaffected peritoneum of women with endometriosis compared to women without endometriosis underlines the importance of the peritoneum in the establishment of endometriosis.

Protein Inhibitor of Activated STAT3 (PIAS3) Is Down-Regulated in Eutopic Endometrium of Women with Endometriosis.

Endometriosis is a major cause of chronic pelvic pain and infertility. Activation of STAT3 appears central to the inflammatory phenotype of eutopic endometrium in women with endometriosis. However, the molecular mechanism by which this occurs remains unknown. Our objective is to determine how STAT3 activity is regulated in endometriosis. Protein inhibitor of activated STAT3 (PIAS3) is a negative regulator of STAT3 activity. We examined the levels of PIAS3 in endometrium from women with and without endometriosis using Western blot analysis and immunohistochemistry. Levels of PIAS3 are significantly lower, in contrast with phosphorylation of STAT3, in women with endometriosis compared to women without endometriosis. Furthermore, induction of endometriosis in the baboon showed a significant reduction of PIAS3 expression during the progression of the disease. Interferon-γ (INFγ) reduces PIAS3 protein levels and increases phospho-STAT3 levels through CXCL10 in endometrial cells, Ishikawa, and 12Z cells. These results suggest that attenuation of PIAS3 causes aberrant activation of STAT3 in endometriosis, leading to inflammatory changes that may impair fertility or cause pain.

Altered expression of progesterone receptor isoforms A and B in human eutopic endometrium in endometriosis patients

Recent data implicate an altered expression of progesterone receptor isoform A (PR-A) and B (PR-B) in the endometrium of endometriosis patients. This prospective exploratory study aimed to precisely determine the PR-A and PR-B expression using immunohistochemical techniques in eutopic endometrium of women with endometriosis compared with disease-free women throughout the menstrual cycle.All symptomatic patients underwent laparoscopy for the diagnosis of endometriosis and histological confirmation of the disease (EO) whereas controls were proven disease-free (CO). In CO samples (n=10) an increased expression of PR-A and PR-B during the proliferative to early secretory phase and a decreased expression of both receptor isoforms during the mid to late secretory phase was ascertained in accordance with previous studies.In patients with endometriosis (n=16) no cycle dependent pattern of PR-A and PR-B expression was identified in contrast to patients without endometriosis. Moreover, in EO samples a huge variety of inter- and intra-individual differences in PR-A and PR-B expression were detected.These data provide further evidence that dysregulation of the PR-A and PR-B expression might contribute to the pathophysiology of endometriosis.

Ten-Eleven Translocation Genes are Downregulated in Endometriosis.

Our previous whole genome expression analysis of endometriomas suggested dysregulation of the ten-eleven translocation genes (TET1, TET2, and TET3), involved in converting 5- methylcytosine to 5-hydroxymethylcytosine (5-hmC). The objective of this study was to validate the expression of TET genes in ectopic and eutopic endometrium and in primary cultures of human endometrial stromal fibroblasts (HESF) during in vitro decidualization and to quantify 5-hmC levels in patients with endometriosis. Blood, eutopic endometrium, and endometriotic tissues were collected at time of gynecologic surgery. HESF cultures were created from eutopic endometrium of women without (HESF-CONTROL) and with endometriosis (HESF-ENDO) and underwent in vitro decidualization. Genomic DNA from blood and tissues underwent quantification of the absolute amount of 5-hmC using ELISA. The expression of TET1, TET2, and TET3 was decreased in endometriosis compared to non-endometriosis control eutopic endometrium. Surprisingly, the global amount of 5-hmC was higher in ectopic endometrium than control eutopic endometrium, while genomic DNA from blood of women with endometriosis contained statistically significantly less 5-hmC than women without endometriosis. Expression of TET1, TET2, and TET3 was decreased in non-decidualized HESFENDO. Upon in vitro decidualization, control HESF showed decreased expression of TET3, while decidualized HESF-ENDO showed no statistically significant change in expression of TET1, TET2, or TET3. These results indicate that the TET genes are downregulated in ectopic endometrium and in HESF-ENDO, and suggest for the first time that TET genes play a role in endometriosis. High global amounts of 5-hmC in endometriotic tissues suggest unique epigenetic regulation in these tissues.

Elevated phosphatase of regenerating liver 3 (PRL-3) promotes cytoskeleton reorganization, cell migration and invasion in endometrial stromal cells from endometrioma

Is phosphatase of regenerating liver-3 (PRL-3) associated with increased motility of endometriotic cells from endometrioma? Elevated PRL-3 promotes cytoskeleton reorganization, cell migration and invasion of endometrial stromal cells (ESCs) from endometrioma. Overexpression of PRL-3 is associated with cancer cell migration, invasion and metastatic phenotype. Primary human ESCs were isolated from eutopic endometrium of women without endometriosis (EuCo, n = 10), with histologically proven endometrioma (EuEM, n = 19) and from the cyst wall of ovarian endometriosis (OvEM, n = 26). The expression of PRL-3 in ESCs derived from EuCo, EuEM and OvEM at different phases of menstrual cycle were compared. The protein and mRNA levels of PRL-3 were examined by western blot and RT-qPCR, respectively. ESCs from OvEM were transfected with/without short hairpin RNA (shRNA) or small interfering RNA (siRNA). Additionally, a plasmid-mediated delivery system was used to achieve PRL-3 overexpression in ESCs from EuEM. The cellular distribution of F-actin and α-tubulin were examined by immunocytochemistry. Cell motility was evaluated by a transwell migration/invasion assay. The protein and mRNA levels of PRL-3 are significantly elevated in ESCs from OvEM compared with EuCo and EuEM. The expression of PRL-3 was not altered between proliferative phase and secretory phase in ESCs from all groups. Knockdown of PRL-3 significantly modified the distribution of F-actin and α-tubulin cytoskeleton, inhibited cell migration and invasion. Endogenous inhibition of PRL-3 attenuated the expression of Ras homolog gene family members A and C (RhoA, RhoC), Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) and matrix metalloproteinase (MMP) 9, but not MMP2 in ESCs from OvEM. Additionally, overexpression of PRL-3 in ESCs from EuEM up-regulates cell migration and invasion, and increases the expression of RhoA, RhoC, ROCK1 and MMP9. Lack of in vivo animal studies is the major limitation of our report. Our results should be further confirmed in a larger cohort of patients and extended to include eutopic and ectopic endometrium from patients with peritoneal endometriosis at different stages of the disease. Our study describes that elevated expression of PRL-3 contributes to the cell motility of ESCs from endometrioma. The results emphasize the importance of metastatic-related factor PRL-3 in the pathogenesis of endometrioma. This work was supported by National Natural Science Foundation of China (No. 81170546) and Zhejiang Medicine Science and Technology Projects (No. Y13H040003). The authors declare no conflict of interest.

Increased uNK Progenitor Cells in Women With Endometriosis and Infertility are Associated With Low Levels of Endometrial Stem Cell Factor.

Uterine natural killer (uNK) cells play a significant role in successful human pregnancy. Having previously demonstrated uNK cell progenitors in human endometrium, we hypothesized that abnormal uNK cell maturation contributes to infertility in women with endometriosis. We aimed to characterize uNK cells at different developmental stages in women with and without endometriosis and to investigate possible mechanisms to explain any differences. We characterized uNK cell development in women with and without endometriosis using flow cytometry, protein array and in vitro experiments. We found increased proportions of uNK cells at developmental stages 1 and 2 in endometrium from women with endometriosis (n = 36; mean = 21.2%) when compared with healthy fertile women (n = 9; mean = 7.0%). Protein array analysis revealed significantly lower levels of stem cell factor (SCF) in the eutopic endometrium of women with endometriosis when compared to healthy women. Addition of SCF to endometrial progenitor cells in vitro restored uNK cell maturation. We have shown that women with endometriosis have low levels of endometrial SCF, which we hypothesize contributes to abnormal maturation of local uNK cell populations. This defect may also compromise embryo implantation and hence contribute to endometriosis-associated infertility. SCF replacement may be a new therapeutic approach.

The Clinical Usefulness of Endometrial Biopsy for the Diagnosis of Endometriosis in the Work up of Women with Infertility = الفائدة السريرية من استخدام خزعة بطانة الرحم لتشخيص بطانة الرحم المهاجرة عند النساء المصابات بالعقم

Background & Aims: The eutopic endometrium in women with endometriosis harbor small nerve fibres. This study aimed to explore the clinical usefulness of office endometrial biopsy to detect endometrial nerve fibres as diagnostic test for endometriosis in the work up of women with infertility and no endometrioma. Material & Methods: A cohort of 28 Jordanian women with infertility and without endometriomas was prospectively followed up for 1 year. All women underwent clinical evaluation and an office endometrial biopsy. Those with positive nerve fibres using immunohistochemistry with PGP9.5 were considered endometriosis patients and underwent planned laparoscopy while those with negative results were considered endometriosis free and underwent assisted reproductive techniques. All patients were followed up at 3, 6 and 12 months. Results: Eighteen women had positive nerve fibres and underwent planned laparoscopy. At laparoscopy 14 patients had endometriosis, two cases had no visible endometriosis (sensitivity 88.9 (16/18). In women with endometriosis and pain symptoms (n- 12) the density of nerve fibres was higher than in those with endometriosis and no pain symptoms (n-6) 2.1 and 0.6 per mm2 respectively (P = 0.005). Women with negative results at biopsy (n-10) underwent either ovulation induction or IVF. In both groups at one year follow up the pregnancy rate was similar (60%). Conclusions: The proposed method may be useful in the work up of patients with infertility and without endometrioma and would allow gynecologists to triage infertility patients and plan for a potentially valuable laparoscopy and will also shorten the delay of endometriosis diagnosis.