PR-A Expression Research Articles

Exploring the Role of MicroRNAs in Progesterone and Estrogen Receptor Expression in Endometriosis.

Background/Objectives: Patients with endometriosis still respond poorly to progestins due to progesterone resistance associated with microRNAs (miRNAs). The aim of this study was to investigate the expression of selected miRNAs, estrogen receptor (ER)α, ERβ, progesterone receptor (PR)-A and PR-B and to determine the target genes of upregulated miRNAs in endometriosis. Methods: In this study, 18 controls, 18 eutopic and 18 ectopic samples were analysed. Profiling and validation of miRNAs associated with functions of endometriosis were performed using next-generation sequencing (NGS) and qRT-PCR. At the same time, the expression of ERα, ERβ, PR-A and PR-B was also determined using qRT-PCR. Target prediction was also performed for miR-199a-3p, miR-1-3p and miR-125b-5p using StarBase. Results: In this study, NGS identified seven significantly differentially expressed miRNAs, of which six miRNAs related to the role of endometriosis were selected for validation by qRT-PCR. The expression of miR-199a-3p, miR-1-3p, miR-146a-5p and miR-125b-5p was upregulated in the ectopic group compared to the eutopic group. Meanwhile, ERα and ERβ were significantly differentially expressed in endometriosis compared to the control group. However, the expressions of PR-A and PR-B showed no significant differences between the groups. The predicted target genes for miR-199a-3p, miR-1-3p and miR-125b-5p are SCD, TAOK1, DDIT4, LASP1, CDK6, TAGLN2, G6PD and ELOVL6. Conclusions: Our findings demonstrated that the expressions of ERα and ERβ might be regulated by miRNAs contributing to progesterone resistance, whereas the binding of miRNAs to target genes could also contribute to the pathogenesis of endometriosis. Therefore, miRNAs could be used as potential biomarkers and for targeted therapy in patients with endometriosis.

How Progesterone Receptor Expression Impacts Platinum Sensitivity in Ovarian Clear Cell Carcinoma: Insights from Clinical and Experimental Perspectives.

Ovarian clear cell carcinoma (OCCC) is often considered a relatively platinum-resistant malignancy. The aim of this study was to explore the influence of progesterone receptor (PR) expression levels on platinum sensitivity and survival outcomes in people with OCCC. A retrospective analysis was conducted with 80 people with OCCC who underwent surgery followed by adjuvant chemotherapy. PR expression was assessed via immunohistochemical (IHC) staining and quantified using the H score. The platinum sensitivity and survival outcomes of patients with weak and strong PR expression were compared. Additionally, cisplatin viability and migration experiments were conducted with OCCC cell lines (ES-2 and TOV-21G) with varying PR isoform expressions. Among the 80 patients, 62 were classified as having platinum-sensitive disease, while 18 had platinum-resistant disease. The mean total PR H- score of platinum-sensitive tumors was significantly higher than that of platinum-resistant tumors (p = 0.002). Although no significant differences in progression-free and overall survival were observed between patients with high and low PR expression, those with high PR expression tended to have longer survival. While PR protein was only weakly detectable in ES-2 and TOV-21G cells, a transfection of the PR-A or PR-B gene resulted in a strong expression of PR-A or PR-B, which led to significantly reduced proliferation and migration in ES-2 and TOV-21G cells. Furthermore, overexpression of PR-A or PR-B enhanced cisplatin cytotoxicity in these cell lines. In conclusion, strong PR expression was associated with improved platinum sensitivity and survival outcomes, consistent with our experimental findings. The potential of PR as a tumor sensitizer to cisplatin in OCCC warrants further investigation.

Abstract 4390: IGFBP-6 is induced by progesterone to regulate progesterone receptor expression in breast cancer cells

Abstract Insulin-like growth factor binding protein-6 (IGFBP-6) is induced by progesterone (P4) in T-47D breast cancer cells. IGFBP-6 regulates insulin-like growth factor 2 (IGF2) by sequestering it to inhibit receptor binding and extend its extracellular half-life. IGF2 binds to insulin-like growth factor 1 receptor or insulin receptor A to promote breast cancer cell proliferation and survival. Progesterone is an ovarian steroid hormone which has complex roles in breast cancer through the actions of progesterone receptor A and B (PR-A and B). Progesterone antagonizes the actions of estradiol (E2), another steroid hormone which promotes proliferation and survival in breast cancer. T-47D cells treated with both P4 and E2 exhibit significantly reduced proliferation compared to treatment with E2 alone. To better understand the role of IGFBP-6 in progesterone signaling, T-47D cells which over-express IGFBP-6 were treated with P4, E2, or both. Overexpressing cells exhibited stronger antagonism compared to controls when co-treated with P4 and E2. RNA-interference was performed to knockdown IGFBP-6 induction in the presence of P4 and E2. Knockdown of IGFBP-6 in the presence of P4 and E2 decreases the antagonistic effects of P4. Expression of both PR-A and PR-B decreases by about 50% when IGFBP-6 is knocked down. These results indicate that IGFBP-6 increases PR expression in response to P4 stimulation. Citation Format: Francisco Javier Lariz, Pacha Botero, Kevin Houston. IGFBP-6 is induced by progesterone to regulate progesterone receptor expression in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4390.

Abstract 3981: Immune targeting progesterone receptor in hormone receptor positive breast cancer

Abstract Background: Hormone receptor positive (HR⁺) breast cancer (BC) is a subtype of BC that is characterized by expression of estrogen receptor (ER) and progesterone receptor (PR). A cornerstone treatment for this subtype of BC is endocrine inhibition therapy (EIT), in which estrogen and ER are suppressed using antagonists to suppress tumor growth. However, patients eventually obtain resistance to EIT. Metastatic HR⁺BC is the deadliest form of BC, with no cure. Current therapies currently only utilize the ER-axis, with no PR-targeted therapies. Little is known about the role of PR in these BCs, but we have found evidence for an immunosuppressive role for PR in HR⁺BC. Yet, it is unclear how PR mediates immunosuppression and if this axis can be therapeutically targeted. We hypothesized that PR confers local immunosuppression through the stimulation of immunosuppressive transcriptional activity and that we can immunologically target this axis using a PR vaccine. Experimental methods: To assess the immune impact of PR, we expressed the isoforms of PR (A, B and A+B) in mouse mammary cancer cells and assessed their capacity to elicit PR signaling. We assessed the impact of PR expression on T cell activity in targeting PR⁺ mammary tumors that express GFP, along with using GFP-specific JEDI CD8⁺ T cells. We explored the ability of PR to protect against GFP xenoantigen immune responses by engrafting PR⁺ mammary cells into immune competent and deficient mice. Lastly, we engineered 1st generation adenoviral vectors encoding mouse and human PR and utilized two antiprogestins (Mifepristone and Onapristone) in vivo. Using our vectors, we vaccinated BALB/c in the footpad and assessed PR-specific immunity using IFNγ ELISPOT. Results: Using PR signaling reporters, we found canonical PR signaling activity in the absence of progesterone (P4) by PR-A and in the presence of P4 by PR-B, but the most profound activation of PR signaling was seen through combined expression of PR-A and B. While expression of these genes did not confer striking cytotoxic T cell resistance in vitro, combination of PR-A+PR-B in 67NR cells permitted successful tumor engraftment in immune competent mice, despite xenoantigen expression. GFP expression did not alter tumor growth in immunodeficient mice, suggesting a key role in modulating local anti-tumor immunity. The use of either Mifepristone or Onapristone significantly suppressed tumor engraftment in immune competent mice in vivo. We found that an Ad-PR vaccine could elicit T cell responses against human PR-specific epitopes, a potential step towards immune targeting these BCs. Conclusions: We found that murine PR-A and PR-B can confer robust P4 signaling that is linked to an immunosuppressive phenotype in vivo. We developed a PR vaccine that elicited PR-specific T cell responses in vivo. We are currently examining the mechanisms that underlie PR-mediated immunosuppression and exploring the potential of PR vaccines in HR⁺BC. Citation Format: Andrea E. Wilson, Junping Wei, Gangjun Lei, Xiao-Yi Yang, Cong-Xiao Liu, Melissa Gajda, Tao Wang, Christy Hagan, Zachary C. Hartman. Immune targeting progesterone receptor in hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3981.

Assessment of Differential Expression of Progesterone Receptors A & B in Endometriosis Undergoing IVF Treatment: A Personalized Approach for Better IVF Success

Objective: Estrogen and Progesterone hormones play a pivotal role in the pathogenesis of Endometriosis. In the present study, relative quantification of PR-A and PR-B expression in eutopic endometrial tissue and endometriotic implants was done. By examining their expression profiles, we aim to gain deeper insights into the molecular mechanisms that can influence IVF success or failure in endometriotic patients. Our study also observed certain ART outcomes in both endometriosis and control groups, to evaluate the IVF success rate. Methods: This study was conducted at MHRT, a tertiary care center, and referral centers from other hospitals. The research encompassed an examination of 125 cases of infertility spanning from January 2019 to January 2023. Finally, biopsy samples were collected from 60 patients with endometriosis during laparoscopic procedures, while control biopsy samples were gathered from 18 patients who exhibited no signs of endometriosis lesions during the same laparoscopic procedures. The biopsy samples were then sent for Progesterone assay and later evaluated for ART outcomes. Results: It was observed from the results of our study that there was a significant downregulation of PR-A in stage III, and IV compared to Stage I and II. However, there was no significant change in PR-B expression in grades I and II, III and IV. Our study also observed certain ART outcomes in both endometriosis and control groups, revealing that women with endometriosis have lower chances of achieving pregnancy than women with other causes of infertility (95% CI, 22/60 vs. 12/18, p-value = 0.024). Conclusion: Our study results shed light on the complex role of progesterone isoforms in endometriosis. Progesterone receptor-A significant downregulation in severe cases challenges existing paradigms and opens new avenues for research.

P-321 increased expression of TGF-β1 contributes to the downregulation of progesterone receptor expression in the eutopic endometrium of infertile women with minimal/mild endometriosis

Abstract Study question Do immune cells regulate progesterone resistance and endometrial receptivity of eutopic endometrial stromal cells (ESCs) by transforming growth factor-β1 (TGF-β1)? Summary answer By activating TGF-β/Smad signaling in eutopic ESCs, elevated TGF-β1 from CD45+ immune cells could attenuate expression of progesterone receptor (PR), and further decrease endometrial receptivity. What is known already Endometriosis is a hormone-dependent disease related to impaired immunoregulation. We characterized the trascriptomic transformation of eutopic endometrium from patients with minimal/mild endometriosis and controls across the menstrual cycle. However, the regulatory mechanism of altered immune microenvironment on eutopic ESCs remains unclear. Study design, size, duration Single-cell RNA transcriptomes were profiled using 10x Genomics platform. Primary culture of eutopic ESCs was performed to explore the effects of TGF-β1 on the expression of Smad / PR, and the in vitro decidualization. Additionally, co-immunoprecipitation (Co-IP) was used to explore the direct interaction between Smad and PR. Participants/materials, setting, methods Endometrial tissue samples were obtained from twenty-four women who were laparoscopically confirmed minimal/mild endometriosis according to the revised American Fertility Society classification. Additionally, four patients who were laparoscopically endometriosis-free were enrolled as controls. All samples were collected in DMEM/F12 medium and processed in one of two ways, either for single-cell sequencing (n = 10) or primary cell culture (n = 14). Main results and the role of chance The results of single-cell RNA sequencing revealed that the percentage of CD45+ cells in eutopic endometrium was increase in both proliferative (26% vs 24.1%) and secretory (19.3% vs 8.3%) stages. Overexpression of TGF-β1 of CD45+ cells was observed in eutopic endometrium of endometriosis in both proliferative and secretory stages. We found attenuate expression of PRB (p = 0.026) and PRA (p = 0.678) after using TGF-β1 in eutopic ESCs by western blot. Similarly, qRT-PCR results showed that the mRNA level of PR (p＜0.001), PRB (p = 0.003) and HOXA10 (p＜0.001) decreased significantly after TGF-β1 treatment, but that increased (p＜0.01) after SB431542 treatment in the eutopic ESCs. Moreover, TGF-β1 has a negative effect on the in vitro decidualization of eutopic ESCs (p = 0.003). And the group with treatment of both TGF-β1 and SB435142 in eutopic ESCs showed significant decidual-like changes with increased prolactin level (p = 0.01). Co-IP showed no physical interaction between the PR and p-Smad3/Smad3 proteins. Limitations, reasons for caution This study focused on CD45+ immune cells and the cytokine, TGF-β1. Further in vivo and in vitro studies are needed to elucidate the specific types of immune cell and the mechanisms that behind endometrial receptivity decrease in endometriosis. Wider implications of the findings Our results demonstrate abnormal activation of CD45+ cells in eutopic endometrium with minimal/mild endometriosis. And overexpression of TGF-β1 was found by CD45+ cells. We found that TGF-β1 suppresses PR expression and decidualization in eutpoic ESCs. These results provide a novel insight into the mechanisms behind progesterone resistance in endometriosis. Trial registration number not applicable

Effect of peripheral neural stimulation with allopregnanolone on ovarian physiology.

Neuroactive steroids can rapidly regulate multiple physiological functions in the central and peripheral nervous systems. The aims of the present study were to determine whether allopregnanolone (ALLO), administered in low nanomolar and high micromolar concentrations, can: (i) induce changes in the ovarian progesterone (P4) and estradiol (E2) release; (ii) modify the ovarian mRNA expression of Hsd3b1 (3β-hydroxysteroid dehydrogenase, 3β-HSD)3β-, Akr1c3 (20α-hydroxysteroid dehydrogenase, 20α-HSD), and Akr1c14 (3α-hydroxy steroid oxidoreductase, 3α-HSOR)); and (iii) modulate the ovarian expression of progesterone receptors A and B, α and β estrogenic receptors, luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). To further characterize ALLO peripheral actions, the effects were evaluated using a superior mesenteric ganglion-ovarian nervous plexus-ovary (SMG-ONP-O) and a denervated ovary (DO) systems. ALLO SMG administration increased P4 concentration in the incubation liquid by decreasing ovarian 20α-HSD mRNA, and it also increased ovarian 3α-HSOR mRNA expression. In addition, ALLO neural peripheral modulation induced an increase in the expression of ovarian LHR, PRA, PRB, and ERα. Direct ALLO administration to the DO decreased E2 and increased P4 concentration in the incubation liquid. The mRNA expression of 3β-HSD decreased and 20α-HSD increased. Further, ALLO in the OD significantly changed ovarian FSHR and PRA expression. This is the first evidence of ALLO's direct effect on ovarian steroidogenesis. Our results provide important insights about how this neuroactive steroid interacts both with the PNS and the ovary, and these findings might help devise some of the pleiotropic effects of neuroactive steroids on female reproduction. Moreover, ALLO modulation of ovarian physiology might help uncover novel treatment approaches for reproductive diseases.

High Expression of PR-A and Low Expression of PR-B is Correlated with Inflammation in Endometrioma Cases

BACKGROUND: Progestin therapy has been commonly used in endometriosis. The regulation of progesterone receptors B (PR-B) greatly affects the success rate of therapy in cases of endometriosis. The presence of tumor necrosis factor (TNF)-a in endometriosis triggers PR-B hypermethylation, decreasing PR-B expression and PR-B/A ratio that induce progesterone resistance. It may also occur in endometrioma. Studies regarding the distribution of PR-A and PR-B with TNF-a expression in endometriosis with endometrioma tissue samples has not been elucidated well. Therefore, this study was conducted to measure and compare the distribution of PR-A and PR-B expression, and to assess the effect of PR-B/A ratio on TNF-a in endometrioma and benign cysts.METHODS: A cross-sectional study was conducted by collecting paraffin blocks of endometriomas and benign cysts as controls, from patients undergoing surgery at Dr. Sardjito Hospital, Yogyakarta. Immunohistochemistry was performed to assess the expressions of PR-A, PR-B, TNF-a and PR-B/A ratio, to compared differences between endometriomas and benign cysts.RESULTS: Twenty-three endometrioma and 22 benign cyst tissue samples were collected. The mean PR-B expression and PR-B/A ratio were found to be lower in endometriomas than benign cysts, and mean expression of PR-A and TNF-a in endometriomas was higher than in benign cysts. However, there were no significant correlations between the expression of PR-A, PR-B, PR-B/A ratio, and TNF-a with endometriosis severity.CONCLUSION: In endometrioma cases, the expression of PR-A and TNF-a was higher, while the expression of PR-B and PR-B/A ratio was lower. However, there was no significant relationship between the ratio of PR-B/A and TNF-a.KEYWORDS: progesterone receptor, tumor necrosis factor-alpha, endometriosis, endometrioma, benign cyst, ovarian cyst

Evaluation of Estrogen Alpha Receptor (ER-Α) and Progesterone-A Receptor (PR-A) Expression in the Oviducts and Uterus of Indonesian Local Rabbits after Induced by Several Method of Pseudo-Pregnancy

The objective of this study was to determine the expression of ER-A and PR-A in the oviduct and uterus of pseudo-pregnant rabbits after induced by several induction methods of pseudo-pregnancy. This study used 30 local rabbits, aged 1 - 1.5 years, body weight of 2.8 - 3.0 kg and clinically healthy. Rabbits were divided into 6 groups (n = 5), namely: K1 (negative control, given 1.5 mL physiological NaCl injection), K2 (positive control, given 100 IU eCG injection and mated with male rabbits, followed by 75 IU hCG injection 3 days later), K3 (given 100 IU eCG injection followed by injection of 75 IU hCG 3 days later), K4 (artificial copulation was performed by inserting a 1 cm long cotton bud into the rabbit’s vagina), K5 (ovulation induction by injection of 50 IU hCG) and K6 (ovulation induction by injection of 5 g GnRH). On the 8th day after treatment, rabbits were euthanized and their oviduct and uterus organs were taken for histological preparation and stained using the immunohistochemical staining. The expression of ER-A and PR-A was valued using the intensity score and analyzed using the Kruskal-Wallis test followed the Mann Whitney U test. The expression ER-A in the infundibulum, ampullary and isthmus of K1 was significantly different (p < 0.05) from K2 - K6 rabbits. The ER-A expression in the endometrium of uterine horn of K1 and K3 was significantly different with K2, K4, K5 and K6 rabbits (p < 0.05). Furthermore, PR-A expression in oviducts of K1 and K3 was lower than that in K2, K3, K5 and K6 (p < 0.05) and the expression in the endometrium, uterine glands and myometrium of K2 - K6 was higher than K1 rabbits (p < 0.05). In conclusion, the hormonal induction could be used to induce pseudopregnancy in local rabbits based on the ER-A and PR-A expressions in oviduct and uterus. HIGHLIGHTS Pseudo-pregnant rabbits are needed for research purposes in the field of reproduction Several methods of induction of pseudo-pregnancy in rabbits have been carried out in this study Pseudo-pregnancy induction cause changes in the concentration of reproductive hormones (progesterone and estrogen) These changes can be detected based on the presence of receptors for these two hormones (PRA and ERα) in reproductive tract of four groups of rabbits and the results were then evaluated GRAPHICAL ABSTRACT

An increased proportion of progesterone receptor A in peripheral B cells from women who ultimately underwent spontaneous preterm birth

The enormous challenge in unraveling the etiology of preterm birth (PTB) is to understand the complex interactions between gestational hormones, the immune system and reproductive tissues. PTB can be divided into spontaneous PTB (sPTB) and medically-indicated PTB, e.g. due to preeclampsia (PE) or HELLP syndrome. Progesterone (P4), important for establishment and maintenance of pregnancy, exerts anti-inflammatory effects. The impact of P4 on B cells and its support of maintaining maternal-fetal tolerance is widely unexplored. Therefore, we aimed to determine whether B cells express the progesterone receptor (PR) and to dissect a possible role of PR+ B cells in PTB.We found enhanced IL-6, IL-21 and TNF-α concentrations in maternal plasma in patients with sPTB and PE/HELLP compared to term delivery (TD), accompanied by enhanced PR-A expression by CD19+ B cells. In a second phase of the study, we recruited patients with imminent PTB (iPTB) and controls. Samples were collected at hospital admission and to a later time point, then divided into iPTB patients who delivered preterm and patients whose PTB was prevented. Within the group of iPTB patients, we observed very clear differences: enhanced levels of pro-inflammatory cytokines and increased percentages of PR-A+CD19+ B cells were found in iPTB patients that delivered preterm compared to patients who did not deliver preterm.We conclude that PTB is associated with the activation of an inflammatory pathway leading to the induction of PR-A by B cells. This might further trigger inflammation, result in the break of maternal-fetal tolerance and induce delivery.

Abstract 5951: Synergistic efficacy of sulforaphane and progesterone against endometrial cancer

Abstract Introduction: Worldwide, endometrial cancer (EC) is the most common gynecological malignancy with increasing rate of incidence and mortality. While progesterone-based hormone therapy is a well-accepted conservative therapeutic option for EC patients, loss of progesterone receptor (PR) in long-term treatment leading to hormonal resistance and disease recurrence is still an area of concern. We demonstrated potent anti-cancerous activity of sulforaphane against EC in vitro and in vivo and hypothesized that it will activate PR and overcome progesterone therapy limitations. The purpose of this study was to evaluate the interaction of sulforaphane and progesterone against EC. Methods: The in vitro synergistic interaction between sulforaphane and progesterone was investigated with isobologram analysis of plotting MTT metabolic viability assay results of EC cells treated with drugs individually and in combination using CompuSyn software. ATP, soft-agar colony formation and invasion assays were performed to evaluate the combined drug effects on cell viability, anchorage independent colony formation and invasion properties, respectively. Mitochondrial membrane potential and caspase-3 activity were measured by JC-1 and caspase-3 detection ELISA kit, respectively. Protein expression was accessed by western blot. Progesterone resistant EC cells were developed by treating cells with 30µM of progesterone for more than 3 months. Results: Sulforaphane demonstrated strong synergy or additive effects with progesterone in the EC cell lines tested. Sulforaphane in combination with progesterone significantly decreased levels of cellular ATP, anchorage-independent colony formation, invasion and mitochondrial membrane potential. Furthermore, combination of sulforaphane and progesterone increased caspase-3 activity as compared to individual drug or controls, suggesting induction of apoptosis. Western blot results demonstrated an increased expression of cleaved PARP, with reduced expression of total caspase-3 and PCNA in the combination groups as compared to control and individual drugs. Sulforaphane alone or in combination showed increased expression of PR-A while it had minimal effects on PR-B. Further, in all EC cell lines, the combination of sulforaphane and progesterone demonstrated upregulation of PR-A/PR-B ratio. The progesterone resistant cells demonstrated less sensitivity to progesterone as compared to parent cell lines, however it was found to be sensitive to sulforaphane. Conclusions: Sulforaphane demonstrated synergistic activity with progesterone leading to increased apoptosis and reduced proliferation. Furthermore, sulforaphane exhibited potent toxicity to progesterone resistance cells. Sulforaphane in combination with progesterone could overcome progesterone therapy limitation by re-activation of PR. Funding: Administrative Suppl (P30CA225520) and PHF (GRF00005456) Citation Format: Rajani Rai, Swati Choudhary, Doris M. Benbrook, Vishal Chandra. Synergistic efficacy of sulforaphane and progesterone against endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5951.

Новая стратегия поиска гормональной терапии пролиферативных процессов эндометрия у пациенток в постменопаузе

The limited efficacy of hormone therapy for endometrial proliferative process (EPP) in postmenopausal patients and its side effects on the immune system functionalities have not been studied in detail. Here we assess the feasibility of hormone therapy for EPP in postmenopausal patients through evaluation of estradiol and progesterone receptor gene expression in endometrial tissue and peripheral blood mononuclear cells (PBMC). The study enrolled 92 postmenopausal patients with EPP, including 37 pts with glandular-fibrous polyps, 7 pts with non-atypical endometrial hyperplasia (EH), 8 pts with atypical endometrial hyperplasia (AEH), 31 pts with moderately differentiated adenocarcinoma and 9 pts with highly differentiated adenocarcinoma. The PBMC isolates and endometrial samples were tested for ER⍺, ERβ, mER, PRA, PRB, mPR and PGRmC1 expression by reverse real time polymerase chain reaction (RT–PCR). Differential changes in PBMC receptor profiles upon in vitro exposure to progesterone or mifepristone were determined for patients with endometrial polyps and healthy women. The results indicate elevated expression of ERα, ERβ, PRA, PRB, mPR and PGRmC1 by endometrial tissues in EH and elevated expression of mER, ER⍺ and PRA by PBMC in AEH, apparently reflecting suppressed functionalities of monocytes, macrophages, Т-cells and natural killer cells. Unaltered expression of the studied genes by PBMC in endometrial adenocarcinoma may reflect the incrementing tumor autonomy. In vitro, mifepristone inhibited ER⍺, ERβ, mPR, PGRmC1, PRA and PRB expression in PBMC isolated from patients with endometrial polyps. We suppose that such effects can mitigate the negative influence of sex steroid hormones on immunocompetent cells.

T-Cadherin, E-Cadherin, PR-A, and ER-α Levels in Deep Infiltrating Endometriosis.

The goal of this study was to compare the T-cadherin, E-cadherin, progesterone receptor (PR), and estrogen receptor (ER) staining levels of deep infiltrating endometriosis (DIE) tissue, ovarian endometriomas and normal endometrial tissues in the same individuals. The tissue sections of both DIE nodule(s) and endometrioma(s) of 15 cases were examined. As a control group, normal endometrial tissue sections of 23 cases were examined. T-cadherin, E-cadherin, ER-α, and PR-A staining levels of DIE, endometrioma tissues, and endometrial tissues were compared immunohistochemically. H -score was calculated to compare the expression of T-cadherin, E-cadherin, ER-α, and PR-A in immunohistochemical staining based on the percentage of cells stained at each intensity level. T-cadherin, E-cadherin, ER, and PR H -score were lowest in DIE tissue and highest in endometrial tissue ( P <0.0001, <0.0001, <0.0001, and <0.0001, respectively). In correlation analysis, a positive correlation was found between T-cadherin, E-cadherin, PR, and ER H -score ( P <0.0001 for each). T-cadherin, E-cadherin, ER, and PR H -score were lowest in DIE tissue and highest in endometrium tissue. We think that examination of DIE tissue and endometrioma tissue from the same individual excludes the possibility of an effect due to different genetic and environmental factors from different individuals. With the help of this exclusion we showed that DIE and endometrioma have different biological properties.

Myocardial Aldosterone Receptor and Aquaporin 1 Up-Regulation Is Associated with Cardiomyocyte Remodeling in Human Heart Failure.

Background: Abnormal aldosterone signaling is a recognized source of cardiovascular damage. Its influence on cardiomyocyte structure, function, and hormonal receptors when associated with heart failure is still unreported. Methods: Twenty-six consecutive patients with heart failure (LVEF < 40%) and normal coronaries and valves underwent left ventricular endomyocardial biopsy (EMB) for evaluation of myocardial substrate. Biopsy samples were processed for histology, electron microscopy, immunohistochemistry, and Western blot analysis of myocardial aldosterone receptor and aquaporin-1 correlated with plasma aldosterone (AD) and renin activity (PRA). Eight patients with virus-negative inflammatory cardiomyopathy (ICM) had a control EMB after 6 months of immunosuppressive therapy and recovery of cardiac function with re-evaluation of cardiomyocyte structure and receptor expression. Results: EMB in addition to the diagnosis of myocarditis (15 cases), dilated cardiomyopathy CM (6), alcohol CM (2), and diabetic CM (3) showed vacuolar degeneration and cloudy swelling of cardiomyocytes corresponding at electron microscopy to ions and water accumulation into cytosol, membrane-bound vesicles, nucleus, and other organelles, and was associated with an increased AD, PRA, and myocardial expression of aldosterone receptor (2.6 fold) and aquaporin 1 (2.7 fold). In the 8 patients recovered from ICM, cardiomyocyte diameter reduced with disappearance of intracellular vacuoles and normalization of cytosol, nucleus, and cell organelles’ electron-density, along with down-regulation of aldosterone receptor and aquaporin-1. Conclusion: Human heart failure is associated with overexpression of myocardial aldosterone receptor and aquaporin-1. These molecular changes are paralleled by intracellular water overloading and cardiomyocyte swelling and dysfunction. Cardiac recovery is accompanied by down-regulation of hormonal receptors and normalization of cell structure and composition.

Differential regulatory effect of progesterone on the proliferation and apoptosis of uterine leiomyoma tissue explants and primary leiomyoma cell cultures.

ObjectiveThe growth of uterine leiomyomas is regulated by progesterone, although the underlying molecular mechanisms are not fully understood.MethodsPrimary leiomyoma cells were isolated by standard method from 16 samples of uterine leiomyoma tissue. Uterine leiomyoma explants and primary leiomyoma cell cultures were exposed to progesterone in concentrations of 0.01 µg/ml, 0.1 µg/ml and 1 µg/ml for 24 h. Cell apoptosis was assessed with Annexin V assays performed in cell cultures by flow cytometry. The expression of PR-A, PR-B, Ki67, Akt, ERK, PTEN and PPARγ mRNAs was estimated in cultured leiomyoma cells and tissue explants by real time RT-PCR.ResultsTreatment with progesterone promoted viability and proliferation of cultured leiomyoma cells in a dose-dependent manner. Low and high doses of progesterone decreased early apoptosis of leiomyoma cells. High concentrations of progesterone increased the number of living cells in Annexin V assays. High doses of progesterone increased the expression of Ki67 mRNA, while low doses increased the expression of PR-A mRNA in cultured leiomyoma cells and tissue explants. In cell cultures, low doses of progesterone increased the expression of PR-B mRNA and the expression of PTEN and PPARγ mRNAs in a dose-dependent manner. Exposure of leiomyoma tissue explants to progesterone led to increased expression of PR-B and ERK mRNAs in a dose-dependent manner.ConclusionsThe effects of progesterone on the apoptosis and proliferation of leiomyoma cells was dose-dependent and different in cell cultures and leiomyoma explants, possibly as a result of impacts derived from the tumor microenvironment.

Нарушения экспрессии эстрогеновых и прогестероновых рецепторов в период окна имплантации в эутопическом эндометрии женщин с бесплодием, ассоциированным с наружным генитальным эндометриозом

Study Objective: To study impaired expression of estrogen (ER) and progesteron (PR) receptor isoforms during the implantation window in eutopic endometrium of infertile women associated with endometriosis genitalis externa (EGE). Study Design: open perspective comparative study. Materials and Methods. The study included 32 patients with endometriosis-associated infertility and implantation failures within the scope of assisted reproductive technology programme (study group), and 33 fertile women with EGE rAFS (stages I to IV) (comparison group). We performed morphological and immunohistochemical examination of endometrium biopsy samples (pipelle biopsy) during an expected implantation window. Study Results. Eutopic endometrium of women with EGE and infertility during the implantation window demonstrated statistically higher (р &lt; 0.05) expression of ERα (19-fold), ERβ (2.3-fold) in stroma and PR-B in glands (1.7-folds) as compared to fertile women win EGE with lower PR-A expression in stroma (2.5-fold). Conclusion. The morphofunctional condition of endometrium and its receptive properties during the implantation window in women with endometriosis-associated infertility reflect the nature of ER (α and β) and PR (А and В) isoforms expression, the abnormalities underlying the endometrium receptivity disorders, with clinical manifestations being absent pregnancy. Keywords: endometriosis-associated infertility; implantation window; endometrium receptivity

THE VALUE OF THE EXPRESSION OF PROGESTERONE RECEPTORS (PR-A AND PR-B) AS PREDICTORS OF RECURRENCE OF ENDOMETRIAL HYPERPLASIA AFTER THERAPY WITH PROGESTINS

The aim of the study. Improving diagnostics and predicting the effectiveness of pathogenetic treatment with progestins of endometrial hyperplasia without atypia, based on the study of the expression of progesterone receptors (PR-A and PR-B) and their ratio in endometrial tissues.Materials and methods. A prospective study was performed in 40 patients of reproductive age with abnormal uterine bleeding, who were diagnosed with endometrial hyperplasia without atypia based on histological examination of endometrial tissue. The control group consisted of 15 patients in whom after treatment there were histological changes to the endometrium of the secretory type. Material for morphological and immunohistochemical studies was obtained by scraping the uterine cavity. The study of progesterone receptors (PRA and PR-B) was performed by immunohistochemistry in the laboratory of immunohistochemistry DDMA. All patients received treatment with micronized progesterone at a dose of 400 mg / day continuously for 6 months. To determine the effect of progestin use, a control study was performed by scraping the uterine cavity after 6 months and the effectiveness of treatment was determined by histological changes of the endometrium.Research results. The treatment showed that 74% of women had histological normalization of the ratio of stromal and epithelial component, 18.7% were diagnosed with simple atypical endometrial hyperplasia (PNGE). Complex atypical endometrial hyperplasia (CNGE) was diagnosed in 7.3% of women. Prior to treatment, low PR-A expression in the endometrial glands (P = 0.013) and stroma (P 1 ratio (19%; P <0.001). According to a study of women with no effect from the therapy, in contrast to women with effective therapy, there was a decrease in the expression of progesterone receptors by 65%, which has a statistically significant relationship with recurrence of endometrial hyperplasia. Conclusions. Treatment of endometrial hyperplasia without atypia with progesterone drugs will be 74% ineffective with low expression of progesterone receptors in endometrial cells. immunohistochemical expression of PR-A and PR-B and their ratio in the stroma and glands during endometrial biopsy before progestin therapy is a valuable prognostic factor for recurrence of endometrial hyperplasia. Therefore, when deciding on the appointment of progestins for the treatment of endometrial hyperplasia without atypia, it is recommended to study the expression of receptors in the endometrium.

Human Endometrial Stromal Cell Differentiation is Stimulated by PPARβ/δ Activation: New Targets for Infertility?

Implantation is a reproductive bottleneck in women, regulated by fluctuations in ovarian steroid hormone concentrations. However, other nuclear receptor ligands are modifiers of endometrial differentiation leading to successful pregnancy. In the present study we analyzed the effects of peroxisome-proliferator-activated receptor β/δ (PPARβ/δ) activation on established cellular biomarkers of human endometrial differentiation (decidualization). The objective of this work is to test the effects of PPARβ/δ ligation on human endometrial cell differentiation. Isolated primary human endometrial stromal cells (ESCs) were treated with synthetic (GW0742) or natural (all trans-retinoic acid, RA) ligands of PPARβ/δ, and also with receptor antagonists (GSK0660, PT-S58, and ST247) in the absence or presence of decidualizing hormones (10 nM estradiol, 100 nM progesterone, and 0.5 mM dibutyryl cAMP [3',5'-cyclic adenosine 5'-monophosphate]). In some cases interleukin (IL)-1β was used as an inflammatory stimulus. Time course and dose-response relationships were evaluated to determine effects on panels of well characterized in vitro biomarkers of decidualization. PPARβ/δ, along with estrogen receptor α (ERα) and PR-A and PR-B, were expressed in human endometrial tissue and isolated ESCs. GW0742 treatment enhanced hormone-mediated ESC decidualization in vitro as manifested by upregulation of prolactin, insulin-like growth factor-binding protein 1, IL-11, and vascular endothelial growth factor (VEGF) secretion and also increased expression of ERα, PR-A and PR-B, and connexin 43 (Cx43). RA treatment also increased VEGF, ERα, PR-A, and PR-B and an active, nonphosphorylated isoform of Cx43. IL-1β and PPARβ/δ antagonists inhibited biomarkers of endometrial differentiation. Ligands that activate PPARβ/δ augment the in vitro expression of biomarkers of ESC decidualization. By contrast, PPARβ/δ antagonists impaired decidualization markers. Drugs activating these receptors may have therapeutic benefits for embryonic implantation.

Clinical association of progesterone receptor isoform A with breast cancer metastasis consistent with its unique mechanistic role in preclinical models

BackgroundLuminal breast cancer (L-BCa) comprises the majority of incurable, distally metastatic breast cancer cases. Estrogen supports growth of L-BCa cells but suppresses invasiveness. Estrogen also induces the progesterone receptor (PR). Invasiveness and metastasis of L-BCa cells is supported by the short PR isoform (PR-A), in response to the range of pre- and post-menopausal plasma hormone levels, by counteracting the effects of estrogen via micro RNA-mediated cross-talk with the estrogen receptor (ER). PR-B directly supports L-BCa invasion and metastasis and also inhibits tumor growth, both only at high progesterone levels. As public datasets on L-BCa tumors cannot distinguish PR-A, this study was designed to seek clinical evidence for the role of PR-A in metastasis in comparison with PR-B and ER.MethodsMeasurement of tumor PR-A, PR-B and ER mRNA expression in 125 treatment-naive primary L-BCa patients with differential node involvement and analysis using linear mixed effects models. Transcriptional activity assays of PR-A and PR-B.ResultsLymph node involvement was strongly associated with PR-A expression (median, 3-fold higher vs. node-negative), independent of age, pathologic type, tumor grade, HER2 and PR-B. PR-B and ER correlated weakly with PR-A, but whereas PR-B and the PR-A/PR-B ratio were not significantly associated with node involvement, ER weakly negatively correlated with node positivity. PR-A was hypersensitive to mifepristone compared with PR-B.ConclusionsTaken together with previous mechanistic studies, the findings provide clinical evidence in support of the role of PR-A in L-BCa metastasis. They also suggest the possibility of developing selective PR-A modulators for future interventions in appropriate clinical situations.

OR20-04 Modeling Uterine Disorders Utilizing Adult Uterine Stem Cells

Endometriosis and uterine fibroids (leiomyomas) are benign gynecological disorders affecting 5-15% of women of reproductive age. They cause a wide range of symptoms including mild to severe pelvic pain and infertility. Due to a paucity of proper study models, hormonal and cellular mechanisms driving the pathology of endometriosis and fibroid development and growth remain unclear. Therefore, in the current study, we established 3D spheroid/organoid cultures from human uterine epithelial and Stro-1+/CD44+ myometrial stem cells and also from cells isolated from normal proliferative phase endometrium. Uterine organoid cultures were derived from endometrial epithelial and myometrial cells isolated from women who were not receiving exogenous hormones at the time of laparoscopy or hysterectomy. They were embedded in Matrigel, and grown in culture media. To determine whether spheroids/organoids were responsive to steroid hormones, the cultures were treated in presence or absence of estradiol (E2), progesterone (P4) or the combination (E2+P4) in serum free culture media. Time-dependent spheroid/organoid-growth curves and morphological analyses were used to define growth characteristics of endometrial and myometrial organoids. Subsequently, immunohistochemical colocalization of steroid hormone receptors (estrogen receptor alpha (ER-α) and progesterone receptor (PR-A\\B), alpha smooth muscle actin (α-SMA; myometrial cell marker), vimentin (stromal cell marker) and E-cadherin (endometrial epithelial cell marker) was assessed. Epithelial organoids expressed only E-cadherin in the absence of hormonal treatment. Myometrial organoids expressed α-SMA and vimentin. No expression of E-cadherin was observed in myometrial organoids. However, we observed the expression of ER-α and PR-A\\B when organoids were treated with E2+P4 in a time-dependent manner. Stro-1+/CD44+ myometrial stem cells differentiated into α-SMA and fibroblast/stromal cells and response to sex hormones. These findings suggest human uterine organoid cultures retained their characteristic cellular responses to E2+P4 and could be maintained long-term in ex vivo culture. Thus, the current 3D uterine organoid systems show high expansion capacity with retention of phenotypical and functional properties, which can be used for uterine pathophysiological studies, drug discoveries and drug repositioning.