Progesterone Receptor Protein Levels Research Articles

Altered luteal expression patterns of genomic and non-genomic progesterone receptors in bitches at different reproductive states

The binding of steroid hormones to their specific receptors is necessary to exert their effects on target cells. Progesterone (P4), a steroid hormone, carries out its effects through both genomic and non-genomic (the cell membrane-associated) receptors. This study aimed to ascertain luteal expression patterns of genomic and non-genomic progesterone receptors in bitches in physiological (early dioestrus and early pregnant) and pathological (pyometra) reproductive states. Luteal tissue was collected from the bitches at early dioestrus (ED, n = 5), early pregnant (EP, n = 5), and pyometra (PY, n = 5). The expression profiles of Steroidogenic Acute Regulator Protein (STAR), Progesterone Receptor (PGR), Membrane Progestin Receptors (PAQR5, PAQR7 and PAQR8), and Progesterone Membrane Components (PGMRC1 and PGMRC2) were examined at the mRNA levels using Real-Time Polymerase Chain Reaction (RT-PCR). Protein levels of PGR, PGMRC1 and PGMRC2 were detected by western blotting (WB). The STAR expression was found in all groups, with a statistical difference observed between EP and PY groups (P < 0.05). The protein level of PGR was determined to be highest in the EP group and lowest in the PY group. The expression of PAQR8 increased in the EP group (P < 0.05). The PAQR5 exhibited high expression in the EP group and low expression in the PY group (P < 0.05). PGRMC1 was more elevated in the EP group and lower in the PY group (P < 0.05). Protein levels of PGMRC1 and PGMRC2 were also observed at the highest expression in EP group. According to the altered expression profiles for examined receptors, we suggest that those progesterone receptors have roles in early pregnancy or pyometra in bitches.

The Efficacy of Vitamin D Supplement in the Expression and Protein Levels of Endometrial Decidualization Factors in Women with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) is a challenging situation for infertility specialists, and its treatment is introduced as a difficult case in the field of assisted reproductive technology (ART). Vitamin D (VD) is one of the supplements that have been suggested to improve the implantation process. In the present study, the effect of VD on the expression and protein levels of VD receptor (VDR), progesterone receptor (PR), prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP-1), and homeobox protein A10 (HOXA10) in the endometrial cells of unknown RIF women with and without VD deficiency were assessed by qRT-PCR and immunohistochemistry. Twelve women with unknown RIF and VD deficiency (≤ 20 ng/ml) and twelve women with unknown RIF without VD deficiency (≥ 30 ng/ml) from 2021 to 2022 were identified. Endometrial specimens were collected in the mid-luteal stage before treatment or pregnancy. In the group with VD deficiency, oral medication of VD 50,000 units was prescribed for 2 to 3 months and their serum levels of VD were re-measured, then an endometrial biopsy at the same stage of the menstrual cycle was performed. The expression and protein levels of VDR, PR, PRL, IGFBP1, and HOXA10 in RIF patients with VD deficiency were lower than the RIF patients without VD deficiency (P value < 0.05). Our findings suggest that VD can play a key role in the pregnancy process, especially during embryo implantation and decidualization of the endometrial cells.IRCT registration number: IRCT20220528055006N1, Registration date: 2022-10-15, Registration timing: retrospective.

Jiawei Shoutai Pill promotes decidualization by regulating the SGK1/ENaC pathway in recurrent spontaneous abortion

Ethnopharmacological relevanceJiawei Shoutai Pill (JWSTW) is a traditional herbal formula for recurrent spontaneous abortion (RSA). Although JWSTW significantly improves the clinical symptoms of RSA patients, its molecular mechanism remains unclear. Aim of studyThis study evaluated the expression and function of the serum/glucocorticoid regulated kinase 1/epithelial sodium channel (SGK1/ENaC) pathway and decidualization level in RSA patients and mice. It also investigated the therapeutic effects and potential mechanisms of JWSTW. Materials and methods30 early RSA patients and 30 normal pregnant women undergoing induced abortion during the same period were included in the study. Decidual tissues were collected, and HE staining, immunohistochemistry, Western blot, and RT-PCR were used to detect protein and mRNA expression levels of SGK1, ENaC-a, estrogen Rreceptor β (ERβ), and progesterone receptor (PR) in patients’ decidual tissues. Protein expression levels of prolactin receptor (PRLR) and insulin-like growth factor binding protein 1 (IGFBP-1) were also detected. A classical RSA mouse model was constructed, and the mice were randomly divided into four groups: normal, model, dydrogesterone (DQYT) (0.33 g/kg/d), and JWSTW (1.66 g/kg/d). The normal and model groups received the same volume of distilled water by gavage for 8 and 14 days after pregnancy. On the 14th day of pregnancy, the embryonic loss rate of each group, the number of offspring born to naturally delivered mice, and the protein or mRNA expression levels of key factors of the SGK1/ENaC pathway (SGK1, ENaC-a, ERβ, and PR), decidual proliferation marker (Ki67), mesenchymal-epithelial transition (E-cadherin and Vimentin), and decidualization markers (PRLR and IGFBP-1) in mouse decidual tissue on the eighth day of pregnancy were observed. ResultsThe decidual tissue structure of RSA patients was abnormal. Immunohistochemical analysis revealed significantly reduced positive expression of SGK1, ENaC-a, ERβ, and PR proteins in the decidual tissue of RSA patients (P < 0.001). Western blot and RT-PCR analyses demonstrated significantly decreased protein and mRNA expression of SGK1, ENaC-a, ERβ, and PR in the decidual tissue of RSA patients (all P < 0.05). Additionally, protein expression of PRLR and IGFBP-1 was significantly reduced (both P < 0.001). The RSA mouse model exhibited a significant increase in embryo loss rate and decreased litter size (both P < 0.001). Treatment with DQYT and JWSTW rescued the embryo loss rate and litter size to varying extents (all P < 0.05). The protein or mRNA expression levels of SGK1, ENaC-a, ERβ, PR, Ki67, E-cadherin, vimentin, PRLR, and IGFBP-1 in RSA mice were improved to different degrees after treatment with DQYT and JWSTW (all P < 0.05). ConclusionsAbnormal SGK1/ENaC signaling pathway regulation is closely associated with early endometrial damage in RSA patients. JWSTW promotes endometrial proliferation and mesenchymal-epithelial transition through the SGK1/ENaC signaling pathway, improving endometrial shedding. Consequently, JWSTW is a potential treatment for RSA.

Fractalkine Improves the Expression of Endometrium Receptivity-Related Genes and Proteins at Desferrioxamine-Induced Iron Deficiency in HEC-1A Cells.

Fractalkine (CX3CL1/FKN) is a unique chemokine belonging to the CX3C chemokine subclass. FKN exists in two forms: a membrane-bound form expressed by both endometrium cells and trophoblasts thought to be implicated in maternal-fetal interaction and a soluble form expressed by endometrium cells. Endometrium receptivity is crucial in embryo implantation and a complex process regulated by large numbers of proteins, e.g., cytokines, progesterone receptor (PR), SOX-17, prostaglandin receptors (PTGER2), and tissue inhibitors of metalloproteinases (TIMPs). It has also been reported that iron is important in fertility and affects the iron status of the mother. Therefore, iron availability in the embryo contributes to fertilization and pregnancy. In this study, we focused on the effect of iron deficiency on the secreted cytokines (IL-6, IL-1β, leukocyte inhibitory factor, TGF-β), chemokines (IL-8, FKN), and other regulatory proteins (bone morphogenic protein 2, activin, follistatin, PR, SOX-17, prostaglandin E2 receptor, TIMP2), and the modifying effect of FKN on the expression of these proteins, which may improve endometrium receptivity. Endometrial iron deficiency was mediated by desferrioxamine (DFO) treatment of HEC-1A cells. FKN was added to the cells 24 h and 48 h after DFO with or without serum for modelling the possible iron dependence of the alterations. Our findings support the hypothesis that FKN ameliorates the effects of anemia on the receptivity-related genes and proteins in HEC-1A cells by increasing the secretion of the receptivity-related cytokines via the fractalkine receptor (CX3CR1). FKN may contribute to cell proliferation and differentiation by regulating activin, follistatin, and BMP2 expressions, and to implantation by altering the protein levels of PR, SOX-17, PTGER2, and TIMP2. FKN mitigates the negative effect of iron deficiency on the receptivity-related genes and proteins of HEC-1A endometrium cells, suggesting its important role in the regulation of endometrium receptivity.

Significance of differential expression of OLFM4 in the development of endometrial adenocarcinoma.

The incidence of endometrial adenocarcinoma (EA) has increased worldwide in recent years due to the widespread use of estrogen therapy and the overall increase in life expectancy. However, we know of no sensitive molecular index that can be used to predict the onset of EA, evaluate the therapeutic effects of treatment agents, or provide prognostic benefit in post-treatment follow-up. To explore the correlation between human olfactomedin 4 (OLFM4) and the clinicopathologic parameters of EA, and to determine the precise involvement of OLFM4 as a related factor in the occurrence and development of EA. We enrolled 61 gynecologic patients for a retrospective study at the Tai'an Central Hospital of Shandong Province from January 1, 2016, to June 30, 2022. We determined the expression levels of estrogen receptor α (ERα), progesterone receptor (PR), and OLFM4 proteins in endometrial tissue with the immunohistochemical S-P staining method, and analyzed the correlations among ERα, PR, and OLFM4 protein expression levels and with the pathologic stage, histologic grade, myometrial invasiveness, and lymphatic metastasis of EA. The expression levels of OLFM4 in EA were higher than in normal endometrium (P = .036). The expression level of OLFM4 protein in stage II-III patients was higher than that in stage I patients (P = .034), and the expression levels of ERα and PR proteins in EA were lower than those in normal endometrial tissue (P = .014 and P = .0005). While we observed no correlation in endometrial tissues of disparate pathologic types between OLFM4 and the expression levels of ERα and PR proteins, we noted a positive correlation between the expression levels of ERα and PR protein. The expression level of OLFM4 protein increased with the malignant degree of endometrial lesions and OLFM4 protein expression was related to the FIGO stage of EA. And OLFM4 protein can be used as 1 of the potential diagnostic factors for endometrial lesions, which is worthy of further study.

Progestogens exhibit progestogen-, promoter- and isoform-specific effects via the progesterone receptor

Hormonal contraceptives (HCs) and hormone replacement therapy (HRT) are therapies designed to target the progesterone receptor (PR) to prevent unwanted pregnancy and to alleviate the symptoms of menopause, respectively, in women. Although these therapies are widely used globally, few studies have investigated in parallel how the transcriptional responses of the progestogens used in these therapies compare to each other via the PR isoforms (PR-A and PR-B). Using dose–response promoter-reporter and endogenous gene expression assays, we compared the transcriptional responses of six widely-used progestogens via each PR isoform. The present study shows that progestogens exhibit progestogen-specific potencies and efficacies via both PR isoforms. In addition, the endogenous gene expression data reveals that progestogens exhibit promoter-specific effects. Furthermore, this study reveals that progestogen responses via PR-A are significantly more potent and less efficacious than those observed via PR-B, and that this is unlikely due to differences in PR protein levels. Correlation analysis revealed that there is no detectable correlation between potency or efficacy of progestogens for PR-B or PR-A versus reported relative binding affinity (RBA) of progestogens for the PR, consistent with complex mechanisms of PR regulation. Taken together, our data show that it cannot be assumed that all progestogens have similar transcriptional responses on all genes. Since the PR plays a role in cognition, regulation of inflammation, mitochondrial function, neurogenesis, female reproduction and disease, the data suggest that these important physiological functions could be differentially affected depending on progestogen, promoter, and ratios of PR isoforms.

Abstract 102: IGFBP6 modulates estrogen receptor alpha phosphorylation and transcriptional activity in breast cancer cells

Abstract Insulin-like growth factors (IGFs) are signaling proteins which bind to insulin-like growth factor 1 receptor (IGF-1R) and induce a signaling cascade to promote proliferation and survival in breast cancer cells. Insulin-Like Growth Factor Binding Protein 6 (IGFBP-6) induces apoptosis by inhibiting IGF-1R upon binding to both IGF-1 and IGF-2, however, the role of IGFBP-6 in breast cancer has not been adequately studied. While IGFBP-6 can bind to both IGF-1 and IGF-2, the binding affinity for IGF2 is 20-fold higher compared to IGF1. To determine if IGFBP-6 modulates breast cancer cells, MCF-7 and T-47D cells were stably transfected with an IGFBP-6 expression plasmid. IGFBP6 over-expression resulted in decreased proliferation rates in both cell lines when compared to empty vector controls. To identify a mechanism of decreased proliferation rates, the activation of the IGF-1R-dependent AKT and MAPK mediators was measured. Reduced phosphorylation of MAPK and AKT was observed in both cell lines over-expressing IGFBP-6 compared to empty vector controls. MAPK and AKT phosphorylate ERα at serine 118 and serine 167 respectively, therefore, ERα phosphorylation at these residues was determined in the presence of IGFBP6 expression. Reduction in phosphorylation was greater in MCF-7 cells than T-47D cells when IGFBP6 was constitutively expressed. To determine if reduced ERα phosphorylation altered expression of known targets of ERα-modulated expression, such as IGF-1R and Progesterone Receptor (PR) was measured. Unexpectedly, IGF-1R and PR protein and transcript levels were greater in IGFBP6 over-expressing cells. Studies have shown that differences in ERα phosphorylation change the recruitment of transcriptional modulators associated with ERα-mediated transcription. These results suggest that despite reducing IGF-1R-dependent cell signaling, compensatory mechanisms may be induced to enhance ERα-mediated expression. Citation Format: Francisco Javier Lariz, Kevin D. Houston. IGFBP6 modulates estrogen receptor alpha phosphorylation and transcriptional activity in breast cancer cells [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 102.

The effect of pre-analytical variables on downstream application and data analysis of human endometrial biopsies.

STUDY QUESTIONWhat are the effects of pre-analytical variables on the downstream analysis of patient-derived endometrial biopsies?SUMMARY ANSWERThere are distinct differences in the protein levels of the master regulator of oxygen homeostasis, hypoxia-inducible factor-1-alpha (HIF1α), and the protein and mRNA levels of three related genes, carbonic anhydrase 9 (CA9), vascular endothelial growth factor A (VEGFA) and progesterone receptor (PR) in human endometrial biopsies, depending on the pre-analytical variables: disease status (cancer vs benign), timing of biopsy (pre- vs post-hysterectomy) and type of biopsy (pipelle vs full-thickness).WHAT IS KNOWN ALREADYPatient-derived biopsies are vital to endometrial research, but pre-analytical variables relating to their collection may affect downstream analysis, as is evident in other tissues.STUDY DESIGN, SIZE, DURATIONA prospective observational study including patients undergoing hysterectomy for endometrial cancer (EC) or benign indications was conducted at a large tertiary gynaecological unit in the UK. Endometrial biopsies were obtained at different time points (pre- or post-hysterectomy) using either a pipelle endometrial sampler or as a full-thickness wedge biopsy.PARTICIPANTS/MATERIALS, SETTING, METHODSThe changes in HIF1α, CA9, VEGFA and PR protein levels were measured by semi-quantitative analysis of immunostaining, and the expression levels of three genes (CA9, VEGFA and PR) were investigated by quantitative real-time PCR, in endometrial biopsies from 43 patients undergoing hysterectomy for EC (n = 22) or benign gynaecological indications (n = 21).MAIN RESULTS AND THE ROLE OF CHANCEAn increase in HIF1α immunostaining was observed in EC versus benign endometrium (functionalis glands) obtained pre-hysterectomy (P < 0.001). An increase in CA9 immunostaining was observed in EC versus benign endometrial functionalis glands at both pre- and post-hysterectomy time points (P = 0.03 and P = 0.003, respectively). Compared with benign endometrial pipelle samples, EC samples demonstrated increased mRNA expression of CA9 (pre-hysterectomy P < 0.001, post-hysterectomy P = 0.008) and VEGFA (pre-hysterectomy P = 0.004, post-hysterectomy P = 0.002). In benign uteri, HIF1α immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.009), VEGFA immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.01) and VEGFA mRNA levels (P = 0.008) were increased in matched post-hysterectomy versus pre-hysterectomy samples. Similarly, in EC, an increase in VEGFA immunoscores (epithelial and stromal) and VEGFA mRNA expression was observed in the matched post-hysterectomy versus pre-hysterectomy biopsies (P = 0.008, P = 0.004 and P = 0.018, respectively). Full-thickness benign post-hysterectomy endometrial biopsies displayed increased VEGFA (P = 0.011) and PR (P = 0.006) mRNA expression compared with time-matched pipelle biopsies.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis descriptive study explores the effect of pre-analytical variables on the expression of four proteins and three hypoxia-related genes in a limited number of endometrial biopsies from patients with EC and benign controls. Due to the small number, it was not possible to investigate other potential variables such as menstrual cycle phase, region-specific differences within the endometrium, grade and stage of cancer, and surgical technicalities.WIDER IMPLICATIONS OF THE FINDINGSCareful consideration of the effects of these pre-analytical variables is essential when interpreting data relating to human endometrial biopsies. A standardized approach to endometrial tissue collection is essential to ensure accurate and clinically transferrable data.STUDY FUNDING/COMPETING INTEREST(S)The authors have no conflicts of interest to declare. The work included in this manuscript was funded by Wellbeing of Women project grants RG1073 and RG2137 (D.K.H.), Wellbeing of Women Entry-Level Scholarship ELS706 and Medical Research Council MR/V007238/1 (A.M./D.K.H.), Liverpool Women’s Hospital Cancer Charity (M.A.) and University of Liverpool (L.B., L.R. and E.N.).

The Flavonoid Baicalein Negatively Regulates Progesterone Target Genes in the Uterus in Vivo.

Baicalein is a flavonoid extracted from the root of Scutellaria baicalensis (Chinese skullcap) and is consumed as part of this botanical dietary supplement to reduce oxidative stress, pain, and inflammation. We previously reported that baicalein can also modify receptor signaling through the progesterone receptor (PR) and glucocorticoid receptor (GR) in vitro, which is interesting due to the well-established roles of both PR and GR in reducing inflammation. To understand the effects of baicalein on PR and GR signaling in vivo in the uterus, ovariectomized CD-1 mice were treated with DMSO, progesterone (P4), baicalein, P4 with baicalein, and P4 with RU486, a PR antagonist, for a week. The uteri were collected for histology and RNA sequencing. Our results showed that baicalein attenuated the antiproliferative effect of P4 on luminal epithelium as well as on the PR target genes HAND2 and ZBTB16. Baicalein did not change levels of PR or GR RNA or protein in the uterus. RNA sequencing data indicated that many transcripts significantly altered by baicalein were regulated in the opposite direction by P4. Similarly, a large portion of GO/KEGG terms and GSEA gene sets were altered in the opposite direction by baicalein as compared to P4 treatment. Treatment of baicalein did not change body weight, organ weight, or blood glucose level. In summary, baicalein functioned as a PR antagonist in vivo and therefore may oppose P4 action under certain conditions such as uterine hyperplasia, fibroids, and uterine cancers.

Irilone, a Red Clover Isoflavone, Combined with Progesterone Enhances PR Signaling through the Estrogen and Glucocorticoid Receptors.

Trifolium pratense L. (red clover) is a popular botanical supplement used for women's health. Irilone isolated from red clover previously demonstrated progestogenic potentiation activity. In this study, irilone enhanced progesterone signaling was determined to not occur due to post-translational phosphorylation or by reducing progesterone receptor (PR) protein levels but instead increased PR protein levels in T47D breast cancer cells, which could be blocked by estrogen receptor (ER) antagonists, suggesting an ER dependent effect. Further, irilone increased luciferase activity from a hormone responsive element in a cell line that lacked ER and PR but expressed the glucocorticoid receptor (GR). A siRNA knockdown of GR in Ishikawa PR-B endometrial cancer cells reduced irilone's ability to enhance progesterone signaling. In an ovariectomized CD-1 mouse model, irilone did not induce uterine epithelial cell proliferation. The mechanism of action of irilone gives insight into PR crosstalk with other steroid hormone receptors, which can be important for understanding botanicals that are used for women's health.

Mechanism of Action of Irilone as a Potentiator of Progesterone Receptor Signaling

Progesterone signaling and its proper regulation is important for reproductive function. When progesterone signaling is dysregulated, gynecological diseases can occur, for example endometriosis, uterine fibroids, and endometrial cancer. While these diseases are treated with progestin therapy, progestins can bind to multiple steroid receptors, exerting side effects of weight gain, immunosuppression, cardiovascular disease, and stroke. Discovering an alternative progestin that is selective for the progesterone receptor (PR) is ideal. One potential source of such an alternative is botanical dietary supplements, which have become increasingly popular among consumers with sales reaching $9.6 billion in 2019. Although botanical supplements are popular, the chemical structures and biological action of botanical supplements would benefit from deeper scientific investigation. Studies of Trifolium pratense L. (red clover), primarily used for the treatment of menopausal symptoms, identified phytoestrogen compounds as the chemicals that mitigate those symptoms. Interestingly, irilone, identified from red clover, potentiated progesterone signaling via a progesterone response element luciferase (PRE/Luc) assay. Potentiation is when a compound has no activity by itself but when combined with another molecule, i.e. progesterone, that compound enhances PR activity. Prior to irilone, a natural compound with the ability to potentiate progesterone signaling had not been previously reported. The purpose of this study was to determine the mechanism of action of irilone. We hypothesized that irilone was potentiating PR by blocking PR degradation and by altering PR post-translational modifications. Irilone was found to potentiate 5 nM P4 using a PRE-luciferase assay in both T47D and Ishikawa PR expressing cells. Since PR is a downstream target gene of ER, we investigated if irilone also had ER activity. Irilone increased expression of an ERE-luciferase reporter gene. Next, we investigated if irilone could stabilize PR degradation and if irilone altered PR phosphorylation via western blot. Irilone was found to increase PR protein levels, but when ER was blocked, this was mitigated. In the presence of P4, irilone did not increase phosphorylation of serine 294 on PR. Future studies will determine if irilone is altering sumoylation of PR, and if irilone can potentiate PR signaling in vivo. Determining how irilone is potentiating progesterone will help us understand PR biology and could be an effective treatment for gynecological diseases by enhancing endogenous progesterone action.

Therapeutic effects and mechanism of ferulic acid and icariin in mammary gland hyperplasia model rats via regulation of the ERK pathway

BackgroundFerulic acid (FA) and icariin (ICA), as the active compounds derived from Angelica sinensis and Epimedium brevicornum Maxim, respectively, have been shown to promote blood circulation, regulate menstruation, exhibit anti-inflammatory effects, and modulate estrogen levels. The aim of this study was to elucidate the possible mechanism of FA, ICA, and FA-ICA on estrogen-induced mammary gland hyperplasia (MGH) in rats.MethodsHematoxylin and eosin (HE) staining was performed to record the pathological changes in breast tissue, and enzyme-linked immunosorbent assay (ELISA) was utilized to determine the serum levels of luteinizing hormone (LH), PRL (prolactin), testosterone (T), estradiol (E2), follicular stimulating hormone (FSH), and progesterone (P). The message RNA (mRNA) expression levels of extracellular signal-regulated kinase 1 (ERK1), extracellular signal-regulated kinase 2 (ERK2), estrogen receptor (ER), and progesterone receptor (PR) were detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. Furthermore, ER, phosphorylated-ER, PR, p-PR, ERK1/2, and p-ERK1/2 protein factors in the ERK signaling pathway were evaluated by Western blotting. Simultaneously, changes in breast height, diameter, body weight, and organ index were all recorded.ResultsWe found that FA and ICA could modulate the degree of breast swelling, and reduce the body weight, thymus index, and uterus index. Furthermore, it could also block the pathological changes of MGH, including the number of mammary lobules, and the proliferation or expansion of acini and ducts. Moreover, treatment with FA and ICA remarkably down-regulated the serum expression levels of LH, PRL, T, and E2, as well as the mRNA expression levels of ERα, PR, ERK1, and ERK2. Additionally, protein levels of ERα, p-ERα, PR, p-PR, ERK1/2, and p-ERK1/2 in breast tissue were down-regulated, however the serum content of FSH and P was up-regulated.ConclusionsOur outcomes revealed that FA and ICA might potentially inhibit ERα, PR, ERK1/2, and their phosphorylated proteins via the ERK signaling pathway, thus indicating a positive feedback control for the degree of breast hyperplasia.

Evaluation of Expression and Phosphorylation of Progesterone Receptor in Endometrial Stromal Cells of Patients with Recurrent Implantation Failure Compared to Healthy Fertile Women

Recurrent implantation failure (RIF) is the repeated failure of good-quality embryos in implantation process following several assisted reproduction cycles. Disruption of the endometrial receptivity is one of the main causes of RIF. Progesterone plays a pivotal role in the endometrial receptivity through the regulation of gene expression pattern by binding to its receptors in the endometrial cells. The aim of this study was to evaluate the expression level of progesterone receptor (PR) and its phosphorylated form in the endometrial stromal cells (eSC) of RIF patients and compare it to the eSC of healthy fertile women as control group. After isolation of the eSC from biopsy samples of RIF patients and healthy fertile women and their characterization, expression levels of PR mRNA, PR protein, and phospho-Ser294 PR protein were evaluated by quantitative real-time PCR and immunofluorescence staining, respectively. The results demonstrated a significant reduction in PR mRNA expression (P < 0.01.) and phospho-Ser294 PR protein (P < 0.05) level in RIF patients compared to the control group. These data for the first time suggest that the expression of PR and its phosphorylated form are impaired in RIF patients. Therefore, designing therapeutic methods for improving PR expression status and its regulation in the endometrium of RIF patients may help in improving the final reproductive outcomes of these cases.

Patterns of sex hormone receptor expression in stimulated endometrium from oocyte donors

In patients with high serum E2 embryo transfer is often postponed, as high E2 levels adversely affect embryo transfer outcome. We aimed to determine if stratified serum oestradiol (E2) and progesterone (P4) levels differentially affect endometrial histology and endometrial oestrogen and progesterone receptor protein levels. Endometrial biopsies were collected from oocyte donors. Samples were divided based on peak serum E2 levels into three groups: (i) low-E2 (n = 33) E2≤2999pg/mL; (ii) mid-E2 (n = 40) E2 3000–4999 pg/mL; and (iii) high-E2 (n = 15) E2≥5000 pg/mL. Oestrogen receptor alpha (ERα) and progesterone receptors A and B (PR) protein levels in endometrial stroma (S), glandular (GE) and luminal (LE) epithelia were assessed by immunohistochemistry. Samples in high-E2 group demonstrated strongest association with accelerated endometrial maturation (2 (1–2); 2 (1–3); and 3 (2.8–3) median days of advancement of endometrial maturation respectively in low, mid, high-E2 groups, p = 0.046). There were significant differences in ERα and PR immunoexpression in S, GE and LE among the groups (p < 0.05). Higher E2 levels were associated with decreased ERα expression (p < 0.017) in GE and LE, and increased PR expression in S and GE (p < 0.011 and p < 0.0001, respectively). Higher serum E2 levels were associated with impaired endometrial steroid hormone receptor expression, higher serum P4 and more advancement of endometrial maturation.

3D Visualization of the Dynamic Bidirectional Talk Between ER/PR and Her2 Pathways.

Background: Extensive amounts of archived formalin fixed paraffin embedded (FFPE) human tumor tissues are the ultimate resource to investigate signaling network underlying tumorigenesis in human. Yet, their usage is severely limited for lacking of suitable protein techniques. In this study, a quantitative, objective, absolute, and high throughput immunoblot method, quantitative dot blot (QDB), was explored to address this issue by investigating the putative relationship between estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) pathways in breast cancer tumorigenesis. Methods: In this descriptive observational retrospective study, ER, PR, Her2, and Ki67 protein levels were measured absolutely and quantitatively in 852 FFPE breast cancer tissues using the QDB method. ER, PR, and Her2 levels were charted on the X, Y, and Z-axes to observe samples distribution in a 3D scatterplot. Results: A “seesaw” relationship between ER/PR and Her2 pathways was observed in ER–PR–Her2 space, characterized by the expression levels of these 3 proteins. Specimens with strong expressions of ER/PR proteins were found spreading along the ER/PR floor while those with strong Her2 expression were found wrapping around the Her2 axis. Those lacking strong expressions of all 3 proteins were found accumulating at the intersection of the ER, PR, and Her2 axes. Few specimens floated in the ER–PR–Her2 space to suggest the lack of co-expression of all 3 proteins simultaneously. Ki67 levels were found to be significantly reduced in specimens spreading in the ER–PR space. Conclusions: The unique distribution of specimens in ER–PR–Her2 space prior to any clinical intervention provided visual support of bidirectional talk between ER/PR and Her2 pathways in breast cancer specimens. Clinical interventions to suppress these 2 pathways alternatively warrant further exploration for breast cancer patients accordingly. Our study also demonstrated that the QDB method is an effective tool to analyze archived FFPE cancer specimens in biomedical research.

Effect of 1,25(OH)2-vitamin D3 on expression and phosphorylation of progesterone receptor in cultured endometrial stromal cells of patients with repeated implantation failure

Repeated implantation failure (RIF) occurs in a condition when good quality embryos fail to implant in the endometrium following several in vitro fertilization (IVF) cycles. Suboptimal endometrial receptivity is one of the main underlying factors that causes this failure. Progesterone is the key regulator of endometrial receptivity which regulates gene expression through binding to its receptors in the endometrial stromal cells (eSC). The aim of this study was to evaluate the effect of 1,25(OH)2-vitamin D3 on progesterone receptor (PR) expression level and its phosphorylation on Ser294 residues in eSC of RIF patients and healthy fertile women. After isolation of the eSC from biopsy samples of RIF patients and healthy fertile women and their characterization, the cells were incubated with vitamin D3 and the expression level of PR mRNA, PR protein and phospho-Ser294 PR protein were evaluated after treatment. The results showed that vitamin D3 treatment increases PR mRNA and protein level and phospho-Ser294 PR protein level in the isolated eSC of both RIF patients and the control group. These results suggest that vitamin D3 may possibly play a key role during the embryo implantation process by affecting the expression pattern and regulatory modifications of the PR in the eSC.

The antiestrogenic effects of black cohosh on BRCA1 and steroid receptors in breast cancer cells.

BackgroundBlack cohosh (BC) is an herbal remedy often used by women to treat symptoms associated with menopause. Research has shown that the molecular activity of BC is associated with estrogen receptor alpha (ER-α) regulation. Progesterone receptor (PR) expression is found to be consistent with ER expression and mutations in the BRCA1 gene, a tumor-suppressor gene, are known to be responsible for about 40%–45% of hereditary breast cancers.PurposeThe objective of this study was to determine the effects of BC alone, as well as in combination with hormones and antihormones, on cell viability and expression of ER-α, PR, and BRCA1 in both T-47D and MCF-7 cell lines.MethodsCells were cultured in charcoal-stripped serum prior to their treatment and subsequent protein extraction. Western blot analyses were performed following a Bio-Rad Bradford protein assay and SDS-PAGE gel electrophoresis, with ECL luminescence and Image Studio Lite software. Cellular viability assays were performed using propidium iodine (PI) staining, and the distribution of fluorescent structures was evaluated through confocal microscopy. RT-qPCR analysis was performed on extracted cellular RNA. All statistical analyses were performed using SPSS software, and data was subjected to Kruskal-Wallis testing, followed by post-hoc analysis using the Mann-Whitney U-test to determine the statistical significance of all findings.ResultsWestern blot analysis displayed significant alterations of ER-α, PR, and BRCA1 protein levels after 24-hour treatment with 80–500 μM BC. BC displayed a concentration-dependent decrease on ER-α and BRCA1 expression, with an 87% reduction of ER-α expression and a 43% of BRCA1 expression in T-47D cells compared to control. After six days of treatment with 400 μM BC, a 50% decrease in cell proliferation was observed. Following 24 hours of co-treatment with 400 μM BC and 10 nM E2, ER-α was downregulated by 90% and BRCA1 expression was reduced by 70% compared to control. The expression of PR, following the same treatment, exhibited similar effects. The proliferative effect of E2 was reduced in the presence of BC.ConclusionBlack Cohosh demonstrates substantial anti-cancer properties, and this study may significantly aid in the understanding of the molecular effects of BC on ER-α, PR, and BRCA1 in breast cancer cells.

The Modified Bushen Antai Recipe Upregulates Estrogen and Progesterone Receptors at the Maternal-Fetal Interface in Pregnant Rats with Mifepristone-Induced Pregnancy Loss.

Background The modified Bushen Antai recipe (BSAT) is a centuries-old traditional Chinese medicine that we use in our center as a therapy against pregnancy loss. Our study aimed to explore the potential benefit and mechanism of BSAT in pregnant rats with mifepristone-induced pregnancy loss. Materials and Methods The signature compounds of the eight BSAT ingredients were analyzed by high-performance liquid chromatography (HPLC). The BSAT group (n = 8) was treated daily with 6.3 ml/kg BSAT from gestation day (D) 0.5 to 10.5 and once with 1.25 mg/kg mifepristone on D 10.5. Normal saline replaced BSAT in the model group (n = 8), and both BSAT and mifepristone in the control group (n = 8). Morphological and histological analyses were performed on D 13.5. Results BSAT contains eight medicinal ingredients including Cuscuta chinensis and Dipsacus asperoides. The HPLC analysis detected the signature compounds of seven medicinal ingredients in the extract. Embryo resorption rate in the BSAT group was significantly lower than that in the model group, although the number of surviving embryos was similar between the two groups. Hematoxylin and eosin (HE) staining suggested that the maximum cross-sectional area of the placenta and the area ratio of the placental labyrinth in the BSAT group were higher than those in the model group. Immunohistochemical (IHC) staining indicated that the expression of ki67, estrogen receptor alpha (ERα), and progesterone receptor (PR) in the placental labyrinth of the BSAT group was higher than that of the model group. Furthermore, the protein levels of ERα, PR, phospho-Akt/Akt, and phospho-Erk1/2/Erk1/2 in the BSAT group were higher than those in the control group. The mRNA levels of ERα and PR in the BSAT group were higher than those in the control group. Conclusions BSAT may induce estrogen and progesterone receptors by phosphorylation via the classic Akt and Erk1/2 signaling pathways in the maternal-fetal interface of pregnant rats, thereby reducing the pregnancy loss rate and improving the live birth rate.

Abstract B58: Progesterone receptor (PR) isoforms drive distinct cell-cell interactions and gene expression programs in human fallopian tube models of early HGSOC

Abstract High-grade serous ovarian carcinoma (HGSOC), the most prevalent and aggressive form of ovarian cancer (OC), contains abundant receptors for the ovarian steroid hormones, estrogen (ER; 76%) and progesterone (PR; 35%). These receptors contribute to the development of breast and reproductive tract cancers via complex mechanisms. Estrogen, acting through ERalpha, is well accepted as a stimulatory hormone while progesterone is thought to be a protective factor that reduces OC risk. Our understanding, however, of the molecular and cell-type specific mechanisms of PR isoform action in the initiation and progression of OC is limited. Therefore, to investigate altered PR isoform signaling in early-stage HGSOC, we utilized p53-dominant negative mutant fallopian tube epithelial (FTE) cells to generate stable cell lines expressing either PR-A or PR-B. Progestin (R5020) treatment of 2D adherent cultures revealed functional PR signaling through MAPK-dependent PR S294 phosphorylation as well as isoform specific expression of PR target genes encoding adhesion molecules (e.g., HEF1), cell cycle regulators (e.g., FOXO1), and glucocorticoid signaling proteins (e.g., CRISPLD2, NDRG1). In order to mimic early dissemination of FTE cells from serous tubal intraepithelial carcinoma (STIC) lesions, a precursor for the majority of HGSOCs, we established a 3D spheroid culture model. Interestingly, we observed that PR expression greatly increased the formation, size, and number of total spheroids compared to PR-negative controls. Progestin treatment resulted in additional enhancement of spheroid size with distinct morphologic differences observed between PR-A and PR-B expressing lines. Furthermore, progestin-treated cells grown as 3D spheroids exhibited increased basal PR protein and PR S294 phosphorylation levels relative to cells maintained in 2D adherent cultures, suggesting that hyperactivation of PRs occurs via cell-cell interactions, in part conferred by 3D architecture. Both PR-A and PR-B spheroids secreted matrix proteins such as fibronectin and other EMT biomarkers (e.g., integrin alpha 5) and progestin treatment modulated the PR isoform-specific expression of these markers. Preliminary RNA-seq analyses, comparing PR-A and PR-B spheroids, revealed differential expression of genes encoding pathways that regulate cell adhesion and cell movement as well as cytokine signaling and cell metabolism. Our results highlight the importance of cell context in understanding PR action and suggest that activation of PR signaling promotes and enhances the formation of nonadherent 3D structures in an isoform-specific manner. Such effects of progesterone are predicted to influence the shedding, aggregation, and dissemination of early FTE lesions. These preliminary findings demonstrate the importance of understanding the impact of steroid hormone receptors, including PR isoforms, on early OC development. Citation Format: Megan I. Seibel, Angela Spartz, Nuri Temiz, Laura J. Mauro, Carol A. Lange. Progesterone receptor (PR) isoforms drive distinct cell-cell interactions and gene expression programs in human fallopian tube models of early HGSOC. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B58.

Sensitivity and Isoform Specificity of 18F-Fluorofuranylnorprogesterone for Measuring Progesterone Receptor Protein Response to Estradiol Challenge in Breast Cancer.

The purpose of this study was to evaluate the ability of 21-18F-fluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione (18F-FFNP) to measure alterations in progesterone receptor (PR) protein level and isoform expression in response to estradiol challenge. Methods: T47D human breast cancer cells and female mice-bearing T47D tumor xenografts were treated with 17β-estradiol (E2) to increase PR expression. 18F-FFNP uptake was measured using cell uptake and tissue biodistribution assays. MDA-MB-231 breast cancer clonal cell lines were generated that express the A or B isoforms of human PR. PR protein levels, transcriptional function, and subcellular localization were determined. In vitro 18F-FFNP binding was measured via saturation and competitive binding curves. In vivo 18F-FFNP uptake was measured using tumor xenografts and positron emission tomography. Statistical significance was determined using analysis of variance and t-tests. Results: After 48 and 72 h of E2, 18F-FFNP uptake in T47D cells was maximally increased compared to both vehicle and 24 h E2 treatment (p<0.0001 vs ethanol; P = 0.02 and P = 0.0002 vs 24 h for 48 and 72 h, respectively). T47D tumor xenografts in mice treated with 72 h E2 had maximal 18F-FFNP uptake compared to ethanol-treated mice (11.3±1.4 vs 5.2±0.81 %ID/g; P = 0.002). Corresponding tumor-to-muscle uptake ratios were 4.1±0.6, 3.9±0.5, and 2.3±0.4 for 48 h E2, 72 h E2, and ethanol-treated mice, respectively. There was no significant preferential 18F-FFNP binding or uptake by PR-A versus PR-B in the PR isoform-specific cell lines and tumor xenografts. Conclusion:18F-FFNP is capable of measuring estrogen-induced shifts in total PR expression in human breast cancer cells and tumor xenografts with equivalent isoform binding.