To characterize the effects of 4-methylumbelliferone (4-MU) on expression of the hyaluronic acid (HA) system and on attachment, migration, and invasion of endometrial epithelial (EECs) and stroma cells (ESCs) to peritoneal mesothelial cells (PMCs), this in vitro study was performed in an Academic Center. De-identified endometrial tissue samples used were from reproductive-aged women. EECs and ESCs isolated from menstrual endometrial biopsies were treated with 4-MU or vehicle. Real-time polymerase chain reaction and western blot were used to assess expression of HA synthases (HAS), hyaluronidase, and standard CD44. Established in vitro assays were used to assess attachment, migration, and invasion with and without treatment with 4-MU. Chi square and Student's t-test were used to analyze the results as appropriate. The addition of 4-MU decreased mRNA and protein expression of HAS 2, HAS 3, and CD44 in EECs and ESCs compared to control. Treatment with 4-MU also decreased attachment, migration, and invasion of EECs and ESCs to PMCs compared to control. 4-MU decreases endometrial cell adhesion, migration, and invasion to PMCs. This effect appears to be mediated by a decrease in HAS 2, HAS 3, and CD44. 4-MU is a potential treatment for endometriosis. Future in vivo studies are needed to evaluate 4-MU as a therapeutic agent for endometriosis.

Uterine fibroids (UFs) are associated with irregular or excessive uterine bleeding, pelvic pain or pressure, or infertility. Ovarian steroid hormones support the growth and maintenance of UFs. Ulipristal acetate (UPA) a selective progesterone receptor (PR) modulator (SPRM) reduce the size of UFs, inhibit ovulation and lead to amenorrhea. Recent liver toxicity concerns with UPA, diminished enthusiasm for its use and reinstate the critical need for a safe, efficacious SPRM to treat UFs. In the current study, we evaluated the efficacy of new SPRM, EC313, for the treatment for UFs using a NOD-SCID mouse model. EC313 treatment resulted in a dose-dependent reduction in the fibroid xenograft weight (p < 0.01). Estradiol (E2) induced proliferation was blocked significantly in EC313-treated xenograft fibroids (p < 0.0001). Uterine weight was reduced by EC313 treatment compared to UPA treatment. ER and PR were reduced in EC313-treated groups compared to controls (p < 0.001) and UPA treatments (p < 0.01). UF specific desmin and collagen were markedly reduced with EC313 treatment. The partial PR agonism and no signs of unopposed estrogenicity makes EC313 a candidate for the long-term treatment for UFs. Docking studies have provided a structure based explanation for the SPRM activity of EC313.

OBJECTIVE:To characterize the production and degradation of hyaluronic acid (HA) in menstrual endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) in women with and without endometriosis. To identify the presence of CD44, the primary receptor of HA, in menstrual EECs and ESCs in women with and without endometriosis.DESIGN:In vitro study.SETTING:Academic center.PATIENT(S):Deidentified patient samples from women with and without endometriosis.INTERVENTIONS:EECs and ESCs were isolated from menstrual endometrial biopsies performed on women with (N = 9) and without (N = 11) endometriosis confirmed by laparoscopy.MAIN OUTCOME MEASURE:Real-time polymerase chain reaction, Western blot, and immunohistochemistry were used to assess hyaluronic acid synthase (HAS) isoforms 1, 2, and 3; hyaluronidase (HYAL) isoforms 1 and 2; and standard CD44. Student t test was used to analyze the results.RESULTS:There was no significant difference in messenger RNA (mRNA) or protein expression of HAS2, HAS3, HYAL1, or HYAL2 in EECs or ESCs from women with or without endometriosis. HAS1 mRNA was variably detected, whereas HAS1 protein was similarly expressed in EECs and ESCs from women with and without endometriosis. Standard CD44 was expressed in both cell types, and expression did not differ in cells from women with or without endometriosis.CONCLUSIONS:The HA system is expressed in eutopic menstrual ESCs and EECs from women with and without endometriosis. There are no differences in expression in HA production or degradation enzymes in EECs or ESCs from women with and without endometriosis. Standard CD44 expression does not differ in eutopic menstrual endometrial cells from women with and without endometriosis.

Hyaluronic Acid (HA) plays a role in the early development of the endometriotic lesion.1 4-Methylumbelliferone (4-MU), a coumarin, has been shown to inhibit endometriotic lesion formation in mice.2 Here, we assessed the effects of 4-MU treatment of endometrial epithelial (EEC) and stromal cell (ESC) on: (1) expression of hyaluronic acid synthases (HAS), and (2) invasion of EECs and ESCs. In vitro studies. EECs and ESCs were isolated from menstrual endometrial biopsies. Cell viability after treatment was previously confirmed. EEC and ESC mRNA expression of HAS 2 and HAS 3 were quantified by RT-PCR with and without 4-MU (2mM). An established in vitro assay using peritoneal mesothelial cell (PMC) monolayers on a Matrigel matrix was used to assess EEC and ESC invasion of 4-MU treated and untreated cells. Results were analyzed with unpaired t-tests. RT-PCR confirmed a significant decrease in HAS 2 and HAS 3 mRNA expression in 4-MU treated EECs and ESCs (p <0.01). EECs invasion to PMCs decreased with the addition of 4-MU compared to control (668 ± 37 vs 1217 ± 51; p<0.0001). ESC invasion to PMCs with addition of 4-MU also decreased compared to control (765 ± 47 vs 1187 ± 64; p=0.04). 4-MU decreases HAS 2 and HAS 3 expression and invasiveness of EECs and ESCs. These findings further suggest that the CD44/HA system is involved in the development of the early endometriotic lesion and may serve as a therapeutic target to prevent the disease.

Endometriosis requires estrogen for development and growth. Levels of estrogen receptor beta (ERβ) in endometriosis are reported to be 100 times higher than normal endometrial tissue and inhibition of ERβ activity by an ERβ selective antagonist suppresses endometriotic lesion growth in mice.1,2 Here, we assessed the role of ERβ in the development of the early endometriotic lesion by examining the effect of ERβ knock down on endometrial stromal cell (ESC) cell attachment and invasion. In vitro study. ERβ was knocked-down (ERβKD) with lipofectamine using shRNA in an immortalized human ESC line (tHESC). Western blot and densitometric analysis were used to assess protein expression. Established in vitro assays were used to assess WT and ERβKD tHESC attachment to peritoneal mesothelial cells (PMCs) and invasion through a monolayer of PMCs. Results were analyzed with unpaired t-tests. tHESCs with ERβKD demonstrated decreased protein expression by Western blot and densitometric analysis. ERβKD attachment to PMCs decreased in comparison to WT (42% vs 67%, respectively; p<0.001). ERβKD invasion through PMCs decreased in ERβKD compared to WT (264 ±16 vs 404 ± 22, respectively; p<0.005). ERβKD decreases tHESCs adhesion to and invasion through PMCs. ERβ may serve as a future therapeutic target to decrease early endometriotic lesion formation.

Menstrual endometrial cells (MECs) from women with endometriosis have increased adhesion and also express higher levels of CD44 variant 6 (v6) than v3, compared to MECs from women without endometriosis.1 Here, we assessed the effects of CD44 standard (CD44s), CD44v3 and CD44v6 overexpression on endometrial epithelial (EECs) and stroma cells (ESCs) in vitro attachment to and invasion through a peritoneal mesothelial cell monolayer (PMC). In vitro studies. Primary EECs and ESCs were isolated from menstrual endometrial biopsies. EECs were immortalized (iEECs) using a telomerase vector, and verified by genotyping. Transient overexpression of CD44 standard and CD44 variants were carried out using Lipofectamine and their expression verified with qRT-PCR. Established in vitro assays were used to assess EEC and ESC attachment to PMCs and invasion through a monolayer of PMCs on a matrigel matrix. Results were analyzed with ANOVA and student t-tests as appropriate. In primary EECs, CD44s, CD44v3 and CD44v6 overexpression increased attachment and invasion compared to plasmid control (p<0.05). In primary ESCs, CD44s, CD44v3 and CD44v6 overexpression did not affect attachment compared to plasmid control. In primary ESCs, CD44v3 overexpression increased invasion (p<0.05), but CD44s and CD44v6 overexpression had no affect on invasion compared to plasmid control. Overexpression of CD44s, CD44v3 and CD44v6 increases adhesiveness and invasiveness of EECs. CD44s, CD44v3 and CD44v6 overexpression did not alter ESC attachment and only CD44v6 increased ESC invasiveness. These findings suggest menstrual endometrial cell type and CD44 variants play a complex role in the development of the early endometriotic lesion.

The Hyaluronan (HA) system is involved in the development of the early endometriotic lesion.1 A coumarin, 4-methylumbelliferone (4-MU), has been shown to decrease hyaluronic acid synthesis and angiogenesis in endometriotic lesions of mice.2 Here, we assessed the effects of 4-MU on endometrial epithelial (EECs) and stroma cells (ESCs): (1) expression of HA synthases (HAS) and hylauronidases (HYAL) (2) HA receptors CD44 and the receptor for HA mediated motility (RHAMM), and (3) attachment to peritoneal mesothelial cells (PMCs). In vitro studies EECs and ESCs were isolated from menstrual endometrial biopsies. Cell viability after treatment with 1, 2 and 4mM doses of 4-MU was assessed in EECs and ESCs using flow cytometry and cell titer glow assays. EEC and ESC mRNA expression of HAS 2, HAS 3, CD44, RHAMM and HYAL2 were quantified by RT-PCR with and without 4-MU (2mM). An established in-vitro assay was used to assess EEC and ESC attachment of 4-MU treated and untreated cells to PMCs. Results were analyzed with paired t-tests. 4-MU slightly decreased cell viability at 2mM and 4mM doses in EECs (10% and 40% respectively). The addition of 4-MU decreased mRNA expression of HAS 2 (p<0.001), HAS 3( p=0.03), CD44 (p<0.001) and RHAMM (p<0.001) in EECs and ESCs compared to control. There was no difference in HYAL2 in EECs and ESCs compared to control. EECs and ESCs attachment to PMCs significantly decreased (61% v 44%, p< 0.001, n=3; 67% vs 21%, p< 0.001, n=3; respectively) with addition of 4-MU(2mM) . 4-MU decreased attachment of EECs and ESCs to PMCs. This effect on the HA system appears to be mediated by a decrease in HAS2, HAS 3, CD44 and RHAMM.

Recent studies have suggested that GnRH agonists (GnRHags) protect ovarian function following chemotherapy. Here, we study the effect of a combination of GnRH antagonist (GnRHan) and GnRHag for gonadal protection from gonadotoxic chemotherapy in adolescent female rats. Cycling Sprague Dawley rats were treated at adolescent age. Thirty female rats were randomized to 5 treatment groups (n = 6/group): (1) placebo, (2) cyclophosphamide (CPA) alone, (3) GnRHan followed by GnRHag with placebo, (4) GnRHan followed by GnRHag with CPA, and (5) GnRHag with CPA. The main outcome measure was live birth rate (LBR), and secondary measures included rat weight, ovarian volume, and follicles. Group 2 had decreased LBR compared to all other groups. Group 4 and 5 had LBR similar to placebo. There was no difference in the ovarian volume. The CPA-alone group had decreased number of antral follicles compared to control. These studies demonstrate that the combination of GnRHan and GnRHag and GnRHag alone preserved fertility in female adolescent rats following gonadotoxic chemotherapy treatment. The addition of a GnRHan to a GnRHag does not confer a greater protective effect.

Sampson's theory suggests that endometrial cells transported retrograde through the fallopian tubes implant on peritoneal mesothelial cells (PMCs) to initiate early endometriotic lesions. Estrogen is a primary regulator of the growth and maintenance of endometriotic implants. Increased estrogen receptor- beta (ER-β) expression has been reported in endometriotic lesions compared to endometrium (Bulun 2012). Overexpression of ER-β has also been reported to increase invasion of endometriotic cells (Han 2015). Here, we examine the effect of ER-β knockdown on the attachment of primary mouse endometrial epithelial cells (EECs) to PMCs. In-vitro study Endometrial tissue and peritoneal mesothelial tissue from WT mice and ER-β knockdown mice was obtained. EECs and PMCs were separated and cultured using established methods (Kirk 1980). ER-β knockdown was confirmed with genotyping. EECs from WT and ER-β knockdown mice were used to assess their attachment to WT PMCs in an established in vitro assay (Lucidi 2005). Results were analyzed with student t- test. Compared to WT EECs (n=12), ER-β knockdown EECs (n=11) had significantly decreased attachment to PMCs in our established in vitro assay (p =0.003). ER-β knockdown significantly decreased attachment of EECs to PMCs in this murine model. These findings, along with prior studies, suggest that ER-β plays a role in the development of endometriotic lesions and may be a target for future therapies for managing endometriosis.

Objective: To determine whether sperm DNA integrity in normozoospermic male partners plays a role in idiopathic recurrent pregnancy loss (RPL).Design: Prospective, cohort study.Setting: Academic tertiary care center.Patient(s): Group I: 26 male partners of women with unexplained RPL. Group II: 31 normozoospermic males with proven fertility.Intervention(s): Semen samples were collected by masturbation after 48-72 hours of abstinence. After liquefaction at room temperature, semen analysis was performed according to World Health Organization standards. Only samples with > 20 x 10(6) spermatozoa/mL with at least 50% progressive sperm motility and 30 % normal morphology were selected for the study. DNA fragmentation of the sperm was assessed with TUNEL assay followed by flow cytometric analysis.Main Outcome Measure(s): Sperm DNA fragmentation in both groups.Result(s): Mean DNA fragmentation (mean +/- SD) was significantly more in men with RPL (36.8 +/- 5) compared with controls (9.4 +/- 2.7).Conclusion(s): Sperm DNA fragmentation may play a role in unexplained RPL despite normal semen analysis parameters. (C) 2016 by American Society for Reproductive Medicine.

Previous studies have shown endometrial cell (EC) CD44 and peritoneal mesothelial cell (PMC)-associated hyaluronan (hyaluronic acid [HA]) are involved in the attachment of endometrial stroma and epithelial cells to peritoneal mesothelium. Here we assess the CD44–HA interaction in the formation of the early endometriotic lesion using CD44–/– (knockout) mice. Using an established murine model and crossover technique, endometrial tissue from donor mice (wild type [WT] and CD44–/–) was used to induce endometriosis in recipient mice (WT and CD44–/–). Endometriotic lesions were visualized by fluorescent microscopy and confirmed by hematoxylin and eosin staining. Early endometriotic lesions were decreased when CD44–/– endometrium was placed in WT recipients and when WT endometrium was placed in CD44–/– recipients (P = .002). Early endometriotic lesions were also significantly decreased when both peritoneal and endometrial tissues lacked CD44 expression (P < .01). These studies demonstrate that both EC and PMC CD44 play a role in the development of early endometriotic lesion.

Sampson’s theory suggests that endometrial cells transported retrograde through the fallopian tubes implant on peritoneal mesothelial cells (PMCs) to initiate the development of the early endometriotic lesions. Our previous studies have shown that estradiol (E2) increases the attachment of EECs to PMCs. Here, we assess the expression of estrogen receptor (ER) isoforms α and β in menstrual endometrial epithelial cells (EEC) and the effects of ER α and β antagonists on EEC attachment to PMCs. In-vitro study. Menstrual endometrium was obtained from women with (n=11) and without (n=8) endometriosis and the EECs were separated using established techniques. EEC ER α and β expression was determined using mRNA extracted from primary EECs and quantitative RT-PCR . An established in-vitro assay was used to assess EEC attachment of estrogen treated and untreated cells to PMCs, with and without ER α [Faslodex (ICI)] and β [R,R tetrahydrochrysene (THC)] antagonists. Results were analyzed with paired t tests, ANOVA and Tukey’s post hoc test. Estrogen significantly increases EEC attachment to PMC compared to controls (p<0.05)1. ERα expression was greater than ERβ in EEC from women with and without endometriosis, and there was no significant difference in ER α/β expression. Both ICI and THC significantly inhibited the estrogen increased attachment to the PMCs (ICI: p= 0.04, THC: p=0.006). The ERα/β ratio was inversely correlated with the β-antagonist inhibition of attachment (R2= 0.91) and linearly correlated with the α-antagonist (R2= 0.95). EECs primarily express ER α and there is no significant difference in their ER α/β expression or ratio when comparing EECs from women with and without endometriosis. Both ICI and THC inhibit estrogen induced attachment of EEC to PMCs and this effect is correlated with the ER α/β ratio. These findings may contribute to future preventative treatment for endometriosis.

Abstract The endometrium is the major target of oncogenesis in the uterus. Endometrial tissue undergoes several cycles of disintegration and repair during women's reproductive years. The involvement of repair mechanisms that include transforming growth factor beta (TGF-beta) pathway may entail epithelial msenchymal transition (EMT) known to be involved in tissue repair. This suggests that the EMT program is inherent in endometrial epithelial cells and may be subverted during the development of invasive disease, whether benign such as endometriosis or malignant such as endometroid endometrial carcinoma. We therefore examined candidate mechanistic modulators of EMT in normal and malignant endometrial epithelial cells as well as endometrial carcinoma samples using cell signaling, gene expression and epigenetic regulation methodologies. Our evidence strikingly show that although both TGF-beta and epidermal growth factor (EGF) stimulated primary normal endometrial epithelial cell invasiveness, EGF was the main inducer of EMT in these cells. EGF also strongly induced EMT in endometrial cancer cells. In normal and malignant cells, Raf-1/MAPK mediated EGF actions. Interestingly, EGF stimulated epithelial adhesion molecule (EpCAM) cleavage and internalization of its intracellular domain EpICD into the nucleus to activate EMT-related genes such as mesenchymal cadherins. This was associated with loss of the epithelial marker E-cadherin and upregulation of its negative regulator Snail1. The involvement of EMT in aggressive endometrial malignancy was further confirmed in a permissive transcriptional epigenetic profile of mesenchymal cadherins in endometrial cancers with poor survival compared to a silenced epigenetic signature in less aggressive tumors. This epigenetic regulation including DNA methylation and histone modification was mediated by EpCAM, as shown in EpCAM-knockdown endometrial cancer cells, suggesting that EpCAM binding to target promoters maintains an open transcriptional conformation. Combined, our data suggest that while plasticity is required for endometrial regeneration during the menstrual cycles, committed epigenetic signatures may alter the path of normal tissue repair to aberrant growth associated with benign tumors or malignant growth. In concert, activation by cell signaling such as EGF/EpCAM may determine the extent of invasiveness and disease progression. Citation Format: Ya-Ting Hsu, Joseph Liu, Peter A. Binkley, Robert S. Schenken, Rajshwar R. Tekmal, Tim H.-M. Huang, Nameer B. Kirma. Parallel EMT pathways mediated by epidermal growth factor, EpCAM and mesenchymal cadherins in benign endometriotic lesions and endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3326. doi:10.1158/1538-7445.AM2014-3326

ObjectiveOvarian steroids are primary regulators of the growth and maintenance of established endometriotic implants; however, little is known about their role in the development of the early endometriotic lesion, and specifically implants on the ovarian stroma and the development of endometriomas. Here, we assessed the effect of estradiol (E2) and progesterone (P4) on endometrial epithelial (EEC) and endometrial stromal cell (ESC) attachment and invasion to normal ovarian surface epithelium (NOSE) cells.DesignIn-vitro study.Materials and MethodsAn immortalized ESC line (HESC) was used and grown to subconfluence. EECs were isolated from endometrial biopsy specimens obtained from women with a laparoscopic diagnosis of endometriosis, and were also grown to subconfluence. Cells were treated with E2 (0.01, 0.05, and 0.1 μg/mL) or P4 (1, 10, and 100 ng/mL) or a combination of E2 (0.1 μg/mL) and P4 (1, 10, and 100 ng/mL). Attachment and invasion of E2, P4, and E2/P4-treated and -untreated cells to NOSE cells was assessed using an established in vitro assay.1,2ResultsE2 increased attachment of ESCs (p<0.001) and EECs (p=0.04). P4 alone decreased attachment, but not significantly (p=0.14). When compared to E2 alone, E2 and P4 decreased attachment of ESCs (p=0.003), but not EECs. E2 increased invasion of EECs (p<0.01), while the combination of E2 and P4 showed a P4 dose-dependent decrease in EEC invasion (p=0.005).ConclusionE2 alone increases attachment of ESCs and EECs to NOSE cells. P4 alone has no significant impact on ESC or EEC attachment. The combination of E2 and P4 shows a decrease in ESC attachment and a decrease in EEC invasion. These findings are consistent with previous findings that E2 promotes attachment of endometrial cells to peritoneal mesothelial cells and this effect is mitigated by higher doses of P4.1. F&S 84:16-21, 2005 2. F&S 90: 1487-95, 2008. ObjectiveOvarian steroids are primary regulators of the growth and maintenance of established endometriotic implants; however, little is known about their role in the development of the early endometriotic lesion, and specifically implants on the ovarian stroma and the development of endometriomas. Here, we assessed the effect of estradiol (E2) and progesterone (P4) on endometrial epithelial (EEC) and endometrial stromal cell (ESC) attachment and invasion to normal ovarian surface epithelium (NOSE) cells. Ovarian steroids are primary regulators of the growth and maintenance of established endometriotic implants; however, little is known about their role in the development of the early endometriotic lesion, and specifically implants on the ovarian stroma and the development of endometriomas. Here, we assessed the effect of estradiol (E2) and progesterone (P4) on endometrial epithelial (EEC) and endometrial stromal cell (ESC) attachment and invasion to normal ovarian surface epithelium (NOSE) cells. DesignIn-vitro study. In-vitro study. Materials and MethodsAn immortalized ESC line (HESC) was used and grown to subconfluence. EECs were isolated from endometrial biopsy specimens obtained from women with a laparoscopic diagnosis of endometriosis, and were also grown to subconfluence. Cells were treated with E2 (0.01, 0.05, and 0.1 μg/mL) or P4 (1, 10, and 100 ng/mL) or a combination of E2 (0.1 μg/mL) and P4 (1, 10, and 100 ng/mL). Attachment and invasion of E2, P4, and E2/P4-treated and -untreated cells to NOSE cells was assessed using an established in vitro assay.1,2 An immortalized ESC line (HESC) was used and grown to subconfluence. EECs were isolated from endometrial biopsy specimens obtained from women with a laparoscopic diagnosis of endometriosis, and were also grown to subconfluence. Cells were treated with E2 (0.01, 0.05, and 0.1 μg/mL) or P4 (1, 10, and 100 ng/mL) or a combination of E2 (0.1 μg/mL) and P4 (1, 10, and 100 ng/mL). Attachment and invasion of E2, P4, and E2/P4-treated and -untreated cells to NOSE cells was assessed using an established in vitro assay.1,2 ResultsE2 increased attachment of ESCs (p<0.001) and EECs (p=0.04). P4 alone decreased attachment, but not significantly (p=0.14). When compared to E2 alone, E2 and P4 decreased attachment of ESCs (p=0.003), but not EECs. E2 increased invasion of EECs (p<0.01), while the combination of E2 and P4 showed a P4 dose-dependent decrease in EEC invasion (p=0.005). E2 increased attachment of ESCs (p<0.001) and EECs (p=0.04). P4 alone decreased attachment, but not significantly (p=0.14). When compared to E2 alone, E2 and P4 decreased attachment of ESCs (p=0.003), but not EECs. E2 increased invasion of EECs (p<0.01), while the combination of E2 and P4 showed a P4 dose-dependent decrease in EEC invasion (p=0.005). ConclusionE2 alone increases attachment of ESCs and EECs to NOSE cells. P4 alone has no significant impact on ESC or EEC attachment. The combination of E2 and P4 shows a decrease in ESC attachment and a decrease in EEC invasion. These findings are consistent with previous findings that E2 promotes attachment of endometrial cells to peritoneal mesothelial cells and this effect is mitigated by higher doses of P4.1. F&S 84:16-21, 2005 2. F&S 90: 1487-95, 2008. E2 alone increases attachment of ESCs and EECs to NOSE cells. P4 alone has no significant impact on ESC or EEC attachment. The combination of E2 and P4 shows a decrease in ESC attachment and a decrease in EEC invasion. These findings are consistent with previous findings that E2 promotes attachment of endometrial cells to peritoneal mesothelial cells and this effect is mitigated by higher doses of P4.

ObjectiveSampson's theory suggests that endometrial cells(ECs) transported retrograde through the fallopian tubes implant on peritoneal mesothelial cells (PMCs) to initiate the development of early endometriotic lesions (ELs). While ovarian steroids are primary regulators of established ELs, little is known about their role in the initial attachment of ECs to PMCs. Here, we assessed the effect of estradiol (E2) and progesterone (P4) on menstrual epithelial cell (EEC) attachment to PMC using an established model.DesignIn-vitro study.Materials and MethodsPrimary endometrial epithelial cells (EECs) and the PMC line LP9 were grown to subconfluence. EECs were treated with E2 (0.1 μg/ml) or a combination of E2 (0.1 μg/ml) and P4 (0.001, 0.01, and 0.1 μg/ml) for 48 hours and compared to untreated controls. EECs proliferation was assessed with the Cell Titer-Glo® Luminescent Cell Viability Assay. Attachment of treated and untreated cells to PMCs was assessed using an established in- vitro assay. Results were analyzed with ANOVA and Tukey's post hoc test.ResultsEEC proliferation was not affected by any steroid treatment. E2 alone and E2 with P4 (0.001 μg/ml) significantly increased attachment of EECs to PMCs (p<0.01) when compared to controls. When compared to controls, and E2 and P4 (0.001 μg/ml), E2 and P4 at higher concentrations (0.01 μg/ml and 0.1 mg) significantly decreased attachment of EECs to PMCs in a dose-dependent fashion.ConclusionE2 alone increases attachment of EECs to PMCs when compared to controls. The combination of E2 and P4 at the lowest concentration increases attachment of EECs to PMCs, while E2 and P4 at higher concentrations decreases attachment. These findings suggest that E2 and P4 are co-regulators of early endometriotic lesions and that P4 in higher doses may inhibit their development. ObjectiveSampson's theory suggests that endometrial cells(ECs) transported retrograde through the fallopian tubes implant on peritoneal mesothelial cells (PMCs) to initiate the development of early endometriotic lesions (ELs). While ovarian steroids are primary regulators of established ELs, little is known about their role in the initial attachment of ECs to PMCs. Here, we assessed the effect of estradiol (E2) and progesterone (P4) on menstrual epithelial cell (EEC) attachment to PMC using an established model. Sampson's theory suggests that endometrial cells(ECs) transported retrograde through the fallopian tubes implant on peritoneal mesothelial cells (PMCs) to initiate the development of early endometriotic lesions (ELs). While ovarian steroids are primary regulators of established ELs, little is known about their role in the initial attachment of ECs to PMCs. Here, we assessed the effect of estradiol (E2) and progesterone (P4) on menstrual epithelial cell (EEC) attachment to PMC using an established model. DesignIn-vitro study. In-vitro study. Materials and MethodsPrimary endometrial epithelial cells (EECs) and the PMC line LP9 were grown to subconfluence. EECs were treated with E2 (0.1 μg/ml) or a combination of E2 (0.1 μg/ml) and P4 (0.001, 0.01, and 0.1 μg/ml) for 48 hours and compared to untreated controls. EECs proliferation was assessed with the Cell Titer-Glo® Luminescent Cell Viability Assay. Attachment of treated and untreated cells to PMCs was assessed using an established in- vitro assay. Results were analyzed with ANOVA and Tukey's post hoc test. Primary endometrial epithelial cells (EECs) and the PMC line LP9 were grown to subconfluence. EECs were treated with E2 (0.1 μg/ml) or a combination of E2 (0.1 μg/ml) and P4 (0.001, 0.01, and 0.1 μg/ml) for 48 hours and compared to untreated controls. EECs proliferation was assessed with the Cell Titer-Glo® Luminescent Cell Viability Assay. Attachment of treated and untreated cells to PMCs was assessed using an established in- vitro assay. Results were analyzed with ANOVA and Tukey's post hoc test. ResultsEEC proliferation was not affected by any steroid treatment. E2 alone and E2 with P4 (0.001 μg/ml) significantly increased attachment of EECs to PMCs (p<0.01) when compared to controls. When compared to controls, and E2 and P4 (0.001 μg/ml), E2 and P4 at higher concentrations (0.01 μg/ml and 0.1 mg) significantly decreased attachment of EECs to PMCs in a dose-dependent fashion. EEC proliferation was not affected by any steroid treatment. E2 alone and E2 with P4 (0.001 μg/ml) significantly increased attachment of EECs to PMCs (p<0.01) when compared to controls. When compared to controls, and E2 and P4 (0.001 μg/ml), E2 and P4 at higher concentrations (0.01 μg/ml and 0.1 mg) significantly decreased attachment of EECs to PMCs in a dose-dependent fashion. ConclusionE2 alone increases attachment of EECs to PMCs when compared to controls. The combination of E2 and P4 at the lowest concentration increases attachment of EECs to PMCs, while E2 and P4 at higher concentrations decreases attachment. These findings suggest that E2 and P4 are co-regulators of early endometriotic lesions and that P4 in higher doses may inhibit their development. E2 alone increases attachment of EECs to PMCs when compared to controls. The combination of E2 and P4 at the lowest concentration increases attachment of EECs to PMCs, while E2 and P4 at higher concentrations decreases attachment. These findings suggest that E2 and P4 are co-regulators of early endometriotic lesions and that P4 in higher doses may inhibit their development.

OBJECTIVE:To investigate the expression and regulation of colony-stimulating factor 1 (CSF-1) and its receptor, C-FMS, in endometriosis.DESIGN:In vivo and vitro study.SETTING:University-based academic medical center.PATIENT(S):Reproductive-age women undergoing surgery for benign conditions.INTERVENTION(S):Peritoneal and endometrial tissue samples were obtained.MAIN OUTCOME MEASURE(S):CSF-1 and C-FMS expression.RESULT(S):Significantly higher CSF-1 levels were found in peritoneal fluid of patients with endometriosis compared with control subjects. Ectopic endometriotic tissue had 3.5-fold and 1.7-fold increases in CSF-1 and C-FMS expression, respectively, compared with eutopic tissue. Coculture of endometrial cells from either established cell lines or patient samples with peritoneal mesothelial cells (PMCs) led to increased expression of CSF-1 and C-FMS. A higher but nonsignificant increase in levels of C-FMS and CSF-1 was found in cocultures of endometrial epithelial cells from patients with endometriosis compared with those without endometriosis.CONCLUSION(S):Increased CSF-1 levels may contribute to endometriosis lesion formation and progression. Elevation in CSF-1 after coculture of endometrial cells with PMCs suggests that endometrial tissue may be a source of peritoneal CSF-1. Increased C-FMS expression in endometrial cells from women with endometriosis cocultured with PMCs suggests that endometrial tissue involved in lesion formation is highly responsive to CSF-1 signaling.

Abstract Ovarian and endometrial cancers are the 5th and 8th overall leading causes of female cancer related deaths in the US, respectively. According to the American Cancer Society, 21,990 new ovarian cancer cases and 46,470 new endometrial cancer cases were diagnosed in 2011. These cancers share a common mechanism of pathogenesis, epithelial-mesenchymal transition (EMT). EMT increases the invasiveness and progression of epithelial tumor cells into more aggressive and metastatic disease. Amplification of growth factor signaling is also common in malignancy. Epidermal growth factor receptor (EGFR) is amplified in 60-80% of ovarian and endometrial cancer and its overexpression is associated with increased invasiveness and poor prognosis. Studies have shown that specific cytokines, including transforming growth factor-β (TGFβ) and epidermal growth factor (EGF), interact with the intracellular MEK/ERK signaling cascade, a pathway involved in several malignant processes including EMT. Raf-1 is an intracellular MAPK signaling molecule that is recruited to membrane bound Ras upon activation by growth factors. Activation of Raf-1 includes phosphorylation at ser 338, which in turn activates the downstream MEK/ERK signaling cascade. Our data showed that EGF phosphorylates Raf-1 at ser 338 and ERK in endometrial cancer cells, which is inhibited by the specific Raf-1 antagonist GW5074. Little is known on the involvement of Raf-1 in the pathogenesis of ovarian or endometrial cancer. Our studies show that inhibition of Raf-1 by GW5074 significantly decreases TGFβ and EGF induced invasiveness of immortalized endometrial epithelial cells (EECs). EGF and to a lesser degree TGFβ, enhanced the survival of EECs under serum starvation. GW5074 inhibited this effect as evidenced by increased apoptosis and caspase 3/7 activity, suggesting that Raf-1 mediates EEC survival. While EGF had minimal effects on inducing ovarian cancer cell line SKOV3, the addition of GW5074 significantly reduced SKOV3 cell growth by half. Our data shows that the addition of EGF, but not TGF-β, to endometrial and ovarian epithelial cells induced EMT as evidenced by the disruption of cell-cell contact, loss of the characteristic “cobblestone” appearance of epithelial cells, increased motility and induction of the mesenchymal marker vimentin. Treatment with GW5074 of cells exposed to EGF reversed these changes. Our FACS analysis showed that EGF decreases the cell surface detection of EPCAM, an epithelial cell surface adhesion molecule, by more than half, further suggesting that EGF is involved in EMT in endometrial cancer cells. Our data suggests that multiple steps in the pathogenesis of both ovarian and endometrial cancers are mediated by Raf-1. Raf-1's involvement in inducing EMT suggests that Raf-1 may be a novel therapeutic target for ovarian and endometrial cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-14. doi:1538-7445.AM2012-LB-14