{"paper_id":"c5cb1755-cdc5-47e3-befb-53df3fa70f7c","body_text":"Edinburgh Research Explorer \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nCharacterization of molecular changes in endometrium\nassociated with chronic use of progesterone receptor\nmodulators: ulipristal acetate vs. mifepristone\nCitation for published version:\nKannan, A, Bhurke, A, Sitruk-Ware, R, G. Lalitkumar, P, Gemzell-danielsson, K, Williams, A, Taylor, RN,\nBagchi, MK & Bagchi, IC 2017, 'Characterization of molecular changes in endometrium associated with\nchronic use of progesterone receptor modulators: ulipristal acetate vs. mifepristone', Reproductive\nSciences, pp. 1933719117746764. https://doi.org/10.1177/1933719117746764\nDigital Object Identifier (DOI):\n10.1177/1933719117746764\nLink:\nLink to publication record in Edinburgh Research Explorer\nDocument Version:\nPeer reviewed version\nPublished In:\nReproductive Sciences\nGeneral rights\nCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)\nand / or other copyright owners and it is a condition of accessing these publications that users recognise and\nabide by the legal requirements associated with these rights.\nTake down policy\nThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer\ncontent complies with UK legislation. If you believe that the public display of this file breaches copyright please\ncontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and\ninvestigate your claim.\nDownload date: 13. Jun. 2026\n\nCharacterization of molecular changes in endometrium associated with chronic use of \nprogesterone receptor modulators: ulipristal acetate vs. mifepristone \nAthilakshmi Kannan1, Arpita Bhurke1, Regine SitrukIWare3, Parameswaran G. Lalitkumar4,\nKristina GemzellIDanielsson4, Alistair R. W. Williams5, Robert N. Taylor6, Milan K Bagchi2,\nan\nd Indrani C Bagchi1#\n1Department of Comparative Biosciences, 2Department of Molecular and Integrative Physiology,\nUn\niversity of Illinois Urbana/Champaign, Urbana, IL, 3Center for Biomedical Research,\nPop\nulation Council, New York, NY, 4Department of Obstetrics and Gynecology, Karolinska\nUniversity Hospital, Stockholm, Sweden, 5Department of Pathology, University of Edinburgh,\n6Department of Obstetrics and Gynecology, Wake Forest School of Medicine, WinstonISalem, \nNor\nth Carolina 27157 \n# To whom correspondence should be addressed: \nIndrani C. Bagchi, EImail: ibagchi@illinois.edu \nPage 1 of 28\n1\nReproductive Sciences\n\nAbstract \nUlipristal acetate (UPA) is a selective progesterone receptor modulator (PRM), which is used as \nan emergency contraceptive in women. Recent studies demonstrated the efficacy of a UPA \ncontraceptive vaginal ring (UPAICVR) as a blocker of ovulation. However the endometrium of \nwomen exposed to UPA over a sixImonth period display glandular changes, termed PRMI\nassociated endometrial changes (PAECs). We, therefore, investigated whether UPAIinduced \nPAECs are associated with altered expression of the transcription factor HAND2 whose down \nregulation is observed in endometrial epithelial hyperplasia and cancer. Our results showed that \nwhile exposure to mifepristone, a wellIknown PRM, leads to suppression of endometrial \nHAND2 expression, longIterm exposure to UPAICVR did not cause down regulation of this \nmarker. Further studies, using human primary endometrial stromal cells, confirmed that whereas \nmifepristoneImediated suppression of HAND2 elevated the levels of its downstream target \nfibroblast growth factor 18, UPA did not significantly alter the expression of this growth factor. \nA rationale for the differential regulation of HAND2 by these PRMs was provided by our \nobservation that mifepristoneIbound progesterone receptors turn over at a faster rate than those \nbound to UPA. Collectively, these results support the selective effects of different PRMs and \nindicate that chronic exposure to UPA does not alter the HAND2 pathway whose dysregulation is \nlinked to complex atypical endometrial hyperplasia and cancer. The results from this study \ninvolving a limited number of clinical samples should pave the way for a larger study to \ndetermine the safety of UPA for longIterm use. \nKey words: Ulipristal acetate; Mifepristone; Contraception; Endometrium; HAND2 \nPage 2 of 28\n2\nReproductive Sciences\n\nIntroduction \nIt is estimated that 225 million women worldwide lack access to effective and acceptable \ncontraceptive methods. Therefore, the development of novel clinically safe and effective \nmethods of fertility control remains a necessity. The steroid hormone progesterone (P) acting \nthrough its nuclear receptor critically controls the ovulatory process as well as endometrial \nfunction in the human. Progesterone receptor modulators (PRM) are synthetic compounds that \ninteract with the progesterone receptor (PR) to suppress ovulation and/or induce endometrial \natrophy, resulting in amenorrhea, a condition that is perceived favorably in many cultures around \nthe world 1. Therefore, the development and use of PRMs as contraceptives is of particular\ninterest. \nUlipristal acetate (UPA), also referred to as VA/CDBI2914, is a new and promising PRM 2I6.\nUPA has been approved as an emergency contraceptive 7, 8 in the United States and abroad and\nas a treatment for heavy menstrual bleeding due to uterine fibroids 9, 10 in Canada and Europe.\nSuccessful use of this PRM as an emergency contraceptive has raised the possibility that a \nsimplified continuous delivery of UPA could improve longIterm contraceptive safety and \nefficacy and compliance. With this goal in mind, a UPA contraceptive vaginal ring (UPAICVR) \nwas designed for longIterm contraceptive use by the Population Council, New York. In a study \nconducted by the Council, healthy women with normal baseline ovulation were randomized to \nreceive UPAICVR for two consecutive 12Iweek treatment periods, followed by a recovery cycle \n11. The results from these studies indicated that the UPAICVR has the potential to become an \neffective longIacting, userIcontrolled contraceptive. However, endometrial biopsies taken at the \nend of the treatment period displayed histological glandular changes, described as PRMI\nassociated endometrial changes (PAECs) 11. While these endometrial changes are considered to\nbe benign due to the lack of cytological atypia12 an inIdepth study is needed to confirm the\nabsence of any endometrial abnormality, including hyperplasia, following chronic PRM use. \nThe endometrium, the innermost layer of the uterus, undergoes proliferation and differentiation \nin a cyclical manner in response to the steroid hormones, 17βIestradiol (E) and P acting via their \ncognate receptors 13I15. While E acting via ERα f unctions as a mitogen and promotes the growth\nand proliferation of the endometrial epithelium in a cyclical fashion during the reproductive \nPage 3 of 28\n3\nReproductive Sciences\n\ncycle, P acting via PR inhibits EIinduced epithelial proliferation and causes differentiation. \nUncontrolled proliferation of the endometrial epithelium results in alterations of glandular \narchitecture (shape and size) and an increase in endometrial glandItoIstroma ratio, leading to \nendometrial hyperplasia 15I17. The majority of cases of endometrial hyperplasia are associated\nwith compromised P signaling that fails to oppose E signaling 17I19.\nWe have previously shown that the transcription factor HAND2 (HeartI and neural crest \nderivativesIexpressed protein 2), which is regulated by the PR present in the endometrial stroma, \nis a key mediator of the wellIknown antiIproliferative effect of P on the endometrial epithelium \n20. HAND2 suppresses the production of several stromal fibroblast growth factors (FGFs), which \nact in a paracrine manner via the FGF receptors to promote epithelial proliferation. Therefore, in \nthe absence of HAND2, the endometrial epithelium undergoes unbridled FGFIinduced \nproliferation that leads to complex atypical hyperplasia. It is of interest to note that the HAND2 \ngene locus is prone to epigenetic alterations. Our recent studies revealed that the HAND2 gene is \na hypermethylated and silenced in endometrial hyperplasia and cancer 21. When compared to\nother frequent DNAIbased alterations in endometrial cancers, such as p53, PTEN, and PIK3CA \nmutations, HAND2 hypermethylation was found to be the most common 21. Since the down\nregulation of HAND2 expression is linked to endometrial hyperplasia and cancer, we examined \nthe expression of this factor in endometrial biopsies of women exposed to UPAICVR for 24 \nweeks. We also compared the endometrial effects of UPA with those of mifepristone, a wellI\nknown PRM. \nMa\nterials and Methods \nEndometrial biopsies \nEndometrial biopsy samples were obtained using either a Pipelle (Cooper Surgical, Trumbull, \nCT, USA) (DR, Chile) or an Explora (Cooper Surgical) (Oregon) device. A portion of wach \nsample was placed for use in 10% neutral buffered formaldehyde for histology and \nimmunohistochemistry studies. \nPage 4 of 28\n4\nReproductive Sciences\n\nIn vitro decidualization of human endometrial stromal cells (HESC) \nOur studies involving primary HESC cultures follow the regulations stated for the protection of \nhuman subjects participating in clinical research and are approved by the institutional review \nboards of Emory University, Wake Forest University (WinstonISalem, North Carolina), and the \nUniversity of Illinois at UrbanaIChampaign (UIUC). Endometrial samples from the early \nproliferative stage of the menstrual cycle were obtained by Pipelle biopsy at Emory University \nand Wake Forest Medical Centers from fertile, regularly cycling volunteers with no sign of \nuterine abnormality, providing written informed consent as described previously 21, 22.\nC\nells were cultured in DMEM/FI12 medium (Invitrogen) supplemented with 5% (vol/vol) fetal \nbovine serum (Hyclone), 50 µg/mL penicillin, and 50 µg/mL streptomycin (Invitrogen). For in \nvitro differentiation, the cells were treated with differentiation cocktail composed of 10 nM E \n(Sigma), 1 µM progesterone (Sigma), 0.5 mM 8IbromoadenosineIcAMP (Sigma), 10 µM UPA \nor mifepristone in DMEM/FI12 medium (Invitrogen) supplemented with 2% (vol/vol) charcoal \ndextranIstripped fetal bovine serum for 0I6 days. At the end of the culture (2 or 6 days), the cells \nwere detached from the plates, counted, and stored at I80°C for RNA extraction. Additionally \nsome cells were fixed for immunocytochemical (ICC) analysis. In some experiments, the cells \nwere treated with differentiation cocktail composed of 10 nM E, 1 µM progesterone, 0.5 mM 8I\nbromoadenosineIcAMP, 5 µM UPA or mifepristone in DMEM/FI12 medium supplemented with \n2% (vol/vol) charcoal dextranIstripped fetal bovine serum for 0I6 days. Cultures were terminated \nat days 2 to 6 for RNA extraction. \nChemicals, reagents, and antibodies \nProgesterone (P), 17βIestradiol (E), naphthol ASIMX phosphate, Fast Blue RR (4I\nbenzoylaminoI 2,5Idimethoxyaniline diazonium), collagenase, pancreatin, dimethyl sulfoxide \n(DMSO), 8Ibromoadenosine 3', 5'Icyclic monophosphate salt (cAMP), and Trypan blue were \npurchased from Sigma. Hanks Balanced Salt Solution (HBSS), dispase, Dulbecco’s modified \nEagle mediumIF12 medium HEPES, no phenol red (DMEM/F12), PenicillinIStreptomycin, and \nFungizone, were purchased from Life Technologies. Fetal bovine serum (FBS) was purchased \nfrom Fisher Scientific. FluoromountIG with DAPI was purchased from eBiosciences. \nPage 5 of 28\n5\nReproductive Sciences\n\nEndometrial sections or endometrial stromal cells were incubated with one or more of the \nfollowing primary antibodies: heartI and neural crest derivativesIexpressed transcript 2 \n(HAND2, 1:250, Santa Cruz Biotechnology antibody SCI9409), FGF18 (1:100, Santa Cruz \nBiotechnology antibody SCI393471), PR (1:100, DAKOIA0098), and PRB22 (1:300, Cell\ns\nignaling CSTI31575). The fluorescentItagged secondary antibodies and normal donkey serum \nwere purchased from Jackson ImmunoResearch. The following secondary antibodies were used: \nrhodamine or Cy3 donkey antiIrabbit, 488 donkey antiIrabbit, 488 donkey antiImouse, 488 \ndonkey antiIgoat, and Cy3 donkey antiIrat. \nImmunohistochemistry (IHC) and immunocytochemistry (ICC) \nParaffinIembedded endometrial biopsy sections were subjected to IHC as described previously. \nTissue sections were deparaffinized in xylene, rehydrated through a graded series of ethanol, and \nwashed in tap water. For most of the immunostaining, antigen retrieval was performed in a \npressure cooker in 10 mM sodium citrate buffer (pH 6.0) for 20 min and then the slides were \ncooled to room temperature. The sections were washed between steps (three times for 5 min \neach) using 1x phosphateIbuffered saline solution containing 0.05% Tween 20 (PBSIT). \nNonspecific binding was inhibited by incubating the sections with 10% normal serum for 1 h at \nroom temperature. After the serum block, sections were incubated overnight at 4°C with the \ndiluted antibody solution in PBSIT containing 1% normal serum. \nLabeling was visualized by incubation with a fluorescentItagged secondary antibody for 1 h at \nroom temperature. All incubations were done using a humidified chamber protected from light. \nSlides were mounted using a mounting solution containing DAPI. Pictures were taken using the \nOlympus BX51 microscope equipped for fluorescent imaging and connected to a Jenoptik \nProgRes C14 digital camera with cImount interface containing a 1.4 Megapixel CCD sensor. \nFluorescent images were processed and merged using Adobe Photoshop Extended CS6 (Adobe \nSystems). HSCOREs were determined as described previously 23.\nF\nor ICC analysis of HESC, cells were fixed in 10% NBF for 10 min, and then washed with PBS. \nCells were then permeabilized using PBS containing 0.1% Triton X for 10 min at room \ntemperature. Nonspecific binding was inhibited by incubating the sections with 10% normal \nPage 6 of 28\n6\nReproductive Sciences\n\nserum for 1 h at room temperature. After the serum block, the cells were incubated overnight at \n4°C with the diluted antibody solution in PBS containing 1% normal serum. Labeling was \nvisualized by incubation with a fluorescentItagged secondary antibody for 1 h at room \ntemperature. One drop of mounting solution containing DAPI was added to each well to stain the \nnucleus. Pictures were taken using the Olympus Ix70 inverted microscope adapted to a \nDiagnostic Instrument digital camera containing a 2.0 Megapixel CCD sensor. Fluorescent \nimages were merged and processed using Adobe Photoshop Extended CS6. \nQuantitative real time PCR analysis (qPCR) \nTotal RNA was isolated from endometrial cells using a standard TRIzolIbased protocol. The \nRNA concentration of each sample was determined at 260 nm using a Nanodrop ND1000 UVI\nVis spectrophotometer (Nanodrop Technologies). RNA samples were reverse transcribed using \nthe High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the \nmanufacturer's instructions. Real time quantitative PCR (qPCR) reactions were carried out using \nSYBRIgreen master mix (Applied Biosystems) in a 7500 Applied Biosystems RealItime PCR \nmachine (Applied Biosystems). For each sample, the mean threshold cycle (Ct) was calculated \nfrom Ct values obtained from three replicates. The normalized ∆Ct in each sample was \ncalculated as mean Ct of target gene subtracted by the mean Ct of the reference gene. The fold \nchange of gene expression in each sample relative to a control was generated using the 2−∆∆Ct \nmathematical model for relative quantification of quantitative PCR. The mean fold induction and \nSEM were calculated from at least three or more independent experiments. The housekeeping \ngene RPLP0 (36B4), which encodes a ribosomal protein, was used as a reference gene. \nStatistical analyses \nExperimental data for studies related to UPAICVR were collected from 12 independent subjects. \nFor each subject, 4 endometrial biopsy samples were obtained. Biopsy 1 was an endometrial \nspecimen obtained before administration of UPAICVR, biopsies 2 and 3 were endometrial \nspecimens obtained after each 12Iweek period in which UPAICVR released UPA daily, and \nbiopsy 4 was obtained following a 4Iweek postItreatment recovery period. Results from \nmifepristone studies were obtained from 6 independent clinical samples. Data related to primary \nHESCs were collected from 3 independent clinical samples, which were subjected to the same \nPage 7 of 28\n7\nReproductive Sciences\n\nexperimental conditions. All numerical data are expressed as mean ± SEM. When experimental \nsamples were compared with control samples, statistical significance between the control and \nexperimental sample was determined using the Student t test. A P value of ≤.05 was considered \nto be significant. \nResults \nExpression of HAND2 is unaltered in human endometrial biopsies exposed to UPA-CVR \nHuman endometrial biopsies were obtained from three different clinics located in the United \nStates, Dominican Republic, and Chile. We have analyzed a total of 12 independent subjects. \nFor each subject, 4 endometrial biopsy samples (biopsies 1I4) were obtained. Biopsy 1 is an \nendometrial specimen obtained before administration of UPAICVR during the luteal phase based \non urine LH determinations. Biopsies 2 and 3 are endometrial specimens obtained after each 12I\nweek period in which UPAICVR released 1.5 mg or 2.5mg UPA daily. Biopsy 4 was obtained \nfollowing a 4Iweek postItreatment recovery period in the luteal phase, determined as above. \nFigure 1 shows representative endometrial samples at baseline, before administration of UPAI\nCVR (panel A), after exposure to UPAICVR (panel B), and in the recovery phase (panel C). \nBaseline samples show normal midIsecretory phase endometrium. Upon exposure to UPAICVR, \nthe glands show variable cystic dilatation, mildly disordered architecture, nonIphysiological \nsecretory appearances, and coexistent mitoses and apoptotic bodies. The stroma is compact, nonI\ndecidualized and contains occasional thickIwalled vessels. These features are characteristic of \nPRMIassociated endometrial changes or PAECs. In the recovery phase, the endometrium \nexhibits normal early secretory phase appearances. \nTo examine the molecular changes in the endometrium following prolonged exposure to UPAI\nCVR, we investigated the expression of HAND2 in the biopsy specimens. An intense nuclear \nstaining specific to HAND2 was observed in the endometrial stromal cells of preItreatment \nbiopsyI1 specimen (Figure 2). This expression of stromal HAND2 remained unaltered in the \nbiopsies exposed to UPA (biopsyI2, and I3, Figs. 2B and C) and in the postItreatment biopsy \nPage 8 of 28\n8\nReproductive Sciences\n\nspecimen (biopsyI4, Fig. 2D). To more accurately quantify the immunohistochemical findings, \nHSCOREs were analyzed. HSCORES of endometrial HAND2 immunostaining revealed no \nsignificant changes across the treatment period (Figure 3). These results indicate that a CVR \nreleasing 1.5 or 2.5mg/day of UPA for 24 weeks does not affect HAND2 expression. \nExpression of HAND2 is reduced in human endometrial biopsies exposed to mifepristone \nWe also analyzed the expression of HAND2 in endometrial biopsies collected from women \nexposed to mifepristone, a wellIknown PRM. In this study, 50 mg of oral mifepristone was \nadministered every other day for 12 weeks. Endometrial biopsies were taken in the secretory \nphase of the last week of mifepristone treatment. Luteal phase biopsies from unexposed women \ndemonstrated robust expression of HAND2 in the nuclei of stromal cells, as expected. In \ncontrast, endometrial biopsies of women treated with mifepristone showed a significant decline \nin the expression of HAND2 (Figure 4). Quantification of HAND2 immunoIpositive cells in the \nstroma revealed greater than 80% reduction in HAND2 expression in mifepristoneIexposed \nbiopsies when compared to unexposed controls. Collectively, these results suggest that UPAI\nCVR and mifepristone have differential effects on endometrial HAND2 expression. It is possible \nthat the differences are due to the pharmacology of the PRM compounds, their doses, duration or \nroute of administration. \nUPA and mifepristone differentially regulate HAND2 and FGF18 expression in cultured \nhuman endometrial stromal cells \nTo directly examine the pharmacological effects of UPA and mifepristone on HAND2 \nexpression in the endometrial stroma under identical study conditions, we utilized a wellI\nestablished human endometrial stromal cell culture system. In this system, undifferentiated \nstromal cells isolated from human endometrial biopsies (HESC) obtained from normal women in \nthe proliferative stage of the menstrual cycle were placed in culture and subjected to \ndecidualization in response to a hormonal mixture containing 10 nM E, 1 µM P, and 0.5 mM 8I\nbromoIcAMP 24, 25. Under the treatment conditions, cells were treated with the hormonal mixture\nw\nith or without 10 µM UPA or mifepristone. HESCs were cultured in the presence of hormones \nPage 9 of 28\n9\nReproductive Sciences\n\nwith or without PRMs for up to six days. HAND2 mRNA expression was reduced when \nendometrial stromal cells were exposed to UPA or mifepristone for two days, compared to cells \nnot treated with PRMs (Figure 5). While UPA exposure reduced HAND2 expression by 20%, \ntreatment with mifepristone resulted in almost 40% reduction in HAND2 expression (P<0.05). \nFurther, the inhibitory effect of mifepristone on HAND2 expression increased in severity with \nlonger duration of treatment. Stromal cells exposed to mifepristone for six days displayed more \nthan 80% reduction in HAND2 expression when compared to untreated control cells. In contrast, \ntreatment with UPA for six days had milder effects, resulting in a 30% reduction in HAND2 \nexpression (P<0.05). Consistent with the RNA profile, immunocytochemical (ICC) analysis \nrevealed similar reductions in HAND2 in the UPA (Figure 6, panel B)I and mifepristone (Figure \n6, panel C) Iexposed endometrial stromal cells compared to UPAI or vehicleItreated stromal \ncells (Figure 6, panel A). \nTo further investigate the differential effects of UPA and mifepristone on HAND2 expression, \nwe reduced the levels of PRMs from 10Ifold to 5Ifold molar excess of P. Human endometrial \nstromal cells were cultured in the presence of hormones with or without 5 µM UPA or \nmifepristone for up to six days. HAND2 mRNA expression was monitored on day 2, day 3, day \n4, day 5, and day 6 after initiation of the culture. As shown in Figure 7, treatment of HESC with \n5 µM UPA did not affect the expression of HAND2 on days 2 to 6 upon initiation of the culture. \nBy contrast, administration of 5 µM mifepristone led to a significant down regulation of HAND2 \nexpression in HESCs. The decline in HAND2 expression was evident on day 2 and continued up \nto day 6 of culture. \nOur previous studies have shown that Hand2 expression in the stroma suppresses the production \nof fibroblast growth factors (FGFs) and inhibits cell proliferation 20. In the absence of Ha nd2,\ncontinued induction of FGFs in the stroma activates FGF receptor (FGFR) signaling in the \nepithelium to promote cell proliferation 20. Consistent with this observation, a recent study \nr\neported a decrease in HAND2 expression and marked increase in the levels of FGF18 in human \nendometrial adenocarcinoma 26. MifepristoneItreated endometrial stromal cells demonstrated a\nm\narked increase in the levels of FGF18 mRNA (Figure 8, upper panel) and protein (Figure 8, \nlower panel) compared to vehicleItreated controls. In contrast, endometrial stromal cells exposed \nPage 10 of 28\n10 \nReproductive Sciences\n\nto UPA did not exhibit alterations in FGF18 expression (Figure 8). Collectively, these results \nsupport our in vivo findings and indicate that endometrial stromal cells cultured with \nmifepristone or UPA under identical in vitro conditions exhibit differential effects on HAND2 \nand FGF18 expression. \nMifepristone and UPA differentially affect PR stability in human endometrial stromal cells \nBoth UPA and mifepristone are known to regulate the function of a tissue by modulating the \nactivity of PR, so it is interesting that endometrial stromal cells display differential gene \nexpression when exposed to the same concentrations of these two PRMs. We considered the \npossibility that the stability of endometrial PR might be regulated differentially by mifepristone \nand UPA. To investigate this possibility, we determined the expression of total PR protein in \nprogesteroneI, UPAI, or mifepristoneItreated HESC by ICC. Cells exposed to progesterone or \nUPA for 6 days displayed prominent nuclear PR staining, while those treated with mifepristone \nshowed markedly reduced levels of PR (Figure 9). \nOur recent studies revealed that the PR isoform PRIB plays a predominant functional role during \nhuman endometrial stromal differentiation by controlling the expression of a large number of \ntarget genes, including HAND2 21. We noted distinct expression of PRIB 22 in the nuclei of\np\nrogesteroneI or UPAItreated stromal cells (Figure 10). In contrast, nuclei of stromal cells \nexposed to mifepristone were mostly devoid of PRIB expression. Taken together, these results \nare consistent with our view that UPA and mifepristone differentially affect PR stability in \nhuman endometrial stromal cells and this is reflected in altered expression of PR target genes, \nsuch as HAND2, in response to these ligands in the endometrial stroma. \nDi\nscussion \nA critical balance of E and P drives proper endometrial stromalIepithelial crosstalk and \nmaintains normal uterine physiology. Disruption of PR function results in unopposed E action, \ncausing epithelial hyperplasia and potentially carcinoma 17I19. HAND2, a PRIregulated gene in\nt\nhe stromal cells, mediates the antiproliferative action of P to regulate endometrial epithelial \nPage 11 of 28\n11 \nReproductive Sciences\n\nfunction. Loss of the antiproliferative actions of P in the uterus has been linked to EIdependent \nendometrial cancer 27. Indeed, our recent study showed that the H AND2 gene is hypermethylated\nin premalignant endometrial lesions compared to normal endometrium and its expression is \nsuppressed in endometrial hyperplasia and cancer 21. HAND2 has therefore emerged as a key\nmolecular alteration in endometrial cancer that could potentially be employed as a biomarker for \nearly detection of endometrial cancer. \nTo determine the clinical utility of UPA as a longIterm contraceptive, it is critical to assess \nwhether this compound, which effectively blocks PR action and ovulation, also alters the critical \nbalance of E and P in the endometrium. Evaluation of endometrial histology following chronic \nUPA treatment revealed the presence glandular changes, known as PAECs, which did not show \nany cytological atypia, a characteristic feature of hyperplasia and cancer. However, routine \nhistological examination of the endometrium may not provide molecular information related to a \nsubtle imbalance of EI and PIdependent signaling that may arise due to PRM exposure. In this \nstudy, we show that the expression of HAND2, which critically regulates the balance of PI and \nEI dependent signaling in the endometrium, is unaffected in women exposed to UPAICVR \ncontinuously for 24 weeks. Since downregulation of endometrial HAND2 has been linked to \ncomplex atypical hyperplasia and cancer, unaltered expression of this factor gives us confidence \nthat exposure to the studied dose of UPA by the vaginal route of administration does not disrupt \nthe critical balance of EI and PI dependent signaling necessary for normal endometrial \nphysiology. \nIn contrast, we found that endometrial biopsies from women treated with mifepristone for 12 \nweeks displayed a dramatic downregulation of HAND2. Differential effects of UPA and \nmifepristone on HAND2 expression were confirmed in endometrial stromal cells cultured under \nidentical conditions, suggesting distinct mechanisms underlie the actions of these PRMs. \nAnalysis of PR in endometrial stromal cells following in vitro exposure to PRMs demonstrated \nthat mifepristone down regulates the PR levels, whereas equivalent molar concentrations of UPA \ndid not have these effects. This suppression of cellular PR levels by mifepristone is consistent \nwith previous reports that addition of mifepristone to a progestogenIonly regimen of \ncontraception leads to downregulation of PRIB 28. Additionally, recent studies have \nPage 12 of 28\n12 \nReproductive Sciences\n\ndemonstrated that administration of mifepristone to an endometrial coIculture system causes \nsuppression of PR expression compared to vehicleItreated controls 29. While the mechanism by\nw\nhich mifepristone causes PR turnover remains unclear, we believe that this downregulation of \nPR is in part responsible for the dramatic suppression of HAND2 expression observed in \nresponse to mifepristone compared to UPA. \nWe have previously shown that HAND2 mediates the antiproliferative effects of P by \nsuppressing the production of the FGF growth factors that mediate the growthIinducing effects \nof E on the endometrial epithelium. In the EIdominant proliferative endometrium, FGFs secreted \nfrom the stroma act on the FGFR(s) in the epithelium to promote proliferation 20. Following\no\nvulation and in response to P production and signaling, HAND2 is induced in stromal cells, \ncausing inhibition of FGF synthesis and attenuation of epithelial proliferation. Disruption of PR \nfunction in the endometrium therefore runs the risk of increasing FGF signaling, leading to \ninappropriate uterine epithelial growth, hyperplasia and cancer. Similar findings were noted in \nthe epithelial glands of rhesus macaques treated with mifepristone 30 . Indeed, a recent study has\ns\nhown downregulation of HAND2 and upregulation of FGF18 in human endometrial \nadenocarcinoma 26. We demonstrate that administration of mifepristone to cultured endometrial\ns\ntromal cells caused inhibition of HAND2 expression and a concomitant enhancement of FGF18 \nexpression. However, treatment of endometrial stromal cells with UPA did not significantly \naffect the expression of either HAND2 or FGF18, further confirming that UPA does not \nsignificantly alter the PIdependent antiproliferative pathways in the endometrium. \nIn summary, this study shows that UPA and mifepristone exhibit differential effects on \nendometrial gene expression in vivo and in vitro, apparently due to differences in stability of PRs \nin response to these PRMs. It also confirms that chronic exposure to UPAICVR over a 24Iweek \nperiod does not lead to adverse effects, such as suppression of the expression of HAND2, which \nis reported to occur in endometrial hyperplasia and cancer. The results from this study involving \na limited number of clinical samples should pave the way for a larger study to determine the \nsafety of UPA for longIterm use. \nPage 13 of 28\n13 \nReproductive Sciences\n\nAcknowledgements: The study was supported in part by a grant from the NICHD/NIH U54 HD \n29990. \nFigure legends \nFig. 1: Endometria of women exposed to UPA-CVR display PRM-associated endometrial \nchanges (PAECs). Haematoxylin & eosin staining of endometrial sections obtained from normal \nmidIsecretory phase endometrium (panel A), UPAICVR releasing 2.5mg UPA daily for two \nconsecutive 12Iweek treatment periods (panel B), and postItreatment recovery period in the \nluteal phase (panel C). Note cystic dilatation, mildly disordered architecture, and nonI\nphysiological secretory appearances in the endometria of women with UPAICVR. These \nendometrial samples are part of the large clinical trial. Representative images are shown. \nFig. 2: Expression of HAND2 in human endometrial biopsies exposed to UPA-CVR for 24 \nweeks. Immunohsitochemical localization of HAND2 in endometrial sections before and after \nexposure to UPAICVR. A total of 12 independent subjects were analyzed and for each subject, 4 \nendometrial biopsy samples were obtained (N=48). Panel A represents endometrial specimen \nobtained during the luteal phase before administration of UPAICVR. Panels B and C indicate \nendometrial specimens obtained after each 12Iweek period with UPAICVR releasing 1.5mg or \n2.5mg UPA daily. Panel D represents endometrial specimen collected during the luteal phase \nfollowing a postItreatment recovery period. Panel E shows endometrial sections from a biopsy \nsample after a 12Iweek exposure to UPAICVR and subjected to IHC protocol omitting the \nprimary antibody. Red staining indicates positive staining for HAND2 in endometrial sections. \nRepresentative images are shown. S and E indicate stroma and epithelium respectively. \nFig. 3: HAND2 expression is unaltered in human endometrial biopsies exposed to UPA-\nCVR. The percentages of the immunostaining positive cells for HAND2 were analyzed by \nImageJ software. The values represent mean ± SEM of twelve independent samples (N=5 for \nUPAICVR releasing 1.5 mg and N=7 for UPAICVR releasing 2.5mg UPA daily) with a total of \nN=48 clinical samples. No obvious doseIresponse effects were noted between the two doses. \nPage 14 of 28\n14 \nReproductive Sciences\n\nFig. 4: Expression of HAND2 is reduced in human endometrial biopsies exposed to \nmifepristone. Upper: Immunohistochemical analysis of HAND2 in human endometrium before \n(panel A) and after administration of 50 mg of oral mifepristone every other day for 12 weeks of \nthe menstrual cycle (panel B). Representative images are shown. Lower: HSCORES of \nHAND2Ipositive cells in the endometrium revealed a significant reduction in HAND2 \nexpression in mifepristoneIexposed biopsies when compared to unexposed controls (N=6). B \nindicates baseline (0 wks) and after 12 weeks (12 wks) indicates end of treatment, respectively, \nand shows a significant decrease (P<0.02).   \nFig. 5: UPA and mifepristone differentially regulate HAND2 mRNA expression in human \nendometrial stromal cells. Primary cultures of human stromal cells were grown in Dulbecco's \nmodified Eagle's medium/FI12 medium containing 5% charcoalIstripped fetal bovine serum. The \ncells were treated with a hormone mixture containing 10 nM E, 1 µM P, 0.5 mM 8IbromoI\ncAMP, and 10 µM UPA, mifepristone or vehicle for 6 days. Cells were harvested 2 days (left \npanel) or 6 days (right panel) after addition of hormone mixture. Total RNA was isolated and \nsubjected to qPCR using primers for HAND2. The level of Rplp0 was used as an internal control \nto normalize gene expression. The values are presented as the mean fold induction ± SEM, \nP<0.05. \nFig. 6: UPA and mifepristone differentially regulate HAND2 protein expression in human \nendometrial stromal cells. Immunocytochemical analysis of HAND2 in stromal cells during in \nvitro decidualization. Panels represent primary cultures of human endometrial stromal cells \ncultured in the absence of UPA or mifepristone (A), in the presence of UPA (B), in the presence \nof mifepristone (C) for 6 days. Representative images from three independent experiments are \nshown. \nFig. 7: UPA and mifepristone differentially regulate HAND2 mRNA expression in human \nendometrial stromal cells. Primary cultures of human stromal cells were treated with a hormone \nmixture containing 10 nM E, 1 µM P, 0.5 mM 8IbromoIcAMP, and 5 µM UPA, mifepristone or \nvehicle for 6 days. Cells were harvested 2, 3, 4, 5, and 6 days after addition of hormone mixture. \nTotal RNA was isolated and subjected to qPCR using primers for HAND2. The level of Rplp0 \nPage 15 of 28\n15 \nReproductive Sciences\n\nwas used as an internal control to normalize gene expression. The values are presented as the \nmean fold induction ± SEM, *P<0.05, **P<0.01. \nFig. 8: Downregulation of FGF18 expression in response to mifepristone in human \nendometrial stromal cells. Human endometrial stromal cells were subjected to differentiation in \nresponse to 0.5 mM 8IbromoIcAMP, 1 µM P, 10 nM E, and 10 µM UPA or mifepristone for 6 \ndays. Upper. Total RNA was isolated and subjected to qPCR using primer for FGF18. YIaxis \nindicates fold induction. The level of Rplp0 was used as an internal control to normalize gene \nexpression. The data are represented as the mean fold induction ± SEM from three separate \nsamples. Lower. Immunocytochemical analysis of FGF18 expression in endometrial stromal in \nthe absence of UPA or mifepristone (left panel), in the presence of UPA (middle panel) and in \nthe presence of mifepristone (right panel). Representative images are shown. \nFig. 9: PR stability in response to UPA or mifepristone in human endometrial stromal cells. \nPrimary cultures of human stromal cells were treated with a hormone mixture containing 10 nM \nE, 1 µM P, 0.5 mM 8IbromoIcAMP, and 10 µM UPA, mifepristone or vehicle for 6 days. \nImmunocytochemical analysis of PR in endometrial stromal in the absence of UPA or \nmifepristone (left panel), in the presence of UPA (middle panel) and in the presence of \nmifepristone (right panel) are shown. Representative images are shown. \nFig. 10: PR-B stability in response to UPA or mifepristone in human endometrial stromal \ncells. Primary cultures of human stromal cells were treated with a hormone mixture containing \n10 nM E, 1 µM P, 0.5 mM 8IbromoIcAMP, and 10 µM UPA, mifepristone or vehicle for 6 days. \nPRIB expression in endometrial stromal in the absence of UPA or mifepristone (left panel), in \nthe presence of UPA (middle panel) and in the presence of mifepristone (right panel) are shown. \nRepresentative images are shown. \nPage 16 of 28\n16 \nReproductive Sciences\n\nFor Peer Review\nReferences \n1 Wagenfeld A, Saunders PT, Whitaker L, Critchley HO: Selective progesterone receptor \nm\nodulators (SPRMs): progesterone receptor action, mode of action on the endometrium and \ntreatment options in gynecological therapies. Expert Opin Ther Targets 2016:1-10. \n2 Glasier A: The rationale for use of Ulipristal Acetate as first line in emergency contraception: \nbiological and clinical evidence. Gynecol Endocrinol 2014;30:688-690. \n3 Glasier AF, Cameron ST, Fine PM, Logan SJ, Casale W, Van Horn J, Sogor L, Blithe DL, Scherrer B, \nMathe H, Jaspart A, Ulmann A, Gainer E: Ulipristal acetate versus levonorgestrel for emergency \ncontraception: a randomised non-inferiority trial and meta-analysis. Lancet 2010;375:555-562. \n4 Gainer EE, Ulmann A: Pharmacologic properties of CDB(VA)-2914. Steroids 2003;68:1005-1011. \n5 Nichols MI: Ulisprisal acetate: a novel molecule and 5-day emergency contraceptive. Obstet \nGynecol 2010;116:1252-1253. \n6 Brache V, Cochon L, Jesam C, Maldonado R, Salvatierra AM, Levy DP, Gainer E, Croxatto HB: \nImmediate pre-ovulatory administration of 30 mg ulipristal acetate significantly delays follicular \nrupture. Hum Reprod 2010;25:2256-2263. \n7 Blithe DL, Nieman LK, Blye RP, Stratton P, Passaro M: Development of the selective \nprogesterone receptor modulator CDB-2914 for clinical indications. 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Eur J Obstet \nGynecol Reprod Biol 2017;208:91-96. \n11 Huang Y, Jensen JT, Brache V, Cochon L, Williams A, Miranda MJ, Croxatto H, Kumar N, Sussman \nH, Hoskin E, Plagianos M, Roberts K, Merkatz R, Blithe D, Sitruk-Ware R: A randomized study on \npharmacodynamic effects of vaginal rings delivering the progesterone receptor modulator \nulipristal acetate: research for a novel estrogen-free, method of contraception. Contraception \n2014;90:565-574. \n12 Mutter GL, Bergeron C, Deligdisch L, Ferenczy A, Glant M, Merino M, Williams AR, Blithe DL: The \nspectrum of endometrial pathology induced by progesterone receptor modulators. Mod Pathol \n2008;21:591-598. \n13 Pawar S, Hantak AM, Bagchi IC, Bagchi MK: Minireview: Steroid-regulated paracrine mechanisms \ncontrolling implantation. Mol Endocrinol 2014;28:1408-1422. \n14 Hantak AM, Bagchi IC, Bagchi MK: Role of uterine stromal-epithelial crosstalk in embryo \nimplantation. Int J Dev Biol 2014;58:139-146. \n15 Chandra V, Kim JJ, Benbrook DM, Dwivedi A, Rai R: Therapeutic options for management of \nendometrial hyperplasia. J Gynecol Oncol 2016;27:e8. \n16 Horn LC, Schnurrbusch U, Bilek K, Hentschel B, Einenkel J: Risk of progression in complex and \natypical endometrial hyperplasia: clinicopathologic analysis in cases with and without \nprogestogen treatment. Int J Gynecol Cancer 2004; 14:348-353. \n17 Daud S, Jalil SS, Griffin M, Ewies AA: Endometrial hyperplasia - the dilemma of management \nremains: a retrospective observational study of 280 women. Eur J Obstet Gynecol Reprod Biol \n2011;159:172-175. \nPage 17 of 28\n17 \nReproductive Sciences\n\nFor Peer Review\n18 Yang S, Thiel KW, Leslie KK: Progesterone: the ultimate endometrial tumor suppressor. Trends \nEndocrinol Metab 2011;22:145-152. \n19 Amant F, Moerman P, Neven P, Timmerman D, Van Limbergen E, Vergote I: Endometrial cancer. \nLancet 2005;366:491-505. \n20 Li Q, Kannan A, DeMayo FJ, Lydon JP, Cooke PS, Yamagishi H, Srivastava D, Bagchi MK, Bagchi IC: \nThe antiproliferative action of progesterone in uterine epithelium is mediated by Hand2. Science \n2011;331:912-916. \n21 Jones A, Teschendorff AE, Li Q, Hayward JD, Kannan A, Mould T, West J, Zikan M, Cibula D, Fiegl \nH, Lee SH, Wik E, Hadwin R, Arora R, Lemech C, Turunen H, Pakarinen P, Jacobs IJ, Salvesen HB, \nBagchi MK, Bagchi IC, Widschwendter M: Role of DNA methylation and epigenetic silencing of \nHAND2 in endometrial cancer development. PLoS Med 2013;10:e1001551. \n22 Whitaker LH, Murray AA, Matthews R, Shaw G, Williams AR, Saunders PT, Critchley HO: Selective \nprogesterone receptor modulator (SPRM) ulipristal acetate (UPA) and its effects on the human \nendometrium. 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Contraception 2016;94:143-151. \n30 Greb RR, Kiesel L, Selbmann AK, Wehrmann M, Hodgen GD, Goodman AL, Wallwiener D: \nDisparate actions of mifepristone (RU 486) on glands and stroma in the primate endometrium. \nHum Reprod 1999;14:198-206. \nPage 18 of 28\n18 \nReproductive Sciences\n\nFig.1 \n254x190mm (72 x 72 DPI) \nPage 19 of 28 Reproductive Sciences\n\nFig.2 \n254x190mm (72 x 72 DPI) \nPage 20 of 28Reproductive Sciences\n\nFig.3 \n254x190mm (72 x 72 DPI) \nPage 21 of 28 Reproductive Sciences\n\nFig.4 \n254x190mm (72 x 72 DPI) \nPage 22 of 28Reproductive Sciences\n\nFig.5 \n254x190mm (72 x 72 DPI) \nPage 23 of 28 Reproductive Sciences\n\nFig.6 \n254x190mm (72 x 72 DPI) \nPage 24 of 28Reproductive Sciences\n\nFig.7 \n254x190mm (72 x 72 DPI) \nPage 25 of 28 Reproductive Sciences\n\nFig.8 \n254x190mm (72 x 72 DPI) \nPage 26 of 28Reproductive Sciences\n\nFig.9 \n254x190mm (72 x 72 DPI) \nPage 27 of 28 Reproductive Sciences\n\nFig.10 \n254x190mm (72 x 72 DPI) \nPage 28 of 28Reproductive Sciences","source_license":"CC0","license_restricted":false}