Effect of long-term treatment of ulipristal acetate on rat ovarian tissue

Effect of long-term treatment of ulipristal acetate on rat ovarian tissue | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of long-term treatment of ulipristal acetate on rat ovarian tissue Mana Hirano, Osamu Wada-Hiraike, Motoko Fukui, Seiji Shibata, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2847062/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ulipristal (UPA), a selective progesterone receptor modulator, has both agonistic and antagonistic effects on progesterone receptors. UPA suppresses ovulation by inhibiting the luteinizing hormone (LH) surge from the pituitary gland; however, the direct effect of UPA on ovarian tissue remains poorly studied. In the present study, we examined the effects of UPA on the ovaries of rats. Rats were treated for 28 d with 4, 20, and 100 mg/kg UPA. UPA treatment increased the number of primordial follicles at each treatment group, with the highest number found in the 4 mg/kg group, and the number of primordial follicles decreasing with increasing dose. The number of primary and antral follicles tended to increase with increasing UPA levels. Furthermore, the decrease in primary follicle number could be attributed to the exhaustion of follicles, but the examination of proliferation markers, oxidative stress markers, and cell death markers revealed no remarkable toxic effects on ovarian tissues. These results suggest that UPA treatment promotes follicle development at each stage but inhibits ovulation by suppressing the LH surge, resulting in an increase in atretic follicles or unruptured luteinized cysts. UPA may not have toxic effects on the ovary because the expression of antioxidant genes and cell death markers was not dramatic in follicles treated with UPA. Taken together, these results suggest that UPA may not have a direct local effect on ovarian follicles. Hence, we hypothesized that prolonged UPA treatment in patients with uterine fibroids may not be harmful and may not decrease future fecundity. Ulipristal acetate ovary follicle development luteinization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ulipristal (UPA) is a selective progesterone receptor modulator (SPRM) that acts as both an agonist and antagonist of the progesterone receptor (PR). It is currently used clinically for the treatment of uterine fibroids [ 1 , 2 ] and emergency contraception [ 3 ]. As an emergency contraceptive, UPA is orally administered at a dose of 30 mg within 120 h after unprotected intercourse. A clinical study indicated that a single 30 mg dose of UPA in healthy women delayed the luteinizing hormone (LH) surge prior to the onset of the LH surge, slowed the LH surge prior to its peak after the onset of the LH surge, and suppressed ovulation for the subsequent 5 d. Ovulation was observed when 30 mg of UPA was administered after the LH peak [ 4 ], but it reportedly alters gene expression and reduces receptivity of the human endometrium, making implantation more difficult [ 5 ]. UPA inhibits the PR-dependent pathway, suppresses ovulation, and possibly serves as contraception [ 6 ]. UPA treatment also suppressed the movement of fallopian tube hairs and muscle contraction, thereby contributing to contraception [ 7 ]. Although a single use of UPA might not have significant impact on ovarian function, the treatment of uterine fibroids using UPA requires long-term administration, and administration of UPA to healthy women for 84 d resulted in anovulation in 82% (5 mg/day) and 80% (10 mg/day) women, and 81% (5 mg/day) and 90% (10 mg/day) of women resulted in amenorrhea after 2 months of treatment [ 8 ]. It has been suggested that UPA could have anti-proliferative functions in uterine fibroid cells, whereas it may not have severe effects on normal uterine smooth muscle tissue, and UPA treatment generally maintains the endogenous secretion of estrogens [ 8 ]. However, long-term use of UPA affects the uterine epithelium, which is known as PR-modulator-associated endometrial change (PAEC) [ 9 ]. The pathophysiology of PAEC remains to be elucidated; however, the activation of PR could be attributed to the occurrence of PAEC. Although it has been reported that UPA administration does not affect the development of fertilized eggs [ 10 ], the direct effects of UPA on ovarian tissue are poorly understood and hardly investigated. Because UPA may affect the PR signaling pathway, we can speculate that UPA may affect ovarian pathophysiology. Although long -term administration of UPA is required, the direct effect of UPA on ovarian tissue remains poorly studied. With the aim to examine the effects of UPA on the ovary, we utilized rats treated with UPA for 28 consecutive days and investigated the effects of UPA administration on the rat ovary. By clarifying the effects of UPA on the ovaries, UPA can be safely administered long-term. Methods Animals Eight-week-old female Crl: CD (SD) rats purchased from Charles River Laboratories Japan Inc. (Kanagawa, Japan) were housed individually in plastic cages embedded with ALPHA-dri (Shepherd Specialty Papers Inc., Kalamazoo, MI, USA), fed CE-2 solid chow (Japan CLEA, Tokyo, Japan), and provided tap water ad libitum. The animal room conditions were as follows: 1) temperature, 20–26°C; 2) relative humidity, 40–75%; 3) ventilation, 15 to 25 air changes per hour; and 4) lighting, 07:00 to 19:00. The rats were orally administered ulipristal daily for 28 d at doses of 0 mg/kg/day (n = 8), 4 mg/kg/day (n = 7), 20 mg/kg/day (n = 8), and 100 mg/kg/day (n = 8), followed by immunization with keyhole limpet hemocyanin on the 14th day of treatment for the antibody production test and sacrificed under isoflurane anesthesia the day after the final administration for various examinations. The experimental protocol was approved by the Animal Research Committee of ASKA Pharmaceutical Co., Ltd. (authorization reference number: K14-026). Hormone assay Blood samples were collected via venipuncture from the abdominal aorta after the rats were euthanized. Blood samples were centrifuged at 2100 × g for 10 min to obtain plasma for hormone assay. Plasma estradiol and progesterone concentrations were measured using the LC-MS/MS method. Briefly, a stable, isotopically labelled internal standard ( 2 H 5 -labelled estradiol and 2 H 9 -labelled progesterone, obtained from C/D/N Isotopes, Quebec, Canada) was used for the determination of estradiol and progesterone. The samples were analyzed using tandem mass spectrometry with an API5000 LC-MS/MS triple quadrupole tandem quadrupole mass spectrometer (AB SCIEX, Framingham, MA, USA) with electrospray ionization in negative mode. The calibration curve for estradiol was linear, ranging from 1 pg/mL to 50 pg/mL. The calibration curve for progesterone was linear, ranging from 1 ng/mL to 1000 ng/mL. The plasma corticosterone concentration was measured using the AssayMax Corticosterone ELISA kit (Assaypro LLC, St Charles, MO, USA). The corticosterone assay was performed according to the manufacturer’s instructions, with each condition performed in duplicate. The data are expressed as the amount of steroid secreted (ng/mL). Tissue collection and histological examination Ovaries and uteri were removed, and wet weights were measured immediately after sacrifice. Rat ovaries, uterus, and vagina were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at a thickness of 5 µm. The sections were stained with Mayer’s hematoxylin and eosin (H&E). H&E-stained specimens were prepared and evaluated using light Leica microscopy (Wetzlar, Germany)). We evaluated the reproductive system according to the internationally accepted nomenclature of the INHAND project [ 11 ]. For the ovaries, each individual was evaluated for the presence of unruptured follicles (the presence of a retained oocyte within a corpus luteum-like structure), corpus luteum cysts (cyst formation in the corpus luteum), follicular cysts (cyst formation in the follicle), reduction of large follicles (larger than follicles with a large oocyte with many layers of cells [ 12 ]), reduction of currently formed corpus luteum (corpus luteum with basophilic luteal cells), and reduction of previously formed corpus luteum (corpus luteum with eosinophilic luteal cells separated by fibrous tissue) at three levels (four levels only for corpus luteum cysts): none, mild, moderate, and severe (only for corpus luteum). The uterus was evaluated for luminal dilation, dilation of the uterine glands, tall columnar epithelial cells, and vacuolation of the epithelium at three levels (none, mild, and moderate). Vaginal secretion was evaluated for mucus degeneration and hyperplasia of the epithelium at three levels (none, mild, and moderate) in each individual. Follicle counts H&E-stained serial sections of the rat ovaries were prepared and counted under an optical Leica microscope (Wetzlar, Germany). We slightly modified the method introduced to count the number of follicles in ovaries [ 13 – 15 ]. Follicle classification was based on the following morphological criteria: primordial (oocytes with a single layer of flattened granulosa cells), primary (oocytes with a single layer of cuboidal or mixed cuboidal/flattened granulosa cells), secondary (oocytes with more than one layer of granulosa cells), and antral (oocytes with multiple layers of granulosa cells and possessing an antral space or spaces), and were further classified as healthy or atretic. Primordial and primary follicles were counted in every serial section, secondary follicles in every 3rd serial section, and antral follicles in every 12th serial section, with care taken to count each of these structures once. The follicle count for each classification for each sample was measured and compared. Immunohistochemistry Paraffin-embedded tissues were subjected to histological analysis and sections of rat ovaries were stained with primary antibodies for Anti-Müllerian Hormone (AMH: GeneTex, Irvine, CA, USA, Cat# GTX42794, 1:100), forkhead box3 (FOXO3a: Cell Signaling Technology, Beverly, MA, USA, Cat# 12829, 1:500), pAkt (Cell Signaling Technology, Cat# 4060, 1:100), catalase (Abcam, Cambridge, UK, Cat# ab16731, 1:200), Superoxide Dismutase 1(SOD1: Abcam, Cat#13499, 1:500), Matrix Metallopeptidase (MMP1: Proteintech, Rosemont, IL, USA, Cat# 10371-2-AP, 1:800), Tissue inhibitor of metalloproteinase (TIMP2: Proteintech, Cat# 17353-1-AP, 1:500), and Ki67 (Invitrogen, Carlsbad, CA, USA, Cat# MA5-14520, 1:750). TUNEL (Roche, Basel, Switzerland, Cat# 11684817910) staining was performed to analyze cell death. The percentage of staining per follicle or corpus luteum was classified into four levels (0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), and 4 (> 75%), and multiplied by the staining intensity (0 negative, 1 weak, 2 intense) to calculate the positive index [ 16 ]. Statistics Data were analyzed using SPSS software (version 28.0, StatsGuild Inc, Urayasu, Chiba, Japan.). A comparative analysis of the means of the four groups was performed using one-way analysis of variance (ANOVA). Significance was set at P > 0.05. Results Plasma hormone concentrations To investigate the effect of the systemic administration of UPA, changes in plasma estradiol, progesterone, and corticosterone levels in response to UPA administration were measured. Plasma estradiol levels were elevated by the administration of UPA (4, 20, and 100 mg/kg) compared to that of the control (0 mg/kg) (Fig. 1 a). In contrast, the plasma progesterone levels showed no significant changes (Fig. 1 b). It is well known that UPA possesses glucocorticoid receptor activation function [ 17 ], and plasma corticosterone levels were examined to investigate the effect of UPA; however, UPA had no significant impact on corticosterone levels (Fig. 1 c). Histological examination To investigate the effect of systemic administration of UPA, the wet weight of representative reproductive organs and the pathological appearance in response to UPA administration were examined. The wet weights of the uteri were unaffected by the administration of UPA (Fig. 2 a). In contrast, dilation of the lumen, dilation of uterine glands, increase in columnar height, and vacuolation of the luminal epithelium were observed after the administration of UPA, although the effect was not dose-dependent (Fig. 2 b–g). We also observed vaginal epithelial hyperplasia after UPA administration (Fig. 2 h–k), and dose-dependent effect on vaginal epithelial hyperplasia was suspected (Fig. 2 i) The wet weight of ovaries was not significantly affected by the different doses of UPA (Fig. 3 a). To investigate the effect of UPA, follicles at each developmental stage were histologically counted and examined (Fig. 3 b). The number of follicles at each developmental stage was unchanged in the control group (0 mg/kg), but the number of primordial follicles was substantially increased by the administration of UPA at 4 and 20 mg/kg doses. The number of primordial follicles tended to decrease with increasing doses of UPA, but the number of primary, secondary, and antral follicles tended to increase with increasing doses of UPA. In the ovary, unruptured follicles, corpus luteum cysts, and follicular cysts were more frequently observed after treatment with UPA, and there was a decrease in the number of large follicles and new and old corpora lutea (Fig. 3 c–h). The degree of findings was more severe in the 20 and 100 mg/kg groups than in the 4 mg/kg group but did not differ between the 20 and 100 mg/kg groups. Ovarian immunohistochemical examination AMH [ 18 ], FOXO3a, and pAkt [ 19 ] are known to be representative of the activation of the primordial ovarian follicle pool, and based on the result that UPA administration markedly elevated the number of primordial follicles, we stained the ovaries and observed the granulosa cells and corpus luteum of each follicle. We evaluated granulosa cells, except for MMP1 and TIMP2, which we evaluated as corpus luteum. AMH-stained granulosa cells of the ovary were pronounced in the 100 mg/kg group, but the other doses were comparable to those of the control (Fig. 4 a). In contrast, FOXO3a staining decreased in the 100 mg/kg group (Fig. 4 b), and tended to be lower depending on the UPA dose. Although pAkt stanining of granulosa cells tended to be higher in UPA 100 mg/kg treatment rats, it did not show significant changes (Fig. 4 c). Ki67 is a representative marker of cellular proliferation. Ki67 staining of each follicle was not expressed in primordial follicles and increased as the follicles developed in the control group. However, UPA treatment decreased the staining of Ki67 in each developmental stage of follicles in granulosa cells; thus, UPA was suspected to decrease the cellular growth of granulosa cells (Fig. 5 a). The cellular death status of granulosa cells was examined using TUNEL staining, and it showed an increasing trend with increasing dose in primordial and primary follicles, but the difference was not noted with all doses of UPA (Fig. 5 b). MMP1 was associated with the regression of corpora lutea, and the expression of MMP1 in ovarian corpus luteum was substantially decreased at 20 and 100 mg/kg (Fig. 6 a). TIMP2, which antagonizes the regression of corpora lutea, tended to increase with UPA administration, although dose-dependency was not observed (Fig. 6 b). The antioxidant stress markers SOD1 and catalase were decreased by UPA in follicles other than primordial follicles, and catalase was decreased by UPA in primordial follicles and decreased by UPA in secondary follicles, and tended to increase with increasing dose (Fig. 6 c and 6 d). Discussion Selective PR modulators (SPRMs) can modulate progesterone pathways. UPA, an SPRM, acts on uterine fibroids and shrinks them. The mechanism of action of UPA in uterine fibroids is related to the higher expression of PRs in uterine fibroids than in normal uterine muscle. UPA suppresses cell proliferation in a dose-dependent manner by inhibiting the expression of PCNA (proliferating cell nuclear antigen), Bcl-2, collagen, VEGF (vascular endothelial growth factor), and adrenomedullin in uterine myoma cells without affecting normal uterine smooth muscle cells and induces apoptosis by increasing the expression of caspase-3, PARP, and matrix metalloproteinases (MMPs) [ 20 ]. SPRMs induce amenorrhea and reduce pain and are used as a treatment for uterine fibroid. Moreover, SPRMs inhibit prostaglandin secretion [ 21 ]. Although SPRM does not affect the normal myometrium and influences on uterine fibroids and endometriosis, its effect on the ovaries remains unknown. The effects of long-term UPA administration on the ovaries were examined. In the PEARL trials, UPA was found to be non-inferior to the GnRH analog in controlling bleeding and shrinking fibroids but did not show lower estrogen levels [ 22 ]. Long-term administration of UPA increases uterine myoma reduction [ 3 ], and this effect persists even after treatment completion. Similar to other SPRMs, UPA causes a characteristic change in the endometrium called PAEC [ 23 ], which is said to disappear within 6 months after the end of UPA administration. When administered before surgery, UPA decreases myoma volume, reduces myoma-related bleeding, and increases hemoglobin levels [ 24 ]. Long-term administration of UPA is considered necessary to control uterine fibroids; however, given that it is administered to women of reproductive age, this study was conducted to determine whether long-term administration would have any adverse effects on the ovaries. The efficacy and safety of long-term UPA administration in myomas have been studied [ 25 , 26 ]. In vitro, UPA induces cell death in uterine myoma cells, but has the remarkable property of causing no noticeable change in the normal myometrium. Therefore, we examined whether UPA also induces cell death in ovarian cells. UPA was administered to rats and its effect on the ovaries was investigated. The blood estradiol level was not decreased by UPA administration, but was notably increased, whereas progesterone and corticosterone levels did not change substantially, suggesting that luteal function and adrenal glands were not severely affected by UPA administration. These results are similar to those of the PEARL trial described above. The number of follicles increased with UPA administration. Primordial follicles were activated and follicle development proceeded, but the number of closed follicles or luteinized unruptured cysts increased. This is owing to the suppression of the LH surge by UPA, thus inhibiting ovulation. The expression of AMH, FOXO3, and pAkt as factors related to primordial follicle activation was examined via immunohistological examination. AMH, which suppresses primordial follicle activation, was significantly increased in the 100 mg/kg group, whereas FOXO3, which also suppressed primordial follicle activation, was significantly decreased at 100 mg/kg. pAkt, the primordial follicle activation, showed a slight increasing trend, but no substantial change was observed. These results suggest that other factors or mechanisms may be involved in primordial follicle activation. UPA treatment decreased the luteal regression marker MMP1 levels and increased the luteal regression antagonist marker TIMP2 levels, suggesting that luteal regression was not suppressed and that mature follicles failed to ovulate, resulting in unruptured luteinized cysts. It has been reported that administration of UPA to uterine fibroids leads to the degradation of hydrogen peroxide and production of reactive oxygen species (ROS) [ 27 ], and UPA with sperm possibly acts as a scavenger of ROS [ 9 ][ 28 ]. In the present study, UPA was administered to normal ovaries, and immunohistological examination revealed that SOD-1 and catalase, which are related to ROS, were decreased by UPA administration in all but primordial follicles. In terms of catalase, SOD-1 tended to decrease from primordial follicles to secondary follicles. The antioxidants SOD1 and catalase tended to decrease with UPA administration, suggesting that ROS may be produced by UPA administration in the ovary; however, this was not a significant change. When 30 mg of UPA was used in humans immediately before ovulation, there was no significant change in Ki67, a proliferation marker, in the endometrium [ 3 ]. In the ovaries of rats treated with UPA for a long period of time, the present study showed no significant changes in Ki67 and TUNNEL, suggesting that UPA may have no relationship with ovarian proliferation or cell death. This is similar to the relationship between the uterine muscle and uterine fibroids. SPRMs act through progesterone receptors and as agonists or antagonists in various target organs. Among them, UPA inhibits the proliferation and induction of apoptosis and cell death pathways in leiomyoma cells, translating to smaller fibroids and lower uterine volumes at the clinical level, with no significant side effects. These results suggest that long-term administration of UPA to the ovary activates primordial follicles and promotes follicle development, but suppresses the LH surge, resulting in ovulation suppression when closed follicles and luteinized unruptured cysts increase. The mechanism by which UPA administration promotes follicle development was not elucidated in this study. It is possible that factors other than AMH, FOXO3, and pAkt are involved in primordial follicle activation. Declarations Ethics approval : This animal experimental protocol was approved by the Animal Research Ethics Committee of ASKA Pharmaceutical Co., Ltd. (authorization reference number: K14-026). Consent to participate, Consent for publication : NA Availability of data and material (data transparency) : Applicable according to the reasonable requirements and the corresponding author is responsible if someone wants to request the data. Competing Interests : Motoko Fukui and Seiji Shibata are employees of ASKA Pharmaceutical Co., Ltd. All other authors have no competing interest. Funding Information: This study was funded by ASKA Pharmaceutical Co., Ltd. Author Contribution: MH, OWH, MF, SS contributed to the conception and design of the study. MH, OWH, MF, SS, MU, AN, and YU contributed to the material preparation, acquisition of data, and analysis and interpretation of data. OWH was the principal investigator and played a significant role in the interpretation of data. The first draft of the manuscript was written by MH, OWH and MF, and KS, MH, KK and YO revised it critically for important intellectual content. All authors have read and approved the final version of the manuscript, and YO gave the approval to submit the latest version. Acknowledgements : The authors thank editage (https://www.editage.jp) for language editing. References Garnock-Jones KP, Duggan ST. Ulipristal Acetate: A Review in Symptomatic Uterine Fibroids. Drugs. 2017;77(15):1665–75. Ekanem E, Talaulikar V. Medical Therapy for Fibroids: What Next for Ulipristal Acetate? Adv Ther. 2021;38(1):137–48. Glasier AF, Cameron ST, Fine PM, Logan SJ, Casale W, Van Horn J, et al. Ulipristal acetate versus levonorgestrel for emergency contraception: a randomised non-inferiority trial and meta-analysis. Lancet. 2010;375(9714):555–62. Brache V, Cochon L, Jesam C, Maldonado R, Salvatierra AM, Levy DP, et al. Immediate pre-ovulatory administration of 30 mg ulipristal acetate significantly delays follicular rupture. Hum Reprod. 2010;25(9):2256–63. Lira-Albarran S, Durand M, Larrea-Schiavon MF, Gonzalez L, Barrera D, Vega C, et al. 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Effects and molecular mechanism of pachymic acid on ferroptosis in renal ischemia reperfusion injury. Mol Med Rep. 2021;23(1). Additional Declarations Competing interest reported. Motoko Fukui and Seiji Shibata are employees of ASKA Pharmaceutical Co., Ltd. All other authors have no competing interest. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2847062","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":203420671,"identity":"05a34e31-0bd6-4c57-bb1f-184735f0c5e1","order_by":0,"name":"Mana Hirano","email":"","orcid":"","institution":"Teikyo University","correspondingAuthor":false,"prefix":"","firstName":"Mana","middleName":"","lastName":"Hirano","suffix":""},{"id":203420673,"identity":"c57f2225-fd4d-41ce-a318-ab0001d00eac","order_by":1,"name":"Osamu Wada-Hiraike","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYPCCAwwMPAyMDxgYJCB8CWK08PAwMBuQrIWNCIVAwM9/+Olm3rY7DPY8Z8yqeXMs8hjYDz9gsNyBW4vkjDSz27xtzxh4eHuAjG0SxQw8aQYMkmdwazG4wQBSebi+h58HrCWxgSEHaFIbHi3nj38DaWHgAWopBmvhf0NAy4EcM4gWoMOYwVokCNgiOSOn7Obcf0AtZ44VS84FammTeGZwAJ9f+PmPb7vx5sxhBvae5I0f3m6rS+znT374WBJPiCEBDgMwxQbEhyUbiNLC/gDOZPxInJZRMApGwSgYGQAAehxM3toqBZsAAAAASUVORK5CYII=","orcid":"","institution":"The University of Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Osamu","middleName":"","lastName":"Wada-Hiraike","suffix":""},{"id":203420674,"identity":"6118d6c1-c0ce-45e3-8833-78ae3ec0a874","order_by":2,"name":"Motoko Fukui","email":"","orcid":"","institution":"ASKA Pharmaceutical Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Motoko","middleName":"","lastName":"Fukui","suffix":""},{"id":203420676,"identity":"99725198-e6a9-4708-8997-20a55496574e","order_by":3,"name":"Seiji Shibata","email":"","orcid":"","institution":"ASKA Pharmaceutical Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Seiji","middleName":"","lastName":"Shibata","suffix":""},{"id":203420678,"identity":"8fe8b7ae-5c66-4e67-a371-b5eae3d1732a","order_by":4,"name":"Mari Uehara","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Mari","middleName":"","lastName":"Uehara","suffix":""},{"id":203420679,"identity":"16a02351-b4a9-45c5-b58a-7578f1b149fe","order_by":5,"name":"Aiko Nagumo","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Aiko","middleName":"","lastName":"Nagumo","suffix":""},{"id":203420680,"identity":"af7b497f-d607-4e15-98c0-8fb59b12ca64","order_by":6,"name":"Yoko Urata","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Yoko","middleName":"","lastName":"Urata","suffix":""},{"id":203420681,"identity":"24ecb800-1b91-403a-915b-0b47218aedb0","order_by":7,"name":"Kenbun Sone","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Kenbun","middleName":"","lastName":"Sone","suffix":""},{"id":203420682,"identity":"08b5fe1a-9f13-4c49-93fa-2abe2aeeacaa","order_by":8,"name":"Miyuki Harada","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Miyuki","middleName":"","lastName":"Harada","suffix":""},{"id":203420683,"identity":"2d43a82e-973a-42ec-a745-a0fa6de20c4d","order_by":9,"name":"Kaori Koga","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Kaori","middleName":"","lastName":"Koga","suffix":""},{"id":203420684,"identity":"5723f0fd-5a20-439b-b47b-7ba2d27725b4","order_by":10,"name":"Yutaka Osuga","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Yutaka","middleName":"","lastName":"Osuga","suffix":""}],"badges":[],"createdAt":"2023-04-22 01:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2847062/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2847062/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":37539664,"identity":"6574e344-33dc-482b-b7c2-208b4b27a61d","added_by":"auto","created_at":"2023-05-26 14:20:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":31988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSecretion of sex steroid hormones after treatment by UPA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma estradiol (1a), progesterone (1b), and corticosterone (1c) in response to UPA are shown.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-2847062/v1/f20043e15ffeb7f3e4f24d0f.png"},{"id":37540700,"identity":"77180187-8546-4e22-a585-1cbbe039c96f","added_by":"auto","created_at":"2023-05-26 14:28:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":784972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of uterus and vagina after treatment by UPA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea: Wet weight of bilateral uteri after UPA treatments is shown. The weight of bilateral uteri did not show any statistically significant difference. Histological aspects of uteruses after UPA treatment are shown. b Luminal dilation (b), dilation of epithelial glands (c), height columnar epithelial cells (d), and vacuolation of epithelia (e) are shown. Representative histological findings of control uterus (f) and the uterus after UPA 20 mg/kg treatment (g) for HE-stained specimens are shown. Histological aspects of vaginas after UPA treatment were also investigated. Mucinous degeneration (h) and epithelial hyperplasia (i) are shown. Representative histological findings of control vagina (j) and the vagina after UPA 20 mg/kg treatment (k) for HE-stained specimens are shown.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-2847062/v1/5a6a88bb4fd218475f09233e.png"},{"id":37539666,"identity":"0098f669-8c3e-424a-90ea-3cad324214cc","added_by":"auto","created_at":"2023-05-26 14:20:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":85424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of ovary after treatment by UPA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wet weight of bilateral ovaries was not significantly different in all groups (a). The number of follicles at each developmental stage were counted (b). The number of primordial follicle was highest at 4 mg/kg and tended to decrease in a dose-dependent manner. The number of primary and antral follicles tended to increase depending on the UPA dose. Pathological findings of the ovaries are shown. Unruptured follicles (c), corpus luteum cysts (d), follicle cysts (e), reduction of large follicles (f), reduction of currently formed corpus luteum (g), and reduction of previously formed corpus luteum (h) were histologically examined and counted.\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-2847062/v1/d198ef7974d47bffd2643c27.png"},{"id":37541567,"identity":"81f95b59-d38f-40fc-9432-b25c448c087e","added_by":"auto","created_at":"2023-05-26 14:36:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOvarian reserve and activation markers of primordial follicles and primary follicles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePositive index of primordial follicles and primary follicles in control and UPA treated ovaries are shown. Positive index of AMH (a), FOXO3a (b), and pAkt (c) were examined.\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-2847062/v1/64bac1cd302a59ccdc3e275e.png"},{"id":37540698,"identity":"c797d0c6-3d78-41bf-aa03-71ce8d3b7726","added_by":"auto","created_at":"2023-05-26 14:28:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":51438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMakers of proliferation and cell death in ovaries\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of Ki67, a representative marker of proliferation, was examined (a), and TUNEL staining (b) was counted in all types of follicles.\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-2847062/v1/2af993978d1bac9b0962f9ff.png"},{"id":37539669,"identity":"12f8b62a-a042-42e7-9e16-71c4690250fd","added_by":"auto","created_at":"2023-05-26 14:20:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":76129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMarkers of luteinization and antioxidative stress genes in ovaries\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMMP1 (a) is associated with the regression of corpora lutea, and TIMP2 (b) antagonizes the regression of corpora lutea. Positivity in corpus luteum was examined and counted. Both SOD1 (c) and Catalase (d) suppresses oxidative stress and positivity was counted in all types of follicles.\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-2847062/v1/add4a6ab050020b6bf89c7b0.png"},{"id":47604311,"identity":"2352a8f1-6d33-481a-bc49-4c0399a304fc","added_by":"auto","created_at":"2023-12-05 04:37:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1386679,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2847062/v1/b354650c-8a5d-4a4f-8b14-3d2e6b070388.pdf"}],"financialInterests":"Competing interest reported. Motoko Fukui and Seiji Shibata are employees of ASKA Pharmaceutical Co., Ltd. All other authors have no competing interest.","formattedTitle":"Effect of long-term treatment of ulipristal acetate on rat ovarian tissue","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUlipristal (UPA) is a selective progesterone receptor modulator (SPRM) that acts as both an agonist and antagonist of the progesterone receptor (PR). It is currently used clinically for the treatment of uterine fibroids [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and emergency contraception [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As an emergency contraceptive, UPA is orally administered at a dose of 30 mg within 120 h after unprotected intercourse. A clinical study indicated that a single 30 mg dose of UPA in healthy women delayed the luteinizing hormone (LH) surge prior to the onset of the LH surge, slowed the LH surge prior to its peak after the onset of the LH surge, and suppressed ovulation for the subsequent 5 d. Ovulation was observed when 30 mg of UPA was administered after the LH peak [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], but it reportedly alters gene expression and reduces receptivity of the human endometrium, making implantation more difficult [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. UPA inhibits the PR-dependent pathway, suppresses ovulation, and possibly serves as contraception [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. UPA treatment also suppressed the movement of fallopian tube hairs and muscle contraction, thereby contributing to contraception [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough a single use of UPA might not have significant impact on ovarian function, the treatment of uterine fibroids using UPA requires long-term administration, and administration of UPA to healthy women for 84 d resulted in anovulation in 82% (5 mg/day) and 80% (10 mg/day) women, and 81% (5 mg/day) and 90% (10 mg/day) of women resulted in amenorrhea after 2 months of treatment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It has been suggested that UPA could have anti-proliferative functions in uterine fibroid cells, whereas it may not have severe effects on normal uterine smooth muscle tissue, and UPA treatment generally maintains the endogenous secretion of estrogens [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, long-term use of UPA affects the uterine epithelium, which is known as PR-modulator-associated endometrial change (PAEC) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The pathophysiology of PAEC remains to be elucidated; however, the activation of PR could be attributed to the occurrence of PAEC. Although it has been reported that UPA administration does not affect the development of fertilized eggs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], the direct effects of UPA on ovarian tissue are poorly understood and hardly investigated. Because UPA may affect the PR signaling pathway, we can speculate that UPA may affect ovarian pathophysiology.\u003c/p\u003e \u003cp\u003eAlthough long -term administration of UPA is required, the direct effect of UPA on ovarian tissue remains poorly studied. With the aim to examine the effects of UPA on the ovary, we utilized rats treated with UPA for 28 consecutive days and investigated the effects of UPA administration on the rat ovary. By clarifying the effects of UPA on the ovaries, UPA can be safely administered long-term.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eEight-week-old female Crl: CD (SD) rats purchased from Charles River Laboratories Japan Inc. (Kanagawa, Japan) were housed individually in plastic cages embedded with ALPHA-dri (Shepherd Specialty Papers Inc., Kalamazoo, MI, USA), fed CE-2 solid chow (Japan CLEA, Tokyo, Japan), and provided tap water ad libitum. The animal room conditions were as follows: 1) temperature, 20\u0026ndash;26\u0026deg;C; 2) relative humidity, 40\u0026ndash;75%; 3) ventilation, 15 to 25 air changes per hour; and 4) lighting, 07:00 to 19:00. The rats were orally administered ulipristal daily for 28 d at doses of 0 mg/kg/day (n\u0026thinsp;=\u0026thinsp;8), 4 mg/kg/day (n\u0026thinsp;=\u0026thinsp;7), 20 mg/kg/day (n\u0026thinsp;=\u0026thinsp;8), and 100 mg/kg/day (n\u0026thinsp;=\u0026thinsp;8), followed by immunization with keyhole limpet hemocyanin on the 14th day of treatment for the antibody production test and sacrificed under isoflurane anesthesia the day after the final administration for various examinations. The experimental protocol was approved by the Animal Research Committee of ASKA Pharmaceutical Co., Ltd. (authorization reference number: K14-026).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHormone assay\u003c/h2\u003e \u003cp\u003eBlood samples were collected via venipuncture from the abdominal aorta after the rats were euthanized. Blood samples were centrifuged at 2100 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min to obtain plasma for hormone assay. Plasma estradiol and progesterone concentrations were measured using the LC-MS/MS method. Briefly, a stable, isotopically labelled internal standard (\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e5\u003c/sub\u003e-labelled estradiol and \u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e9\u003c/sub\u003e-labelled progesterone, obtained from C/D/N Isotopes, Quebec, Canada) was used for the determination of estradiol and progesterone. The samples were analyzed using tandem mass spectrometry with an API5000 LC-MS/MS triple quadrupole tandem quadrupole mass spectrometer (AB SCIEX, Framingham, MA, USA) with electrospray ionization in negative mode. The calibration curve for estradiol was linear, ranging from 1 pg/mL to 50 pg/mL. The calibration curve for progesterone was linear, ranging from 1 ng/mL to 1000 ng/mL. The plasma corticosterone concentration was measured using the AssayMax Corticosterone ELISA kit (Assaypro LLC, St Charles, MO, USA). The corticosterone assay was performed according to the manufacturer\u0026rsquo;s instructions, with each condition performed in duplicate. The data are expressed as the amount of steroid secreted (ng/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTissue collection and histological examination\u003c/h2\u003e \u003cp\u003eOvaries and uteri were removed, and wet weights were measured immediately after sacrifice. Rat ovaries, uterus, and vagina were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at a thickness of 5 \u0026micro;m. The sections were stained with Mayer\u0026rsquo;s hematoxylin and eosin (H\u0026amp;E). H\u0026amp;E-stained specimens were prepared and evaluated using light Leica microscopy (Wetzlar, Germany)). We evaluated the reproductive system according to the internationally accepted nomenclature of the INHAND project [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For the ovaries, each individual was evaluated for the presence of unruptured follicles (the presence of a retained oocyte within a corpus luteum-like structure), corpus luteum cysts (cyst formation in the corpus luteum), follicular cysts (cyst formation in the follicle), reduction of large follicles (larger than follicles with a large oocyte with many layers of cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]), reduction of currently formed corpus luteum (corpus luteum with basophilic luteal cells), and reduction of previously formed corpus luteum (corpus luteum with eosinophilic luteal cells separated by fibrous tissue) at three levels (four levels only for corpus luteum cysts): none, mild, moderate, and severe (only for corpus luteum). The uterus was evaluated for luminal dilation, dilation of the uterine glands, tall columnar epithelial cells, and vacuolation of the epithelium at three levels (none, mild, and moderate). Vaginal secretion was evaluated for mucus degeneration and hyperplasia of the epithelium at three levels (none, mild, and moderate) in each individual.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFollicle counts\u003c/h2\u003e \u003cp\u003eH\u0026amp;E-stained serial sections of the rat ovaries were prepared and counted under an optical Leica microscope (Wetzlar, Germany). We slightly modified the method introduced to count the number of follicles in ovaries [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Follicle classification was based on the following morphological criteria: primordial (oocytes with a single layer of flattened granulosa cells), primary (oocytes with a single layer of cuboidal or mixed cuboidal/flattened granulosa cells), secondary (oocytes with more than one layer of granulosa cells), and antral (oocytes with multiple layers of granulosa cells and possessing an antral space or spaces), and were further classified as healthy or atretic. Primordial and primary follicles were counted in every serial section, secondary follicles in every 3rd serial section, and antral follicles in every 12th serial section, with care taken to count each of these structures once. The follicle count for each classification for each sample was measured and compared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin-embedded tissues were subjected to histological analysis and sections of rat ovaries were stained with primary antibodies for Anti-M\u0026uuml;llerian Hormone (AMH: GeneTex, Irvine, CA, USA, Cat# GTX42794, 1:100), forkhead box3 (FOXO3a: Cell Signaling Technology, Beverly, MA, USA, Cat# 12829, 1:500), pAkt (Cell Signaling Technology, Cat# 4060, 1:100), catalase (Abcam, Cambridge, UK, Cat# ab16731, 1:200), Superoxide Dismutase 1(SOD1: Abcam, Cat#13499, 1:500), Matrix Metallopeptidase (MMP1: Proteintech, Rosemont, IL, USA, Cat# 10371-2-AP, 1:800), Tissue inhibitor of metalloproteinase (TIMP2: Proteintech, Cat# 17353-1-AP, 1:500), and Ki67 (Invitrogen, Carlsbad, CA, USA, Cat# MA5-14520, 1:750). TUNEL (Roche, Basel, Switzerland, Cat# 11684817910) staining was performed to analyze cell death. The percentage of staining per follicle or corpus luteum was classified into four levels (0 (0\u0026ndash;5%), 1 (6\u0026ndash;25%), 2 (26\u0026ndash;50%), 3 (51\u0026ndash;75%), and 4 (\u0026gt;\u0026thinsp;75%), and multiplied by the staining intensity (0 negative, 1 weak, 2 intense) to calculate the positive index [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eData were analyzed using SPSS software (version 28.0, StatsGuild Inc, Urayasu, Chiba, Japan.). A comparative analysis of the means of the four groups was performed using one-way analysis of variance (ANOVA). Significance was set at P\u0026thinsp;\u0026gt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePlasma hormone concentrations\u003c/h2\u003e \u003cp\u003eTo investigate the effect of the systemic administration of UPA, changes in plasma estradiol, progesterone, and corticosterone levels in response to UPA administration were measured. Plasma estradiol levels were elevated by the administration of UPA (4, 20, and 100 mg/kg) compared to that of the control (0 mg/kg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In contrast, the plasma progesterone levels showed no significant changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). It is well known that UPA possesses glucocorticoid receptor activation function [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and plasma corticosterone levels were examined to investigate the effect of UPA; however, UPA had no significant impact on corticosterone levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistological examination\u003c/h2\u003e \u003cp\u003eTo investigate the effect of systemic administration of UPA, the wet weight of representative reproductive organs and the pathological appearance in response to UPA administration were examined. The wet weights of the uteri were unaffected by the administration of UPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In contrast, dilation of the lumen, dilation of uterine glands, increase in columnar height, and vacuolation of the luminal epithelium were observed after the administration of UPA, although the effect was not dose-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u0026ndash;g). We also observed vaginal epithelial hyperplasia after UPA administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh\u0026ndash;k), and dose-dependent effect on vaginal epithelial hyperplasia was suspected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe wet weight of ovaries was not significantly affected by the different doses of UPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). To investigate the effect of UPA, follicles at each developmental stage were histologically counted and examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The number of follicles at each developmental stage was unchanged in the control group (0 mg/kg), but the number of primordial follicles was substantially increased by the administration of UPA at 4 and 20 mg/kg doses. The number of primordial follicles tended to decrease with increasing doses of UPA, but the number of primary, secondary, and antral follicles tended to increase with increasing doses of UPA. In the ovary, unruptured follicles, corpus luteum cysts, and follicular cysts were more frequently observed after treatment with UPA, and there was a decrease in the number of large follicles and new and old corpora lutea (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u0026ndash;h). The degree of findings was more severe in the 20 and 100 mg/kg groups than in the 4 mg/kg group but did not differ between the 20 and 100 mg/kg groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOvarian immunohistochemical examination\u003c/h2\u003e \u003cp\u003eAMH [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], FOXO3a, and pAkt [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] are known to be representative of the activation of the primordial ovarian follicle pool, and based on the result that UPA administration markedly elevated the number of primordial follicles, we stained the ovaries and observed the granulosa cells and corpus luteum of each follicle. We evaluated granulosa cells, except for MMP1 and TIMP2, which we evaluated as corpus luteum. AMH-stained granulosa cells of the ovary were pronounced in the 100 mg/kg group, but the other doses were comparable to those of the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In contrast, FOXO3a staining decreased in the 100 mg/kg group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), and tended to be lower depending on the UPA dose. Although pAkt stanining of granulosa cells tended to be higher in UPA 100 mg/kg treatment rats, it did not show significant changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKi67 is a representative marker of cellular proliferation. Ki67 staining of each follicle was not expressed in primordial follicles and increased as the follicles developed in the control group. However, UPA treatment decreased the staining of Ki67 in each developmental stage of follicles in granulosa cells; thus, UPA was suspected to decrease the cellular growth of granulosa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The cellular death status of granulosa cells was examined using TUNEL staining, and it showed an increasing trend with increasing dose in primordial and primary follicles, but the difference was not noted with all doses of UPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMMP1 was associated with the regression of corpora lutea, and the expression of MMP1 in ovarian corpus luteum was substantially decreased at 20 and 100 mg/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). TIMP2, which antagonizes the regression of corpora lutea, tended to increase with UPA administration, although dose-dependency was not observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The antioxidant stress markers SOD1 and catalase were decreased by UPA in follicles other than primordial follicles, and catalase was decreased by UPA in primordial follicles and decreased by UPA in secondary follicles, and tended to increase with increasing dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSelective PR modulators (SPRMs) can modulate progesterone pathways. UPA, an SPRM, acts on uterine fibroids and shrinks them. The mechanism of action of UPA in uterine fibroids is related to the higher expression of PRs in uterine fibroids than in normal uterine muscle. UPA suppresses cell proliferation in a dose-dependent manner by inhibiting the expression of PCNA (proliferating cell nuclear antigen), Bcl-2, collagen, VEGF (vascular endothelial growth factor), and adrenomedullin in uterine myoma cells without affecting normal uterine smooth muscle cells and induces apoptosis by increasing the expression of caspase-3, PARP, and matrix metalloproteinases (MMPs) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. SPRMs induce amenorrhea and reduce pain and are used as a treatment for uterine fibroid. Moreover, SPRMs inhibit prostaglandin secretion [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Although SPRM does not affect the normal myometrium and influences on uterine fibroids and endometriosis, its effect on the ovaries remains unknown. The effects of long-term UPA administration on the ovaries were examined.\u003c/p\u003e \u003cp\u003eIn the PEARL trials, UPA was found to be non-inferior to the GnRH analog in controlling bleeding and shrinking fibroids but did not show lower estrogen levels [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Long-term administration of UPA increases uterine myoma reduction [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and this effect persists even after treatment completion. Similar to other SPRMs, UPA causes a characteristic change in the endometrium called PAEC [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], which is said to disappear within 6 months after the end of UPA administration.\u003c/p\u003e \u003cp\u003eWhen administered before surgery, UPA decreases myoma volume, reduces myoma-related bleeding, and increases hemoglobin levels [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Long-term administration of UPA is considered necessary to control uterine fibroids; however, given that it is administered to women of reproductive age, this study was conducted to determine whether long-term administration would have any adverse effects on the ovaries. The efficacy and safety of long-term UPA administration in myomas have been studied [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In vitro, UPA induces cell death in uterine myoma cells, but has the remarkable property of causing no noticeable change in the normal myometrium. Therefore, we examined whether UPA also induces cell death in ovarian cells.\u003c/p\u003e \u003cp\u003eUPA was administered to rats and its effect on the ovaries was investigated. The blood estradiol level was not decreased by UPA administration, but was notably increased, whereas progesterone and corticosterone levels did not change substantially, suggesting that luteal function and adrenal glands were not severely affected by UPA administration. These results are similar to those of the PEARL trial described above. The number of follicles increased with UPA administration. Primordial follicles were activated and follicle development proceeded, but the number of closed follicles or luteinized unruptured cysts increased. This is owing to the suppression of the LH surge by UPA, thus inhibiting ovulation.\u003c/p\u003e \u003cp\u003eThe expression of AMH, FOXO3, and pAkt as factors related to primordial follicle activation was examined via immunohistological examination. AMH, which suppresses primordial follicle activation, was significantly increased in the 100 mg/kg group, whereas FOXO3, which also suppressed primordial follicle activation, was significantly decreased at 100 mg/kg. pAkt, the primordial follicle activation, showed a slight increasing trend, but no substantial change was observed. These results suggest that other factors or mechanisms may be involved in primordial follicle activation.\u003c/p\u003e \u003cp\u003eUPA treatment decreased the luteal regression marker MMP1 levels and increased the luteal regression antagonist marker TIMP2 levels, suggesting that luteal regression was not suppressed and that mature follicles failed to ovulate, resulting in unruptured luteinized cysts. It has been reported that administration of UPA to uterine fibroids leads to the degradation of hydrogen peroxide and production of reactive oxygen species (ROS) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and UPA with sperm possibly acts as a scavenger of ROS [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e][\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the present study, UPA was administered to normal ovaries, and immunohistological examination revealed that SOD-1 and catalase, which are related to ROS, were decreased by UPA administration in all but primordial follicles. In terms of catalase, SOD-1 tended to decrease from primordial follicles to secondary follicles. The antioxidants SOD1 and catalase tended to decrease with UPA administration, suggesting that ROS may be produced by UPA administration in the ovary; however, this was not a significant change.\u003c/p\u003e \u003cp\u003eWhen 30 mg of UPA was used in humans immediately before ovulation, there was no significant change in Ki67, a proliferation marker, in the endometrium [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In the ovaries of rats treated with UPA for a long period of time, the present study showed no significant changes in Ki67 and TUNNEL, suggesting that UPA may have no relationship with ovarian proliferation or cell death. This is similar to the relationship between the uterine muscle and uterine fibroids. SPRMs act through progesterone receptors and as agonists or antagonists in various target organs. Among them, UPA inhibits the proliferation and induction of apoptosis and cell death pathways in leiomyoma cells, translating to smaller fibroids and lower uterine volumes at the clinical level, with no significant side effects.\u003c/p\u003e \u003cp\u003eThese results suggest that long-term administration of UPA to the ovary activates primordial follicles and promotes follicle development, but suppresses the LH surge, resulting in ovulation suppression when closed follicles and luteinized unruptured cysts increase. The mechanism by which UPA administration promotes follicle development was not elucidated in this study. It is possible that factors other than AMH, FOXO3, and pAkt are involved in primordial follicle activation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e: This animal experimental protocol was approved by the Animal Research Ethics Committee of ASKA Pharmaceutical Co., Ltd. (authorization reference number: K14-026).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate, Consent for publication\u003c/strong\u003e: NA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material (data transparency)\u003c/strong\u003e: Applicable according to the reasonable requirements and the corresponding author is responsible if someone wants to request the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e: Motoko Fukui\u0026nbsp;and Seiji Shibata are employees of ASKA Pharmaceutical Co., Ltd. All other authors have no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Information:\u0026nbsp;\u003c/strong\u003eThis study was funded by ASKA Pharmaceutical Co., Ltd.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution:\u0026nbsp;\u003c/strong\u003eMH, OWH, MF, SS contributed to the conception and design of the study. MH, OWH, MF, SS, MU, AN, and YU\u0026nbsp;contributed to the material preparation,\u0026nbsp;acquisition of data, and analysis and interpretation of data.\u0026nbsp;OWH was the principal investigator and played a significant role in the interpretation of data.\u0026nbsp;The first draft of the manuscript was written by MH, OWH and MF, and KS, MH, KK and YO\u0026nbsp;revised it critically for important intellectual content. All authors have read and approved the final version of the manuscript, and YO gave the approval to submit the latest version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: The authors thank editage (https://www.editage.jp) for language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGarnock-Jones KP, Duggan ST. Ulipristal Acetate: A Review in Symptomatic Uterine Fibroids. Drugs. 2017;77(15):1665\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEkanem E, Talaulikar V. Medical Therapy for Fibroids: What Next for Ulipristal Acetate? Adv Ther. 2021;38(1):137\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlasier AF, Cameron ST, Fine PM, Logan SJ, Casale W, Van Horn J, et al. Ulipristal acetate versus levonorgestrel for emergency contraception: a randomised non-inferiority trial and meta-analysis. Lancet. 2010;375(9714):555\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrache V, Cochon L, Jesam C, Maldonado R, Salvatierra AM, Levy DP, et al. Immediate pre-ovulatory administration of 30 mg ulipristal acetate significantly delays follicular rupture. Hum Reprod. 2010;25(9):2256\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLira-Albarran S, Durand M, Larrea-Schiavon MF, Gonzalez L, Barrera D, Vega C, et al. Ulipristal acetate administration at mid-cycle changes gene expression profiling of endometrial biopsies taken during the receptive period of the human menstrual cycle. Mol Cell Endocrinol. 2017;447:1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNallasamy S, Kim J, Sitruk-Ware R, Bagchi M, Bagchi I. Ulipristal blocks ovulation by inhibiting progesterone receptor-dependent pathways intrinsic to the ovary. Reprod Sci. 2013;20(4):371\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi HW, Liao SB, Yeung WS, Ng EH, O WS, Ho PC. Ulipristal acetate resembles mifepristone in modulating human fallopian tube function. Hum Reprod. 2014;29(10):2156\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChabbert-Buffet N, Pintiaux-Kairis A, Bouchard P, Group VAS. Effects of the progesterone receptor modulator VA2914 in a continuous low dose on the hypothalamic-pituitary-ovarian axis and endometrium in normal women: a prospective, randomized, placebo-controlled trial. J Clin Endocrinol Metab. 2007;92(9):3582\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunuce MJ, Cicare J, Zumoffen C, Caille A, Ghersevich S, Bahamondes L. Effects of ulipristal acetate on sperm DNA fragmentation during in vitro incubation. Eur J Contracept Reprod Health Care. 2013;18(5):355\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGomez-Elias MD, Munuce MJ, Bahamondes L, Cuasnicu PS, Cohen DJ. In vitro and in vivo effects of ulipristal acetate on fertilization and early embryo development in mice. Hum Reprod. 2016;31(1):53\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDixon D, Alison R, Bach U, Colman K, Foley GL, Harleman JH, et al. Nonproliferative and proliferative lesions of the rat and mouse female reproductive system. J Toxicol Pathol. 2014;27(3\u0026ndash;4 Suppl):1S\u0026ndash;107S.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePedersen T, Peters H. Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil. 1968;17(3):555\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishra B, Ortiz L, Luderer U. Charged iron particles, components of space radiation, destroy ovarian follicles. Hum Reprod. 2016;31(8):1816\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkino N, Wada-Hiraike O, Isono W, Terao H, Honjo H, Miyamoto Y, et al. Activation of Nrf2/Keap1 pathway by oral Dimethylfumarate administration alleviates oxidative stress and age-associated infertility might be delayed in the mouse ovary. Reprod Biol Endocrinol. 2019;17(1):23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim J, Lawson GW, Nakamura BN, Ortiz L, Hur JA, Kavanagh TJ, et al. Glutathione-deficient mice have increased sensitivity to transplacental benzo[a]pyrene-induced premature ovarian failure and ovarian tumorigenesis. Cancer Res. 2013;73(2):908\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol. 1999;17(5):1474\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWagner BL, Pollio G, Giangrande P, Webster JC, Breslin M, Mais DE, et al. The novel progesterone receptor antagonists RTI 3021-012 and RTI 3021-022 exhibit complex glucocorticoid receptor antagonist activities: implications for the development of dissociated antiprogestins. Endocrinology. 1999;140(3):1449\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLa Marca A, Broekmans FJ, Volpe A, Fauser BC, Macklon NS. Table ESIGfRE\u0026ndash;AR. Anti-Mullerian hormone (AMH): what do we still need to know? Hum Reprod. 2009;24(9):2264\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Kawamura K, Cheng Y, Liu S, Klein C, Liu S, et al. Activation of dormant ovarian follicles to generate mature eggs. Proc Natl Acad Sci U S A. 2010;107(22):10280\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCourtoy GE, Donnez J, Marbaix E, Dolmans MM. In vivo mechanisms of uterine myoma volume reduction with ulipristal acetate treatment. Fertil Steril. 2015;104(2):426\u0026ndash;34. e1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGemzell-Danielsson K, Hamberg M. The effect of antiprogestin (RU 486) and prostaglandin biosynthesis inhibitor (naproxen) on uterine fluid prostaglandin F2 alpha concentrations. Hum Reprod. 1994;9(9):1626\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonnez J, Tomaszewski J, Vazquez F, Bouchard P, Lemieszczuk B, Baro F, et al. Ulipristal acetate versus leuprolide acetate for uterine fibroids. N Engl J Med. 2012;366(5):421\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhitaker LH, Murray AA, Matthews R, Shaw G, Williams AR, Saunders PT, et al. Selective progesterone receptor modulator (SPRM) ulipristal acetate (UPA) and its effects on the human endometrium. Hum Reprod. 2017;32(3):531\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLethaby A, Puscasiu L, Vollenhoven B. Preoperative medical therapy before surgery for uterine fibroids. Cochrane Database Syst Rev. 2017;11:CD000547.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonnez J, Hudecek R, Donnez O, Matule D, Arhendt HJ, Zatik J, et al. Efficacy and safety of repeated use of ulipristal acetate in uterine fibroids. Fertil Steril. 2015;103(2):519\u0026ndash;27. e3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonnez J, Donnez O, Matule D, Ahrendt HJ, Hudecek R, Zatik J, et al. Long-term medical management of uterine fibroids with ulipristal acetate. Fertil Steril. 2016;105(1):165\u0026ndash;73. e4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUra B, Monasta L, De Spelorzi YCC, Arrigoni G, Franchin C, Biffi S et al. Proteins involved in oxidative stress in leiomyoma tissues treated with ulipristal acetate. Mol Med Rep. 2021;23(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang GP, Liao YJ, Huang LL, Zeng XJ, Liao XH. Effects and molecular mechanism of pachymic acid on ferroptosis in renal ischemia reperfusion injury. Mol Med Rep. 2021;23(1).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ulipristal acetate, ovary, follicle development, luteinization","lastPublishedDoi":"10.21203/rs.3.rs-2847062/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2847062/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUlipristal (UPA), a selective progesterone receptor modulator, has both agonistic and antagonistic effects on progesterone receptors. UPA suppresses ovulation by inhibiting the luteinizing hormone (LH) surge from the pituitary gland; however, the direct effect of UPA on ovarian tissue remains poorly studied. In the present study, we examined the effects of UPA on the ovaries of rats.\u003c/p\u003e \u003cp\u003eRats were treated for 28 d with 4, 20, and 100 mg/kg UPA. UPA treatment increased the number of primordial follicles at each treatment group, with the highest number found in the 4 mg/kg group, and the number of primordial follicles decreasing with increasing dose. The number of primary and antral follicles tended to increase with increasing UPA levels. Furthermore, the decrease in primary follicle number could be attributed to the exhaustion of follicles, but the examination of proliferation markers, oxidative stress markers, and cell death markers revealed no remarkable toxic effects on ovarian tissues. These results suggest that UPA treatment promotes follicle development at each stage but inhibits ovulation by suppressing the LH surge, resulting in an increase in atretic follicles or unruptured luteinized cysts. UPA may not have toxic effects on the ovary because the expression of antioxidant genes and cell death markers was not dramatic in follicles treated with UPA. Taken together, these results suggest that UPA may not have a direct local effect on ovarian follicles. Hence, we hypothesized that prolonged UPA treatment in patients with uterine fibroids may not be harmful and may not decrease future fecundity.\u003c/p\u003e","manuscriptTitle":"Effect of long-term treatment of ulipristal acetate on rat ovarian tissue","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-26 14:20:36","doi":"10.21203/rs.3.rs-2847062/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9521359-ffed-4fe3-b484-73016118348a","owner":[],"postedDate":"May 26th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-12-05T04:29:22+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-26 14:20:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2847062","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2847062","identity":"rs-2847062","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}