Stanozolol, an Anabolic-Androgenic Steroid, Modulates Expression of Receptors and Disrupts Uterine Histoarchitecture in Mus musculus

Stanozolol, an Anabolic-Androgenic Steroid, Modulates Expression of Receptors and Disrupts Uterine Histoarchitecture in Mus musculus | 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 Stanozolol, an Anabolic-Androgenic Steroid, Modulates Expression of Receptors and Disrupts Uterine Histoarchitecture in Mus musculus Praveenkumar S. Kondaguli, Nafisa H. Balasinor, Chaitra R. Sharma, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7580090/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Abuse of Anabolic-Androgenic Steroids (AAS) by professional and recreational athletes for endurance performance and physique is increasing globally, consequently resulting in secondary pathophysiological effects. The present investigation aims to know the efficacy of one of the AAS, stanozolol, on uterine physiology. A total of 15 female mice were assigned to three experimental groups (n=5). ST was dosed subcutaneously (low-dose, 0.5 mg/kg bwt; high-dose, 5.0 mg/ kg bwt or 1% alcohol-baseline control) for 30 days, and treatment was withdrawn on the 31 st day. Morphometric evaluation of uterus demonstrated aberrant endometrial luminal epithelial proliferation, marked by irregular branching with papillary formations. Prolonged administration of ST results in disruption of uterine organization, reflecting its deleterious impact on uterine histoarchitecture. In high-dose-treated mice, immunoexpression of ERα was downregulated in the endometrial compartment while significantly upregulated in myometrium, concomitant with increased myometrial thickness leading to myometrial hypertrophy. Results indicate the androgenic-to-anabolic efficacy of ST in myometrial compartment. ST, being a derivative of DHT, does not convert into estrogen; instead it may bind to ERα in the myometrium, suggesting a compartment-specific effect of ST on murine uterus. Furthermore, substantial hyperexpression of PRLR in both endometrium and myometrium, indicates a potential stimulatory effect of ST on AR-mediated release of PRL. High-dose ST induced a significant increase in circulating T and PRL levels, while decreasing E 2 concentrations, which reflects an altered hormonal milieu. It is inferred that prolonged treatment of ST resulted in aberrant endometrial proliferation, histoarchitectural anomalies, compartment-specific differential immunoexpression of hormone receptors in the endometrium and myometrium, and altered serum hormonal milieu. Mouse Estrogen receptorα Androgen receptor Prolactin receptor Serum hormone levels Stanozolol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Anabolic-androgenic steroids (AAS) comprise a large category of synthetic analogs of natural male hormone testosterone, originally developed to maximize anabolic potency and minimize androgenic effects (Hoffman and Ratamess, 2006; Oberlander et al., 2012; Piacentino et al., 2015; Huang and Basaria 2018). Due to their diverse biological actions, AAS have been used in the treatment of a variety of pathological conditions like pulmonary disorders, renal disorders, aplastic anemia, hypogonadism, chronic debilitating illness, trauma, and postoperative recovery (Basaria et al., 2001; Shahidi, 2001; Evans, 2004; Kicman, 2008; Woerdeman and de Ronde, 2011; Piacentino et al., 2015; Kloner et al., 2016). However, over the last several decades, AAS have been abused as ergogenic aids by athletes and bodybuilders to enhance performance by augmenting muscular development, strength and endurance (Wagner, 1989; Windsor and Dumitru, 1989; Maravelias et al., 2005). Regrettably, the use of AAS, is becoming popular among adolescent girls and also women athletes, consequently resulting in various secondary pathophysiological effects. The widespread abuse of AAS at supraphysiological doses poses a significant global concern, and comprehensive elucidation of their impact on female reproductive physiology and fertility at the molecular level remains a critical area for investigation. It has been reported that increased levels of androgens by exogenous administration of androgens and/or its derivatives induce detrimental effects on the uterine endometrium in women (Okon et al.,1998; Tuckermann et al., 2000; Zhang et al., 2007). It is worth mentioning here that over the last two decades, many women have been affected by infertility and reproductive tract dysfunction such as endometriosis, uterine cancer (endometrial cancer), anovulation, implantation failure and early pregnancy loss. The primary function of the uterus is to support pregnancy with hormonally responsive endometrium that facilitates embryo attachment, implantation, and maintenance of a conducive environment for fetal development. Hence, successful implantation and maintenance of pregnancy till term rely upon ideal cross talk/signals between the ovary, good quality embryos and a receptive endometrium. In a preliminary experiment from our laboratory, we investigated the efficacy of ST on the maintenance of pregnancy in mice, which revealed that administration of ST resulted in the interruption of pregnancy (Jayamma et al., 2012). A significant reduction noted in the number of corpora lutea, secondary and antral follicles, partial resorption of the embryo/fetus with placental scars and absence of viable embryos/fetus, suggests that ST caused an imbalance in progesterone and estrogen secretion by the ovary, leading to the interruption of pregnancy principally due to the deficiency of luteal hormones (Jayamma et al., 2012). In another experiment, the effect of ST was evaluated on the implantation and maintenance of pregnancy in mice (Sharma et al., 2021). The low-dose ST-treated mice maintained gestation until term with reduced litter size, while high-dose-treated mice revealed vaginal plug at frequent intervals, indicating conception failure. In high-dose-treated mice, decreased serum levels of estradiol and progesterone, accompanied by increased testosterone, and downregulated endometrial expression of ERα and PR, suggest a deficiency of steroid hormones and their respective receptors. Decreased ovarian expression of ERα, hyperexpression of PRLR, AR and abated progesterone secretion led to luteal dysfunction, hindered signalling to LIF and Hoxa 10, consequently attenuating endometrial receptivity during the implantation window, resulting in implantation failure (Sharma et al., 2021). Hence, the current investigation aims to gain further insights into the impact of one of the anabolic-androgenic steroids stanozolol (ST) [17β-hydroxy-17α-methyl-androstano (3, 2-c) pyrazole] , on the female reproductive tract, emphasizing the preparedness of the uterus for successful implantation in the mouse, Mus musculus, with the following objectives: To assess the morphological and histomorphometric analysis of the uterus post- administration of low and high doses of ST. To analyze the efficacy of ST on spatiotemporal immunoexpression of estrogen receptor α (ERα), androgen receptor (AR), and prolactin receptor (PRLR) in the mouse to examine the readiness of its uterus for successful implantation. Materials and Methods Experimental animals, study design and hormone treatment All protocols in this experiment adhered to the CPCSEA guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (No.639/GO/02/a/CPCSEA) at the Department of Zoology, Karnatak University, Dharwad. In total 15 sexually mature (3 months old) female Swiss albino ‘strains’ (weighing 25-30 gm) exhibiting regular estrous cyclicity were obtained from the mouse breeding center maintained in the Department of Zoology, Karnatak University, Dharwad. All the mice were housed in individual polypropylene cages and maintained a 12 h light: dark cycle at 27±1˚C with 40-50% RH, food (pelleted diet, Goldmohur, Lipton, India), and water supplied ad libitum . Stanozolol (ST) was obtained from Sigma-Aldrich, USA. The mice were randomly assigned to three experimental groups containing 5 animals in each group. ST was dosed subcutaneously [0.5 mg/kg bwt (low dose of ST); 5.0 mg/kg bwt (high dose of ST); or 1% alcohol (vehicle control)] for 30 consecutive days. The body weight of all animals was recorded at the beginning of the experiment (initial body weight) and on the day of autopsy (final body weight). On the 31st day, animals were sacrificed. The uteri were excised, weighed, fixed in Bouin’s fluid/4% paraformaldehyde (PFA), and processed for routine histology and immunohistochemistry. Histoarchitecture and histomorphometrical kinetics of uterus Bouin’s fixed tissue samples were dehydrated in a series of increasing concentrations of ethanol (10% - 100%), cleared in benzene, and embedded in paraffin (Fisher Scientific). Moulded blocks of uterine tissues were sectioned at 5µm on a rotational microtome (Leica RM 2025) and subjected to standard histology. Morphometric analysis was performed by calibrated Procam software (Olympus BX5I microscope). A total of twenty-five readings from each animal (both the control and treated group) were considered for the following parameters, and are depicted in Fig. 1. Uterine diameter: mean diameter (D) of X and Y axis (X-major axis, Y-minor axis passing through the center): D=(X+Y)/2 (in µm) Luminal epithelial cell height: from the apical (luminal) surface to the basement membrane separating the epithelium from the stroma. The thickness of the endometrium: from the longest luminal epithelial surface to the beginning of the circular layer of the myometrium. The thickness of myometrium: the thickness of both circular and longitudinal layers of myometrium was measured. Immunohistochemical analysis for estrogen receptor alpha (ERα), prolactin receptor (PRLR), and androgen receptor (AR) Tissue sections were deparaffinized in xylene and rehydrated in descending concentrations of ethanol. This was followed by antigen retrieval in trisodium citrate buffer (pH=6.0, C3674 Sigma-Aldrich, St. Louis, MO, USA) for 20 min in the oven at 90˚C, followed by cooling at ambient temperature. Endogenous peroxidase was inactivated with 3% hydrogen peroxide (H 2 O 2 ). Nonspecific binding was blocked by 5% BSA (A3059, Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the sections were incubated with rabbit polyclonal antibodies specific for ERα (host: rabbit; 1:100; MC-20, cat. no sc-542); AR (host: rabbit; 1:100; N-20, cat. no. sc-816); PRLR (host: rabbit; 1:200; M-170, cat. no sc-30225) from Santa Cruz Biotechnology, USA at 4˚C, overnight. As a negative control, primary antibodies were replaced with antibody diluents (1% BSA). After 3 washes in PBS and PBS-T the sections were incubated with the Goat anti-rabbit IgG conjugated to HRP (sc-2004, Santa Cruz Biotechnology, Dallas, Texas, USA). Staining was visualized with 3, 3’-diaminobenzidine tetra-hydrochloride hydrate (DAB; D5637, Sigma-Aldrich, St. Louis, MO, USA). Finally, sections were counterstained with Harris-Hematoxylin (39411, Fisher Scientific). Images were acquired using Nikon Eclipse 80i with ACT-2U software (Nikon Corporation, Tokyo, Japan). Quantitative evaluation of immunoexpression of ERα, AR and PRLR The immunohistochemical staining intensities of ERα, AR, and PRLR in different compartments of the uterus were evaluated by measuring their optical densities (ODs). Briefly, the images were captured with Nikon microscope 90E with ACT-2U software at 200X magnification. The OD was evaluated using ImageJ (Fiji) software. Each image was first deconvoluted using the H/DAB vector into three different colored images (i.e., green, brown, and blue). The brown DAB image was converted to grayscale and its intensity was recorded by measuring the mean integrated intensities (mean grey value). At least five fields of each histological compartment were recorded in each section, and five sections per animal were evaluated. The intensity numbers were converted into the OD using the following equation: (OD = log (max intensity/mean intensity), where the max intensity = 250 and the mean intensity = mean grey value. The results were exported to a Microsoft Excel spreadsheet. The ODs of ERα, AR, and PRLR were evaluated in the luminal and glandular epithelium, stroma, circular and longitudinal layers of myometrium of each tissue section. Quantification of serum steroid hormone The blood was collected from jugular vein of both control and treated mice; and centrifuged (2000 g for 10 min at 4 °C) to extract the serum, which was then kept at -80 °C until analyzed. Electrochemiluminescence immunoassay (ECLIA) was used to assess the concentrations of testosterone (T), estradiol (E 2 ) and prolactin (PRL) using a Cobas e411 completely automated electrochemiluminescence immunity analyzer (Cobas; Roche Diagnostics, GmbH, Mannheim, Germany). To measure the serum hormone levels, a proprietary serum assay kit (Elecsys Estradiol III, test no. 1370; Elecsys Testosterone II, test no. 111; Elecsys Prolactin II test no. 131) by Roche Diagnostics, Indianapolis, Indiana, USA) was utilized. A PerkinElmer JANUS Automated Liquid Handling System was used to move the samples into Roche sample cups. The secondary antibody labeled with a ruthenium complex was then added to streptavidin-coated microparticles. Each test was run following the manufacturer's three-level assay control. The system software of the Roche/Hitachi Cobas e 411 control unit calculated the hormone concentration after the hormone assay measurement and was immediately recorded on the Windows-based computer-aided evaluation program (WinCAEv). All tests had intra- and inter-assay errors of less than 10% and 15%, respectively. Statistical analysis All statistical tests were performed using SPSS version 20. The initial and final body weight difference was compared using paired t-test. Uterine diameter, luminal epithelial cell height, thickness of endometrium and myometrium, optical density (OD), and serum steroid hormone levels between different treatment and control groups were evaluated using a one-way analysis of variances (ANOVA) technique to know the significant difference between different treated groups. The multiple comparison test (Tukey’s HSD post hoc test) was carried out to determine which treatment group significantly differed from the control group. Numerical values are presented as the mean ± standard error (SE). Both statistical tests were two-sided tests with a 5% & 1% levels of significance ( P < 0.05 & P < 0.01). Results Effect of ST on body weight The results revealed a significant increase in body weight of both LD (t 8 = 5.993; p < 0.01) and HD (t 8 = 9.428; p < 0.01) treated mice when compared to initial weight (Fig. 2). Administration of ST for 30 consecutive days leads to alterations in uterine histomorphology Histoarchitecture of Uterus: Endometrium: The luminal epithelium of control mice consists of simple columnar cells that extend into endometrial tubular glands within the loosely arranged reticular connective tissue of endometrial stroma (Figs. 3A & B). A significant increase in the height of luminal epithelium in LD ( p < 0.05) and HD ST-treated ( p < 0.001) mice was noticed when compared to control group (F 2,12 = 97.953, p < 0.001) (Fig. 5B). The endometrial luminal epithelium of LD treated mice exhibited cryptic, wavy and irregularly branched with widened lumen (Fig. 3C & D). Also, the endometrial luminal epithelium of HD treated mice exhibited tortuous and irregular branching giving a papillary appearance (Figs. 3E, F). While this hypertrophy of luminal epithelium and its lining resulted in detachment of endometrial portion in two HD treated mice (Fig. 4). Diameter of uterus: Uterine diameter decreased significantly in both LD ( p < 0.05) and HD treated groups ( p < 0.01) compared to control animal group (F 2,12 = 8.485, p 0.05) and a significant increase in HD ( p < 0.05) ST-treated group were observed (F 2,12 =4.747; p < 0.05); (Fig. 5C). Myometrium: The control myometrium was composed of an inner circular and outer longitudinal layer of smooth muscle fibers separated by highly vascular connective tissue (Figs. 3A, B). A substantial increase in thickness of circular myometrium in both low ( p < 0.05) and high dose ( p < 0.001) was noticed (F 2,12 =24.690, p 0.05) and a significant increase ( p < 0.001) were observed in HD treated animals (F 2,12 = 41.738, p < 0.001); (Fig. 5E). Immunolocalization of ERα in mouse uterus upon ST treatment Endometrium: Immunohistochemical signal for ERα was found in nuclei of endometrial luminal and glandular epithelial cells, stromal cells, and myometrium (circular and longitudinal muscle layer) in the uterus of control animals (Fig. 6A). A significant decrease in luminal epithelial expression of ERα was noticed in both LD and HD ( p < 0.001) ST treated groups when compared to control (F 2,12 = 52.714; p < 0.001). Glandular epithelium exhibited a notable reduction in ERα expression in both LD and HD ST-treated mice ( p < 0.05), (F 2,12 = 7.168; p < 0.01). Besides, the intensity of immunoreactions for ERα was significantly down-regulated in the endometrial stromal cells of both LD and HD ( p < 0.001) treatment groups (F 2,12 = 26.091; p < 0.001); (Figs. 6B & C; Fig. 9A). Myometrim: However, the intensity of ERα in the myometrial layer was substantially higher in both treatment groups in comparison to control group. Its intensity was dramatically augmented in the circular layer of myometrium in both LD and HD treatment groups ( p < 0.001), (F 2,12 = 92.342; p < 0.001). A noticeable increase in the longitudinal layer of myometrial expression of ERα was witnessed in LD ( p < 0.01) and in HD ( p < 0.001) ST-treated mice (F 2,12 = 45.650; p < 0.001); (Figs. 6B & C; Fig. 9A). Immunolocalization of AR in mouse uterus following ST treatment Endometrium: The endometrial and myometrial expression of AR was observed in the uterine compartments of control mice (Fig. 8A). No noticeable difference in AR expression was observed in luminal epithelium of both treatment groups ( p > 0.05) when compared to control (F 2,12 = 0.213; p > 0.05). Whereas, an insignificant increase in glandular epithelium of LD ( p > 0.05) and a significant increase in HD ( p < 0.05) were observed (F 2,12 = 4.106; p < 0.05). ST treatment up-surged the expression of AR in the endometrial stroma of LD ( p < 0.05) and in HD ( p < 0.001) ST treated mice (F 2,12 = 23.310; p 0.05) and a significant increase in HD ( p < 0.001) treatment group was observed when compared to control (F 2,12 = 16.098; p < 0.001). An upregulation of AR was visualized in longitudinal myometrium in LD ( p < 0.05) and HD ( p < 0.01) treatment groups (F 2,12 = 9.739; p < 0.01); (Figs. 7B & C; Fig. 9B). Immunolocalization of PRLR in mouse uterus upon ST treatment Endometrium: The immunolocalization of PRLR noticed mainly in the inner wall of the plasma membrane of luminal and glandular epithelial cells, and its expression in stromal cells was below the detection level in the control uterus. Very few myometrial cells expressed PRLR protein (Fig. 8A). The endometrial immunoexpression of PRLR deepened in the luminal epithelium of LD ( p < 0.01) and HD treated mice ( p < 0.001); (F 2,12 = 101.097; p < 0.001) and also, in glandular epithelial cells of LD and HD treated mice ( p < 0.001); (F 2,12 = 23.805; p < 0.001) when compared to control (Figs. 8B & C; Fig. 9C). Likewise, PRLR protein expressed deeply in stromal cells of both LD and HD treated groups ( p < 0.001) than the control (F 2,12 = 47.557; p < 0.001); (Figs. 8B & C; Fig. 9C). Myometrium: The myometrial immunoexpression of PRLR was augmented in both circular layer of low and high dose ( p < 0.001); (F 2,12 = 79.061; p < 0.001) and in longitudinal layer of both low and high dose treated mice ( p < 0.001); (F 2,12 = 73.871; p < 0.001) when compared to control (Figs. 8B & C; Fig. 9C). Effect of ST treatment on serum levels of Testosterone (T), Estradiol (E 2 ) & Prolactin (PRL) hormones Electrochemiluminescence immunoassay (ECLIA) was utilized to quantify the serum levels of T, E2 & PRL. Treatment of ST for 30 days caused a significant increase in serum T level in both LD ( p < 0.01) and HD ( p < 0.001) treatment groups (F 2,12 = 57.285; p 0.05) and a significant decline in HD ( p < 0.05) group were noticed (F 2,12 = 4.603; p < 0.05), (Fig. 10B). A substantial upregulation of PRL was observed in LD ( p < 0.05) and HD ( p < 0.01) treated mice in comparison to control group (F 2,12 = 13.069; p < 0.01); (Fig. 10C). Discussion Anabolic-androgenic steroids (AAS) cause adverse effects on a broad spectrum of organs, including the reproductive and neuroendocrine systems. The morphometric and histoarchitecture of the uterus were meticulously analysed i.e., in a compartment-specific manner – a) Endometrium (luminal epithelium, glandular epithelium and stroma) and b) Myometrium (circular and longitudinal muscle layer) to understand the efficacy of ST on the murine uterus. Prolonged treatment with ST resulted in a significant increase in body weight, primarily due to enhanced food intake, as witnessed in the feeding behavior of mice. The elevated body's energy demands might have caused increased food intake. The current investigation reveals a dose-dependent increase in body weight, which is likely attributable to the anabolic efficacy of ST to augment muscle mass. Such an increase in the growth/body weight following AAS treatment has been reported previously in rodents (Camargo et al., 2009; Brasil et al., 2015; Saddick 2018). The endometrium of mouse consists of epithelial (luminal and glandular) and stromal compartments that play distinct roles in the maintenance of hormonal microenvironment of the uterus. Uterine morphometric analysis reveals profound alterations in its morphology, specifically an aberrant endometrial proliferation induced irregular branching giving a papillary appearance in high-dose ST-treated groups, corroborating the earlier report by Far et al., 2007. While this proliferation of endometrium resulted in detachment of its portion in two high-dose ST-treated mice. The observed results suggest that prolonged treatment with ST for one month might have altered the ovarian signal to the uterus leading to its disorganization, reflecting the deleterious impact of STon uterine histoarchitecture. Parallel to our results, testosterone and some other AASs (17α- methyltestosterone and Nandrolone Decanoate) have been shown to alter uterine morphology and physiology in mice (Papaconstantinou et al., 2002; Simitsidellis et al., 2016) and rats (Camargo et al., 2009; 2014; Chuffa et al., 2011; Saddick 2018; Gerez et al., 2005). Interestingly, the high dose of ST is more effective in increasing myometrial thickness, indicating the dose-dependent androgenic to anabolic efficacy of this AAS in the myometrium region. Similarly, reports on rodents suggest that androgens stimulate the myometrium to a greater extent, whereas the endometrial cells are less responsive, suggesting stanozolol may exert a direct effect on the uterine tissue, potentially impacting its histoarchitecture and physiology (Nantermet et al., 2005). Further, androgens such as testosterone and 5α DHT have uterotrophic and anti-uterotrophic effects in rats and mice (Armstrong et al., 1976; Nantermet et al., 2005). Immunolocalization of Erα in the uterine compartments: The immunoexpression of ERα was downregulated in the endometrium and dramatically increased in the myometrium of both low and high-dose ST-treated mice. The observed down-regulation of immunoexpression of ERα in the endometrium signifying its negative impact on uterine receptivity, which supports our earlier study on implantation; where it was observed that high-dose ST downregulated the endometrial and ovarian immunoexpression of ERα as well as serum E 2 levels leading to impaired endometrial receptivity resulting in implantation failure during the implantation window (Sharma et al., 2021). Besides, it is worth mentioning here that the increased thickness of myometrium mentioned (in the above paragraph) and upregulated myometrial expression of ERα upon ST treatment indicate the androgenic to enhanced anabolic efficacy of this AAS. The observed result suggests the non-genomic action of this AAS in myometrial compartment. Since, ST is a derivative of DHT which is a non-aromatizable androgen that does not convert into estrogen, instead, it may bind to ERα in the myometrium, resulting in its hypertrophy. This differential immunoexpression of ERα in the regions of endometrium and myometrium indicates the compartment-specific effect of ST in the murine uterus. Further, it has already been reported that at doses greater than 100 nM, DHT can bind to ERα (Rochefort and Garcia., 1976 ; Ekena et al., 1998). Immunoexpression of AR protein in the uterine compartments: Androgen receptors (AR) are mainly localized in the uterine endometrial stromal cells and any effects of androgens in the epithelium are mediated directly through epithelial ER or indirectly through stromal AR or ER (Takeda et al., 1990; Pelletier et al., 2000). In the present investigation, prolonged treatment of ST augmented stromal immunoexpression of AR in the endometrium, suggesting AR-dependent mechanisms in this compartment. It is interesting to note that high-dose ST-treated mice exhibited endometrial proliferation and irregular branching, giving a papillary appearance. These observed results on the endometrial epithelial proliferation per se may be induced by the up-regulated expression of AR in stromal cells, and agree well with the findings that stromal AR stimulates IGF-I expression, which increases luminal epithelial proliferation (Kowalski et al., 2004). Similar to our results, chronic treatment with DHT increases AR-dependent epithelial cell proliferation in ovariectomized mice (Simitsidellis et al., 2016). Further, it is important to note that the observed up-regulated endometrial AR expression may render the endometrium more sensitive to androgen and contribute to poor reproductive performance, which supports our earlier report that high dose of ST impedes embryo implantation by attenuating endometrial receptivity in mice (Sharma et al., 2021). Similarly, high androgen levels have a negative impact on endometrium leading to infertility and miscarriage in women (Tuckerman et al., 2000). Immunoexpression of PRLR in the uterine compartments: Prolactin (PRL) is a pleiotropic polypeptide hormone of leuteotrophic complex. In rodents during pregnancy, PRL plays a crucial role in promoting the luteal production of P4, not by stimulating its synthesis, but by preventing its metabolism (Stouffer and Hearn 1998; Binart et al., 2000). In the present investigation, high-dose ST treatment resulted in pronounced upregulation of PRLR and concomitant hyperexpression of AR in both endometrium and myometrium (Pl see above para), indicating a potential stimulatory effect of ST on the AR-mediated PRLR immunoexpression and release of PRL. Because ST is a derivative of DHT—a non-aromatizable androgen—the E 2 -mediated increase of PRL may not be expected. Furthermore, the observed remarkable hyperexpression of PRLR and downregulation of ERα leads to luteal dysfunction by the ovary, which may result in pregnancy failure (if conceived), which is a concern and supports our earlier report (Sharma et al., 2021). Our results corroborate a report that pronounced up-regulation of prl8 and prlr genes in the uterus of DHT-treated mice (Simitsidellis et al., 2016). Evidences suggest that significant elevation of PRL levels observed in pathological hyperprolactinemia affect GnRH signaling, subsequently inhibiting FSH and LH secretion, thus impairing ovarian steroidogenesis, which consequently leads to infertility (Bouchard et al., 1985; Zinaman et al., 1995; Oner et al., 2013). Moreover, hyperprolactinemia can result in endometrial glandular hyperplasia and even endometrial adenomyosis in mice, rabbits and pigs (Chilton et al., 1988; Young et al., 1989; Rossi et al., 2002). Serum hormone levels: Testosterone (T), Estradiol (E 2 ) and Prolactin (PRL). In the present study, serum testosterone T, E 2, and PRL levels were measured, which revealed an elevation in serum T, PRL and down-regulated E 2 levels. Prolonged treatment of ST for one month may have interfered with the hypothalamic-pituitary-gonadal axis (HPG axis), altering hypothalamic and hypophyseal signals to the ovary, leading to impaired hormone secretion and consequently affecting the hormonal milieu. A recent report suggests that the use of AAS compounds can inhibit the pulsatile secretion of gonadotropin-releasing hormone in the hypothalamus, disrupting the release of ovarian hormones, which are crucial for follicular development and ovulation (Karila et al., 2024). This hormonal imbalance resulted in perturbations in the menstrual cycle, such as spaniomenorrhea and amenorrhea, anovulation, ultimately resulting in infertility (Saadedine et al., 2023; Karila et al., 2024). Likewise, the excess androgens in both women and rodents led to disruptions in the reproductive axis, resulting in decreased serum E2 levels, as well as LH and FSH (Saddick, 2018; Anawalt, 2019). Further, it was reported that seven of the nine female weight lifters who self-administered testosterone and anabolic steroids exhibited a 30-fold increase in serum T levels and a suppression of FSH (Malarkey et al., 1991). Moreover, the downregulation of serum E 2 levels in the present study may be attributed to the fact that ST is a derivative of DHT which is a non-aromatizable androgen that does not serve as a precursor for the ovarian production of estrogen, consequently resulting in reduced circulating estrogen levels. Conclusion It is inferred that prolonged treatment of ST resulted in aberrant endometrial proliferation, histoarchitectural anomalies, compartment-specific differential immunoexpression of hormone receptors in the endometrium and myometrium, and altered serum hormonal milieu. Declarations Acknowledgment: One of the authors (LSI) thanks Indian National Science Academy, New Delhi for the INSA Visiting Scientist Award to visit NIRRH, Mumbai. CRS thanks DST, New Delhi for the award of INSPIRE fellowship. PSK thanks KUD for the University Research Scholarship. All experiments were conducted in accordance with the regulations of CPCSEA guidelines and the Institutional Animal Ethical Committee No.639/GO/02/a/CPCSEA of the Karnatak University, Dharwad, Karnataka, India. References Anawalt BD (2019) Diagnosis and Management of Anabolic Androgenic Steroid Use. 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Endocrinol 157:2116-2128. https://doi.org/10.1210/en.2015-2032 Stouffer, R.L., Hearn, J.P. (1998) Endocrinology of the Transition from Menstrual Cyclicity to Establishment of Pregnancy in Primates. In: Bazer, F.W. (eds) Endocrinology of Pregnancy. Contemporary Endocrinology, vol 9. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-4612-1804-3_2 Takeda H, Chodak G, Mutchnik S, Nakamoto T, Chang C (1990) Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor. J Endocrinol 126:17-25. https://doi.org/10.1677/joe.0.1260017 Tuckerman EM, Okon MA, Li TC, Laird SM (2000) Do androgens have a direct effect on endometrial function? An in vitro study. Fertil Steril 74:771-779. https://doi.org/10.1016/S0015-0282(00)00711-1 Wagner JC (1989) Abuse of drugs used to enhance athletic performance. Am J Hosp Pharm 46:2059–2067. https://doi.org/10.1093/ajhp/46.10.2059 Windsor R, Dumitru D (1989) Prevalence of anabolic steroid use by male and female adolescents. Med Sci Sports Exerc 21:494–497. https://doi.org/10.1249/00005768-198910000-00002 Woerdeman J, De Ronde W (2011) Therapeutic effects of anabolic androgenic steroids on chronic diseases associated with muscle wasting. Expert Opin Investig Drugs 20(1):87-97. https://doi.org/10.1517/13543784.2011.544651 Young KH, Kraeling RR, Bazer FW (1989) Effects of prolactin on conceptus survival and uterine secretory activity in pigs. J Reprod Fertil 86(2):713-722. https://doi.org/10.1530/jrf.0.0860713 Zhang J, Bricker L, Wray S, Quenby S (2007) Poor uterine contractility in obese women. BJOG Int J Obstet Gynaecol 114(3):343–348. https://doi.org/10.1111/j.1471-0528.2006.01233.x Zinaman MJ, Cartledge T, Tomai T, Tippett P, Merriam GR (1995) Pulsatile GnRH stimulates normal cyclic ovarian function in amenorrheic lactating postpartum women. J Clin Endocrinol Metab 80: 2088-2093. https://doi.org/10.1210/jcem.80.7.7608260 Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstractLSI.png Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 14 Oct, 2025 Reviews received at journal 14 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers agreed at journal 03 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviewers agreed at journal 01 Oct, 2025 Reviews received at journal 18 Sep, 2025 Reviewers agreed at journal 11 Sep, 2025 Reviewers invited by journal 10 Sep, 2025 Editor assigned by journal 10 Sep, 2025 Submission checks completed at journal 10 Sep, 2025 First submitted to journal 10 Sep, 2025 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. 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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-7580090","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515567297,"identity":"7e8c2208-2816-472e-ac3b-0f20629a6791","order_by":0,"name":"Praveenkumar S. Kondaguli","email":"","orcid":"","institution":"Karnatak University","correspondingAuthor":false,"prefix":"","firstName":"Praveenkumar","middleName":"S.","lastName":"Kondaguli","suffix":""},{"id":515567298,"identity":"43844b39-ba43-41e7-871a-d0d6d4133e06","order_by":1,"name":"Nafisa H. Balasinor","email":"","orcid":"","institution":"National Institute for Research in Reproductive and Child Health","correspondingAuthor":false,"prefix":"","firstName":"Nafisa","middleName":"H.","lastName":"Balasinor","suffix":""},{"id":515567299,"identity":"c20bba74-8829-4e23-9491-3e91d428e4b3","order_by":2,"name":"Chaitra R. Sharma","email":"","orcid":"","institution":"Karnatak University","correspondingAuthor":false,"prefix":"","firstName":"Chaitra","middleName":"R.","lastName":"Sharma","suffix":""},{"id":515567300,"identity":"6453af88-f1c1-45af-9459-2e5a560955aa","order_by":3,"name":"Laxmi S. Inamdar (Doddamani)","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYBACPiCWYGBjBnMOfGBgSCCohQ1Zy8EZJGth5iFKC/8BxhsfyqzlddvPHjxs22aXx8/ewPjhYw4eLRIJzJYzzqUbbjuTl3A4ty25WLLnALPkzG34tDCwSfO2HWbcdiDHAKiFOXHDjQQ2Zl58WvgPgLXYbzv/xuCwZVs9EVoYEsBaErfdANrCCGQQ1gL1S/K2G28MDvacO544s+dgM16/8ENDzHbb+RzjDz/KqhP72ZsPfviIRwtQ0wcEm5ENTDbgU48O/pCieBSMglEwCkYKAAARzVKxO9o2dQAAAABJRU5ErkJggg==","orcid":"","institution":"Karnatak University","correspondingAuthor":true,"prefix":"","firstName":"Laxmi","middleName":"S. Inamdar","lastName":"(Doddamani)","suffix":""}],"badges":[],"createdAt":"2025-09-10 07:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7580090/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7580090/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91530646,"identity":"9e094311-1bf3-4434-996c-cccb79038a29","added_by":"auto","created_at":"2025-09-17 11:59:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":931247,"visible":true,"origin":"","legend":"\u003cp\u003eDiagrammatic representation of the Histomorphometric evaluation of mouse uterus.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/6dcc1f38e63c21e78caf6813.png"},{"id":91530649,"identity":"7127cf72-4a93-4cb1-ae56-b109e6a9cde9","added_by":"auto","created_at":"2025-09-17 11:59:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of ST on body weight in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreatment of ST for 30 days leads to a rise in body weight. Both doses of ST (low-dose and high-dose) led to a significant increase in final body weight when compared to initial body weight.\u003c/p\u003e","description":"","filename":"Fig.2Bodywt.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/efa4c28d38e0e1e93730e7c5.png"},{"id":91530654,"identity":"bbaaf784-3588-4c5b-a6a1-977fc7bc2e26","added_by":"auto","created_at":"2025-09-17 11:59:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24586856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn-vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e treatment of both LD and HD-ST leads to profound alterations in the histoarchitecture of mouse uterus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) T.S. of the uterus of the control mouse.\u003c/p\u003e\n\u003cp\u003eB) Magnified view of Fig. A.\u003c/p\u003e\n\u003cp\u003eC) T.S. of the uterus of low-dose treated mice depicting cryptic, wavy and irregular branching of the endometrial luminal epithelium proliferation\u003c/p\u003e\n\u003cp\u003eD) Higher magnification of Fig. C.\u003c/p\u003e\n\u003cp\u003eE) T.S. of the uterus of high-dose treated mice unveiling irregular branched proliferation of endometrial epithelium giving a tortuous appearance, and hypertrophy of myometrium.\u003c/p\u003e\n\u003cp\u003eF) Enlarged view of Fig. E.\u003c/p\u003e\n\u003cp\u003eE – Endometrium; LE - Luminal epithelium; Ge - Glandular epithelium; S-Stroma; M – Myometrium; L - Lumen\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eScale bar – A, C \u0026amp; E= 100µm; B, D, \u0026amp; F =40µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/b89f769679d41f7b44cb9208.png"},{"id":91530647,"identity":"74b9a075-fb7e-4c09-a252-cfaa6d2203bc","added_by":"auto","created_at":"2025-09-17 11:59:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1396548,"visible":true,"origin":"","legend":"\u003cp\u003eNote, treatment of high-dose ST leads to proliferation of endometrial epithelium, resulting in detachment of the endometrial portion in the uterine lumen (red arrow), which is a rare case observed in two mice.\u003c/p\u003e\n\u003cp\u003eE – Endometrium; M – Myometrium; \u003cem\u003eScale bar – 100µm\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.4.DetachedEndometrium.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/3ab8e2b6a2c6921f2b211e58.png"},{"id":91530648,"identity":"5f584bba-7d11-45c8-9e0e-05c004d107a0","added_by":"auto","created_at":"2025-09-17 11:59:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":267179,"visible":true,"origin":"","legend":"\u003cp\u003eTreatment of ST altered uterine Morphometry.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA)\u003c/strong\u003eBoth low- and high-dose ST attribute to a decrease in uterine diameter.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB) \u003c/strong\u003eLuminal epithelial cell height increased significantly in LD and HD-treated mice.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC)\u003c/strong\u003e Treatment of ST leads to an insignificant increase in endometrium in LD and a significant increase in HD-treated mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003eA significant increase in the thickness of circular layer of myometrium LD and HD- treated mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003eAn insignificant increase in the thickness of longitudinal layer of myometrium in LD and a significant increase in HD-treated mice.\u003c/p\u003e\n\u003cp\u003eAsterisks represent significance level (\u003cstrong\u003e*\u003c/strong\u003e p \u0026lt; 0.05, \u003cstrong\u003e**\u003c/strong\u003e p \u0026lt; 0.01, \u003cstrong\u003e***\u003c/strong\u003e p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig.5.EndometrialThickness.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/5b3dd886a1d1a34628f4de4a.png"},{"id":91530657,"identity":"d1c8163d-f491-416a-b4cd-c8bfa5cd9af1","added_by":"auto","created_at":"2025-09-17 11:59:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":27224163,"visible":true,"origin":"","legend":"\u003cp\u003eSpatiotemporal changes in the immunoexpression pattern of ERα in the uterine compartment of both LD- and HD-treated mice. The dramatic changes in the immunoexpression of ERα in the uterine compartment can be noted.\u003c/p\u003e\n\u003cp\u003eA) T.S. of the uterus of control mice - Expression of ERα was noticed in luminal, glandular epithelial, stromal, and distinct albeit low levels in myometrial cells.\u003c/p\u003e\n\u003cp\u003eB) Treatment with ST resulted in downregulation of ERα in the endometrium and a significant increase in its expression in the circular layer of myometrium of LD-treated mice.\u003c/p\u003e\n\u003cp\u003eC) High-dose leads to enhanced immunostaining of ERα in both circular as well as longitudinal layers of the myometrium, accompanied by down-regulation of its expression in the endometrium.\u003c/p\u003e\n\u003cp\u003eE – Endometrium; LE - Luminal epithelium; Ge - Glandular epithelium; S-Stroma; CM – \u0026nbsp;Circular myometrium; LM – Longitudinal myometrium;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eScale bar-50µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.6.ERalpha.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/b38b884f1ec4c02e55207d01.png"},{"id":91530655,"identity":"ecf75845-33c9-4486-aa31-53764586e783","added_by":"auto","created_at":"2025-09-17 11:59:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":23097869,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative photomicrograph showing altered AR immunoreactivity in ST-treated mouse uterus.\u003c/p\u003e\n\u003cp\u003eA) Expression of AR was restricted to stromal cells and distinct albeit the low level of AR in myometrial cells in the vehicle group.\u003c/p\u003e\n\u003cp\u003eB) Treatment of ST resulted in up-regulation of AR immunostaining in the endometrial stromal cells, circular and longitudinal layers of myometrium of LD group\u003c/p\u003e\n\u003cp\u003eC) High-dose ST also augments AR immunoexpression in the endometrial stromal cells, circular and longitudinal layers of myometrium.\u003c/p\u003e\n\u003cp\u003eE – Endometrium; LE - Luminal epithelium; Ge - Glandular epithelium; S-Stroma; CM – Circular myometrium; LM – Longitudinal myometrium;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eScale bar-50µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.7.AR.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/4396044cdd80f808979fbff9.png"},{"id":91530656,"identity":"4910e7c5-6d77-4d76-954f-2d4d0b250345","added_by":"auto","created_at":"2025-09-17 11:59:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":24969462,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative micrograph of PRLR immunostaining in ST-treated mouse uterus.\u003c/p\u003e\n\u003cp\u003eA) Expression of PRLR was restricted to stromal cells and a few myometrial cells in the vehicle group.\u003c/p\u003e\n\u003cp\u003eB) The upregulation of PRLR immunostaining in the endometrium and its hyperexpression in myometrium can be visualized in LD-treated mice.\u003c/p\u003e\n\u003cp\u003eC) High-dose ST leads to hyperexpression of PRLR in both endometrium and myometrium compartments.\u003c/p\u003e\n\u003cp\u003eE – Endometrium; LE - Luminal epithelium; Ge - Glandular epithelium; S-Stroma; CM – Circular myometrium; LM – Longitudinal myometrium;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eScale bar-50µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.8.PRLR.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/04f2a106bb9292e31eec063e.png"},{"id":91530652,"identity":"981a563c-f961-4a69-b180-2ffe2ad13b3b","added_by":"auto","created_at":"2025-09-17 11:59:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":261096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA to C\u003c/strong\u003e Depiction of the impact of ST (Low- and High-dose) on the immunoexpression of ERα, AR and PRLR intensity in different uterine compartments. LE - Luminal epithelium; GE - Glandular epithelium; ST - Stromal Cells; CM - Circular myometrium; LM - Longitudinal myometrium.\u003c/p\u003e\n\u003cp\u003eAsterisks represent significance level (* p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/a6cc8fef5b467d0f531ac1a7.png"},{"id":91531132,"identity":"d4150e9e-81cf-4390-8074-934200050d32","added_by":"auto","created_at":"2025-09-17 12:07:26","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":153880,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA to C \u003c/strong\u003eGraphs summarizing the serum levels of testosterone (T), estradiol (E\u003csub\u003e2\u003c/sub\u003e) and prolactin (PRL) in the control and treated mice following ST treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eA remarkable upregulation of serum T level was noted in LD and HD-treated mice. \u003cstrong\u003eB) \u003c/strong\u003eA fall in the concentration of serum\u003cstrong\u003e \u003c/strong\u003eE\u003csub\u003e2\u003c/sub\u003e was witnessed in HD-treated mice. \u003cstrong\u003eC)\u003c/strong\u003e An augmented PRL concentration in LD and in HD-treated group can be seen.\u003c/p\u003e\n\u003cp\u003eAsterisks represent significance level (\u003cstrong\u003e*\u003c/strong\u003e p \u0026lt; 0.05, \u003cstrong\u003e**\u003c/strong\u003e p \u0026lt; 0.01, \u003cstrong\u003e***\u003c/strong\u003e p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig.10Serumhormonelevel.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/ba77968769012a09f9e5a2e2.png"},{"id":91532366,"identity":"7708b1dc-ef8f-4c1b-b477-922549139649","added_by":"auto","created_at":"2025-09-17 12:16:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":92569082,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/034e037a-722f-4fad-a022-aae089445e87.pdf"},{"id":91531133,"identity":"56cb5465-28ee-456c-9a0f-7dc84a8a822f","added_by":"auto","created_at":"2025-09-17 12:07:26","extension":"png","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":286880,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstractLSI.png","url":"https://assets-eu.researchsquare.com/files/rs-7580090/v1/be5fb497c051c5623b4679f7.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stanozolol, an Anabolic-Androgenic Steroid, Modulates Expression of Receptors and Disrupts Uterine Histoarchitecture in Mus musculus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnabolic-androgenic steroids (AAS) comprise a large category of synthetic analogs of natural male hormone testosterone, originally developed to maximize anabolic potency and minimize androgenic effects (Hoffman and Ratamess, 2006; Oberlander et al., 2012; Piacentino et al., 2015; Huang and Basaria 2018).\u0026nbsp;Due to their diverse biological actions, AAS have been used in the treatment of a variety of pathological conditions like pulmonary disorders, renal disorders, aplastic anemia, hypogonadism, chronic debilitating illness, trauma, and postoperative recovery (Basaria et al., 2001; Shahidi, 2001; Evans, 2004; Kicman, 2008; Woerdeman and de Ronde, 2011; Piacentino et al., 2015; Kloner et al., 2016). However, over the last several decades, AAS have been abused as ergogenic aids by athletes and bodybuilders to enhance performance by augmenting muscular development, strength and endurance (Wagner, 1989; Windsor and Dumitru, 1989; Maravelias et al., 2005). Regrettably, the use of AAS, is becoming popular among adolescent girls and also women athletes, consequently\u0026nbsp;resulting in various secondary pathophysiological effects. The widespread abuse of AAS at supraphysiological doses poses a significant global concern, and comprehensive elucidation of their impact on female reproductive physiology and fertility at the molecular level remains a critical area for investigation.\u003c/p\u003e\n\u003cp\u003eIt has been reported that increased levels of androgens by exogenous administration of androgens and/or its derivatives induce detrimental effects on the uterine endometrium in women (Okon et al.,1998; Tuckermann et al., 2000; Zhang et al., 2007). \u0026nbsp;It is worth mentioning here that over the last two decades, many women have been affected by infertility and reproductive tract dysfunction such as endometriosis, uterine cancer (endometrial cancer), anovulation, implantation failure and early pregnancy loss.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe primary function of the uterus is to support pregnancy with hormonally responsive endometrium that facilitates embryo attachment, implantation, and maintenance of a conducive environment for fetal development. Hence, successful implantation and maintenance of pregnancy till term rely upon ideal cross talk/signals between the ovary, good quality embryos and a receptive endometrium. In a preliminary experiment from our laboratory, we investigated the efficacy of ST on the maintenance of pregnancy in mice, which revealed that administration of ST resulted in the interruption of pregnancy (Jayamma et al., 2012). \u0026nbsp;A significant reduction noted in the number of corpora lutea, secondary and antral follicles, partial resorption of the embryo/fetus with placental scars and absence of viable embryos/fetus, suggests that ST caused an imbalance in progesterone and estrogen secretion by the ovary, leading to the interruption of pregnancy principally due to the deficiency of luteal hormones (Jayamma et al., 2012). In another experiment, the effect of ST was evaluated on the implantation and maintenance of pregnancy in mice\u0026nbsp;(Sharma et al., 2021).\u0026nbsp;The low-dose ST-treated mice maintained gestation until term with reduced litter size, while high-dose-treated mice revealed vaginal plug at frequent intervals, indicating conception failure.\u0026nbsp;In high-dose-treated mice, decreased serum levels of estradiol and progesterone, accompanied by increased testosterone, and downregulated endometrial expression of ER\u0026alpha; and PR, suggest a deficiency of steroid hormones and their respective receptors. Decreased ovarian expression of ER\u0026alpha;, hyperexpression of PRLR, AR and abated progesterone secretion led to luteal dysfunction, hindered signalling to LIF and Hoxa 10, consequently attenuating endometrial receptivity during the implantation window, resulting in implantation failure (Sharma et al., 2021).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHence, the current investigation aims to gain further insights into the impact of one of the anabolic-androgenic steroids\u0026nbsp;\u003c/em\u003e\u003cem\u003estanozolol (ST)\u0026nbsp;\u003c/em\u003e\u003cem\u003e[17\u0026beta;-hydroxy-17\u0026alpha;-methyl-androstano (3, 2-c) pyrazole]\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eon the female reproductive tract, emphasizing the preparedness of the uterus for successful implantation in the mouse,\u0026nbsp;\u003c/em\u003e\u003cem\u003eMus musculus,\u0026nbsp;\u003c/em\u003e\u003cem\u003ewith the following objectives:\u003c/em\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eTo assess the morphological and histomorphometric analysis of the uterus post- administration of low and high doses of ST.\u003c/li\u003e\n \u003cli\u003eTo analyze the efficacy of ST on spatiotemporal immunoexpression of estrogen receptor \u0026alpha; (ER\u0026alpha;), androgen receptor (AR), and prolactin receptor (PRLR) in the mouse to examine the readiness of its uterus for successful implantation.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExperimental animals, study design and hormone treatment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll protocols in this experiment adhered to the CPCSEA guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (No.639/GO/02/a/CPCSEA) at the Department of Zoology, Karnatak University, Dharwad. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn total 15 sexually mature (3 months old) female Swiss albino \u0026lsquo;strains\u0026rsquo; (weighing 25-30 gm) exhibiting regular estrous cyclicity were obtained from the mouse breeding center maintained in the Department of Zoology, Karnatak University, Dharwad. All the mice were housed in individual polypropylene cages and maintained a 12 h light: dark cycle at 27\u0026plusmn;1˚C with 40-50% RH, food (pelleted diet, Goldmohur, Lipton, India), and water supplied \u003cem\u003ead libitum\u003c/em\u003e. \u0026nbsp; Stanozolol (ST) was obtained from Sigma-Aldrich, USA. The mice were randomly assigned to three experimental groups containing 5 animals in each group. ST was dosed subcutaneously [0.5 mg/kg bwt (low dose of ST); 5.0 mg/kg bwt (high dose of ST); or 1% alcohol (vehicle control)] for 30 consecutive days. The body weight of all animals was recorded at the beginning of the experiment (initial body weight) and on the day of autopsy (final body weight). On the 31st day, animals were sacrificed. The uteri were excised, weighed, fixed in Bouin\u0026rsquo;s fluid/4% paraformaldehyde (PFA), and processed for routine histology and immunohistochemistry.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistoarchitecture and histomorphometrical kinetics of uterus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBouin\u0026rsquo;s fixed tissue samples were dehydrated in a series of increasing concentrations of ethanol (10% - 100%), cleared in benzene, and embedded in paraffin (Fisher Scientific).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMoulded blocks of uterine tissues were sectioned at 5\u0026micro;m on a rotational microtome (Leica RM 2025) and subjected to standard histology. Morphometric analysis was performed by calibrated Procam software (Olympus BX5I microscope). A total of twenty-five readings from each animal (both the control and treated group) were considered for the following parameters, and are depicted in Fig. 1.\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eUterine diameter: mean diameter (D) of X and Y axis (X-major axis, Y-minor axis passing through the center): D=(X+Y)/2 (in \u0026micro;m)\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLuminal epithelial cell height: \u0026nbsp;from the apical (luminal) surface to the basement membrane separating the epithelium from the stroma.\u003c/li\u003e\n \u003cli\u003eThe thickness of the endometrium: from the longest luminal epithelial surface to the beginning of the circular layer of the myometrium.\u003c/li\u003e\n \u003cli\u003eThe thickness of myometrium: the thickness of both circular and longitudinal layers of myometrium was measured.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunohistochemical analysis for estrogen receptor alpha (ER\u0026alpha;), prolactin receptor (PRLR), and androgen receptor (AR)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue sections were deparaffinized in xylene and rehydrated in descending concentrations of ethanol. This was followed by antigen retrieval in trisodium citrate buffer (pH=6.0, C3674 Sigma-Aldrich, St. Louis, MO, USA) for 20 min in the oven at 90˚C, followed by cooling at ambient temperature. Endogenous peroxidase was inactivated with 3% hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). Nonspecific binding was blocked by 5% BSA (A3059, Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the sections were incubated with rabbit polyclonal antibodies specific for ER\u0026alpha; (host: rabbit; 1:100; MC-20, cat. no sc-542); AR (host: rabbit; 1:100; N-20, cat. no. sc-816); PRLR (host: rabbit; 1:200; M-170, cat. no sc-30225) from Santa Cruz Biotechnology, USA at 4˚C, overnight. As a negative control, primary antibodies were replaced with antibody diluents (1% BSA). \u0026nbsp; After 3 washes in PBS and PBS-T the sections were incubated with the Goat anti-rabbit IgG conjugated to HRP (sc-2004, Santa Cruz Biotechnology, Dallas, Texas, USA). Staining was visualized with 3, 3\u0026rsquo;-diaminobenzidine tetra-hydrochloride hydrate (DAB; D5637, Sigma-Aldrich, St. Louis, MO, USA). Finally, sections were counterstained with Harris-Hematoxylin (39411, Fisher Scientific). Images were acquired using Nikon Eclipse 80i with ACT-2U software (Nikon Corporation, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantitative evaluation of immunoexpression of ER\u0026alpha;, AR and PRLR\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunohistochemical staining intensities of\u0026nbsp;ER\u0026alpha;, AR, and PRLR in different compartments of the uterus were evaluated by measuring their optical densities (ODs).\u0026nbsp;Briefly, the images were captured with Nikon microscope 90E with ACT-2U software\u0026nbsp;at 200X magnification. The OD was evaluated using ImageJ (Fiji) software. Each image was first deconvoluted using the H/DAB vector into three different colored images (i.e., green, brown, and blue). The brown DAB image was\u0026nbsp;converted to grayscale and its intensity was recorded by measuring the mean integrated intensities (mean grey value).\u0026nbsp;At least five fields of each histological compartment were recorded in each section, and five sections per animal were evaluated. The intensity numbers were converted into the OD using the following equation: (OD\u0026thinsp;=\u0026thinsp;log (max intensity/mean intensity), where the max intensity\u0026thinsp;=\u0026thinsp;250 and the mean intensity\u0026thinsp;=\u0026thinsp;mean grey value. The results were exported to a Microsoft Excel spreadsheet. The ODs of ER\u0026alpha;, AR, and PRLR were evaluated in the luminal and glandular epithelium, stroma, circular and longitudinal layers of myometrium of each tissue section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantification of serum steroid hormone\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe blood was collected from jugular vein of both control and treated mice; and centrifuged (2000 g for 10 min at 4 \u0026deg;C) to extract the serum, which was then kept at -80 \u0026deg;C until analyzed. Electrochemiluminescence immunoassay (ECLIA) was used to assess the concentrations of testosterone (T), estradiol (E\u003csub\u003e2\u003c/sub\u003e) and prolactin (PRL) using a Cobas e411 completely automated electrochemiluminescence immunity analyzer (Cobas; Roche Diagnostics, GmbH, Mannheim, Germany). To measure the serum hormone levels, a proprietary serum assay kit (Elecsys Estradiol III, test no. 1370; Elecsys Testosterone II, test no. 111; Elecsys Prolactin II test no. 131) by Roche Diagnostics, Indianapolis, Indiana, USA) was utilized. A PerkinElmer JANUS Automated Liquid Handling System was used to move the samples into Roche sample cups. The secondary antibody labeled with a ruthenium complex was then added to streptavidin-coated microparticles. Each test was run following the manufacturer\u0026apos;s three-level assay control. The system software of the Roche/Hitachi Cobas e 411 control unit calculated the hormone concentration after the hormone assay measurement and was immediately recorded on the Windows-based computer-aided evaluation program (WinCAEv). All tests had intra- and inter-assay errors of less than 10% and 15%, respectively.\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Statistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical tests were performed using SPSS version 20. The initial and final body weight difference was compared using paired t-test. Uterine diameter, luminal epithelial cell height, thickness of endometrium and myometrium, optical density (OD), and serum steroid hormone levels between different treatment and control groups were evaluated using a one-way analysis of variances (ANOVA) technique to know the significant difference between different treated groups. The multiple comparison test (Tukey\u0026rsquo;s HSD post hoc test) was carried out to determine which treatment group significantly differed from the control group. Numerical values are presented as the mean \u0026plusmn; standard error (SE). Both statistical tests were two-sided tests with a 5% \u0026amp; 1% levels of significance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 \u0026amp; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Effect of ST on body weight\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results revealed a significant increase in body weight of both LD (t\u003csub\u003e8\u003c/sub\u003e = 5.993; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01) and HD (t\u003csub\u003e8\u003c/sub\u003e = 9.428; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01) treated mice when compared to initial weight (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Administration of ST for 30 consecutive days leads to alterations in uterine histomorphology\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistoarchitecture of Uterus:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndometrium:\u0026nbsp;\u003c/strong\u003eThe luminal epithelium of control mice consists of simple columnar cells that extend into endometrial tubular glands within the loosely arranged reticular connective tissue of endometrial stroma (Figs. 3A \u0026amp; B). A significant increase in the height of luminal epithelium in LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) and HD ST-treated (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) mice was noticed when compared to control group (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 97.953, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) (Fig. 5B). The endometrial luminal epithelium of LD treated mice exhibited cryptic, wavy and irregularly branched with widened lumen (Fig. 3C \u0026amp; D). Also, the endometrial luminal epithelium of HD treated mice exhibited tortuous and irregular branching giving a papillary appearance (Figs. 3E, F).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWhile this hypertrophy of luminal epithelium and its lining resulted in detachment of endometrial portion in two HD treated mice (Fig. 4).\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiameter of uterus:\u003c/strong\u003e Uterine diameter\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edecreased significantly in both LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) and HD treated groups (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01) compared to control animal group (F\u003csub\u003e2,12\u003c/sub\u003e= 8.485, \u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.05); (Fig. 5A).\u003c/p\u003e\n\u003cp\u003eFurther, in comparison to the control group, an insignificant increase in thickness of the endometrium in LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05) and a significant increase in HD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) ST-treated group were observed (F\u003csub\u003e2,12\u003c/sub\u003e=4.747; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05); (Fig. 5C). \u0026nbsp;\u003cstrong\u003eMyometrium:\u0026nbsp;\u003c/strong\u003eThe control myometrium was composed of an inner circular and outer longitudinal layer of smooth muscle fibers separated by highly vascular connective tissue (Figs. 3A, B). A substantial increase in thickness of circular myometrium in both low (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) and high dose (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) was noticed (F\u003csub\u003e2,12\u003c/sub\u003e=24.690, \u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.001); (Fig. 5D).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAn insignificant increase in longitudinal layer of the myometrium in LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05) and a significant increase (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) were observed in HD treated animals (F\u003csub\u003e2,12\u003c/sub\u003e= 41.738, \u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.001); (Fig. 5E).\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunolocalization of ER\u0026alpha; in mouse uterus upon ST treatment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndometrium:\u0026nbsp;\u003c/strong\u003eImmunohistochemical signal for ER\u0026alpha; was found in nuclei of endometrial luminal and glandular epithelial cells, stromal cells, and myometrium (circular and longitudinal muscle layer) in the uterus of control animals (Fig. 6A). A significant decrease in luminal epithelial expression of ER\u0026alpha; was noticed in both LD and HD (\u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.001) ST treated groups when compared to control (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 52.714; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). Glandular epithelium exhibited a notable reduction in ER\u0026alpha; expression in both LD and HD ST-treated mice (\u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.05), (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 7.168; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01). Besides, the intensity of immunoreactions for ER\u0026alpha; was significantly down-regulated in the endometrial stromal cells of both LD and HD (\u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.001) treatment groups (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 26.091; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001); (Figs. 6B \u0026amp; C; Fig. 9A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMyometrim:\u0026nbsp;\u003c/strong\u003eHowever, the intensity of ER\u0026alpha; in the myometrial layer was substantially higher in both treatment groups in comparison to control group. Its intensity was dramatically augmented in the circular layer of myometrium in both LD and HD treatment groups (\u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.001), (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 92.342; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). A noticeable increase in the longitudinal layer of myometrial expression of ER\u0026alpha; was witnessed in LD (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) and in HD (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) ST-treated mice (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 45.650; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001); (Figs. 6B \u0026amp; C; Fig. 9A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunolocalization of AR in mouse uterus following ST treatment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndometrium:\u003c/strong\u003eThe endometrial and myometrial expression of AR was observed in the uterine compartments of control mice\u0026nbsp;(Fig. 8A). No noticeable difference in AR expression was observed in luminal epithelium of both treatment groups (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05) when compared to control (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 0.213; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05). Whereas, an insignificant increase in glandular epithelium of LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05) and a significant increase in HD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) were observed (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 4.106; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05). ST treatment up-surged the expression of AR in the endometrial stroma of LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) and in HD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) ST treated mice (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 23.310; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001); (Figs. 7B \u0026amp; C; Fig. 9B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMyometrium:\u0026nbsp;\u003c/strong\u003eAn insignificant increase in the immunoexpression of AR in circular layer of LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05) and a significant increase in HD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) treatment group was observed when compared to control (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 16.098; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). An upregulation of AR was visualized in longitudinal myometrium in LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) and HD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01) treatment groups (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 9.739; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01); (Figs. 7B \u0026amp; C; Fig. 9B). \u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunolocalization of PRLR in mouse uterus upon ST treatment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndometrium:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunolocalization of PRLR noticed mainly in the inner wall of the plasma membrane of luminal and glandular epithelial cells, and its expression in stromal cells was below the detection level in the control uterus. Very few myometrial cells expressed PRLR protein (Fig. 8A).\u003c/p\u003e\n\u003cp\u003eThe endometrial immunoexpression of PRLR deepened\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ein the luminal epithelium of LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01) and HD treated mice (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001); (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 101.097; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) and also, in glandular epithelial cells of LD and HD treated mice (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001); (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 23.805; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) when compared to control (Figs. 8B \u0026amp; C; Fig. 9C).\u003c/p\u003e\n\u003cp\u003eLikewise, PRLR protein expressed deeply in stromal cells of both LD and HD treated groups (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) than the control (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 47.557; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001); (Figs. 8B \u0026amp; C; Fig. 9C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMyometrium:\u003c/strong\u003eThe myometrial immunoexpression of PRLR was augmented in both circular layer of low and high dose (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001); (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 79.061; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) and in longitudinal layer of both low and high dose treated mice (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001); (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 73.871; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) when compared to control (Figs. 8B \u0026amp; C; Fig. 9C).\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Effect of ST treatment on serum levels of Testosterone (T), Estradiol (E\u003csub\u003e2\u003c/sub\u003e) \u0026amp; Prolactin (PRL) hormones\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElectrochemiluminescence immunoassay (ECLIA) was utilized to quantify the serum levels of T, E2 \u0026amp; PRL. Treatment of ST for 30 days caused a significant increase in serum T level in both LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01) and HD (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) treatment groups (F\u003csub\u003e2,12\u0026nbsp;\u003c/sub\u003e= 57.285; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001); (Fig. 10A). An insignificant decrease in serum E2 level in LD (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05) and a significant decline in HD (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) group were noticed (F\u003csub\u003e2,12\u003c/sub\u003e= 4.603; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05),\u0026nbsp;(Fig. 10B). A substantial upregulation of PRL was observed in LD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05)\u0026nbsp;and HD (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01)\u0026nbsp;treated mice in comparison to control group\u0026nbsp;(F\u003csub\u003e2,12\u003c/sub\u003e= 13.069; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01); (Fig. 10C).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAnabolic-androgenic steroids (AAS) cause adverse effects on a broad spectrum of organs, including the reproductive and neuroendocrine systems.\u0026nbsp;The\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003emorphometric and histoarchitecture of the uterus were meticulously analysed i.e., in a compartment-specific manner \u0026ndash; a) Endometrium (luminal epithelium, glandular epithelium and stroma) and b) Myometrium (circular and longitudinal muscle layer) to understand the efficacy of ST on the murine uterus.\u003c/p\u003e\n\u003cp\u003eProlonged treatment with ST resulted in a significant increase in body weight, primarily due to enhanced food intake, as witnessed in the feeding behavior of mice. The elevated body\u0026apos;s energy demands might have caused increased food intake. The current investigation reveals a dose-dependent increase in body weight, which is likely attributable to the anabolic efficacy of ST to augment muscle mass. Such an increase in the growth/body weight following AAS treatment has been reported previously in rodents (Camargo et al., 2009; Brasil et al., 2015; Saddick 2018).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe endometrium of mouse consists of epithelial (luminal and glandular) and stromal compartments that play distinct roles in the maintenance of hormonal microenvironment of the uterus. \u0026nbsp;Uterine morphometric analysis reveals profound alterations in its morphology, specifically an aberrant endometrial proliferation induced irregular branching giving a papillary appearance in high-dose ST-treated groups, corroborating the earlier report by Far et al., 2007. While this proliferation of endometrium resulted in detachment of its portion in two high-dose ST-treated mice. The observed results suggest that\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eprolonged treatment with ST for one month might have altered the ovarian signal to the uterus leading to its disorganization, reflecting the deleterious impact of STon uterine histoarchitecture. Parallel to our results, testosterone and some other AASs (17\u0026alpha;- methyltestosterone and Nandrolone Decanoate) have been shown to alter uterine morphology and physiology in mice (Papaconstantinou et al., 2002; Simitsidellis et al., 2016) and rats (Camargo et al., 2009; 2014; Chuffa et al., 2011; Saddick 2018; Gerez et al., 2005). Interestingly, the high dose of ST is more effective in increasing myometrial thickness, indicating the dose-dependent androgenic to anabolic efficacy of this AAS in the myometrium region. \u0026nbsp;Similarly, reports on rodents suggest that androgens stimulate the myometrium to a greater extent, whereas the endometrial cells are less responsive, suggesting stanozolol may exert a direct effect on the uterine tissue, potentially impacting its histoarchitecture and physiology (Nantermet et al., 2005). Further, androgens such as testosterone and 5\u0026alpha; DHT have uterotrophic and anti-uterotrophic effects in rats and mice (Armstrong et al., 1976; Nantermet et al., 2005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunolocalization of Er\u0026alpha; in the uterine compartments:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunoexpression of ER\u0026alpha; was downregulated in the endometrium and dramatically increased in the myometrium of both low and high-dose ST-treated mice. The observed down-regulation of immunoexpression of ER\u0026alpha; in the endometrium signifying its negative impact on uterine receptivity, which supports our earlier study on implantation; where it was observed that high-dose ST downregulated the endometrial and ovarian immunoexpression of ER\u0026alpha; as well as serum E\u003csub\u003e2\u003c/sub\u003e levels leading to impaired endometrial receptivity resulting in implantation failure during the implantation window (Sharma et al., 2021). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBesides, it is\u0026nbsp;worth mentioning here that the increased thickness of myometrium mentioned (in the above paragraph) and upregulated myometrial expression of ER\u0026alpha; upon ST treatment indicate the androgenic to enhanced anabolic efficacy of this AAS. The observed result suggests the non-genomic action of this AAS in myometrial compartment. Since, ST is a derivative of DHT which is a non-aromatizable androgen that does not convert into estrogen, instead, it may bind to ER\u0026alpha; in the myometrium, resulting in its\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ehypertrophy. This differential immunoexpression of ER\u0026alpha; in the regions of endometrium and myometrium indicates the compartment-specific effect of ST in the murine uterus.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFurther, it has already been reported that at doses greater than 100 nM, DHT can bind to ER\u0026alpha; (Rochefort and Garcia., 1976 ; Ekena et al., 1998).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunoexpression of AR protein in the uterine compartments:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAndrogen receptors (AR) are mainly localized in the uterine endometrial stromal cells and any effects of androgens in the epithelium are mediated directly through epithelial ER or indirectly through stromal AR or ER (Takeda et al., 1990; Pelletier et al., 2000). \u0026nbsp; In the present investigation, prolonged treatment of ST augmented stromal immunoexpression of AR in the endometrium, suggesting\u0026nbsp;AR-dependent mechanisms in this compartment.\u0026nbsp;It is interesting to note that\u0026nbsp;high-dose ST-treated mice exhibited endometrial proliferation and irregular branching, giving a papillary appearance. These observed results on the endometrial epithelial proliferation \u003cem\u003eper se\u003c/em\u003e may be induced by the up-regulated expression of AR in stromal cells, and agree well with the\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efindings that stromal AR stimulates IGF-I expression, which increases luminal epithelial proliferation\u0026nbsp;(Kowalski et al., 2004). \u0026nbsp;Similar to our results,\u0026nbsp;chronic treatment with DHT increases AR-dependent epithelial cell proliferation in ovariectomized mice (Simitsidellis et al., 2016). Further, it is important to note that the observed up-regulated endometrial AR expression may render the endometrium more sensitive to androgen and contribute to poor reproductive performance, which supports our earlier report that high dose of ST impedes embryo implantation by attenuating endometrial receptivity in mice (Sharma et al., 2021). \u0026nbsp;Similarly,\u0026nbsp;high androgen levels have a negative impact on endometrium leading to infertility and miscarriage in women (Tuckerman et al., 2000).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunoexpression of PRLR in the uterine compartments:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProlactin (PRL) is a pleiotropic polypeptide hormone of leuteotrophic complex. In rodents during pregnancy,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ePRL plays a crucial role in promoting the luteal production of P4, not by stimulating its synthesis, but by preventing its metabolism (Stouffer and Hearn 1998; Binart et al., 2000). In the present investigation, high-dose ST treatment resulted in pronounced upregulation of PRLR and concomitant hyperexpression of AR in both endometrium and myometrium (Pl see above para), indicating a potential stimulatory effect of ST on the AR-mediated PRLR immunoexpression and release of \u0026nbsp;PRL. Because ST is a derivative of DHT\u0026mdash;a non-aromatizable androgen\u0026mdash;the E\u003csub\u003e2\u003c/sub\u003e-mediated increase of PRL may not be expected. Furthermore, the observed remarkable hyperexpression of PRLR and downregulation of ER\u0026alpha; leads to luteal dysfunction by the ovary, which may result in pregnancy failure (if conceived), which is a concern and supports our earlier report (Sharma et al., 2021). Our results corroborate a report that pronounced up-regulation of \u003cem\u003eprl8\u003c/em\u003e and \u003cem\u003eprlr\u003c/em\u003e genes in the uterus of DHT-treated mice (Simitsidellis et al., 2016). Evidences suggest that significant elevation of PRL levels observed in pathological hyperprolactinemia affect GnRH signaling, subsequently inhibiting FSH and LH secretion, thus impairing ovarian steroidogenesis, which consequently leads to infertility (Bouchard et al., 1985; Zinaman et al., 1995; Oner et al., 2013). Moreover, hyperprolactinemia can result in endometrial glandular hyperplasia and even endometrial adenomyosis in mice, rabbits and pigs (Chilton et al., 1988; Young et al., 1989; Rossi et al., 2002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerum hormone levels:\u0026nbsp;\u003c/strong\u003eTestosterone (T), Estradiol (E\u003csub\u003e2\u003c/sub\u003e) and Prolactin (PRL).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, serum testosterone T, E\u003csub\u003e2,\u0026nbsp;\u003c/sub\u003eand PRL levels were measured, which revealed an elevation in serum T, PRL and down-regulated E\u003csub\u003e2\u003c/sub\u003e levels.\u0026nbsp;Prolonged treatment of ST for one month may have interfered with the hypothalamic-pituitary-gonadal axis (HPG axis), altering hypothalamic and hypophyseal signals to the ovary, leading to impaired hormone secretion and consequently affecting the hormonal milieu. A recent report suggests that the use of AAS compounds can inhibit the pulsatile secretion of gonadotropin-releasing hormone in the hypothalamus, disrupting the release of ovarian hormones, which are crucial for follicular development and ovulation (Karila et al., 2024). This hormonal imbalance resulted in perturbations in the menstrual cycle, such as spaniomenorrhea and amenorrhea, anovulation, ultimately resulting in infertility (Saadedine et al., 2023; Karila et al., 2024).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eLikewise, the excess androgens in both women and rodents led to disruptions in the reproductive axis, resulting in decreased serum E2 levels, as well as LH and FSH (Saddick, 2018; Anawalt, 2019). Further, it was reported that seven of the nine female weight lifters who self-administered testosterone and anabolic steroids exhibited a 30-fold increase in serum T levels and a suppression of FSH (Malarkey et al., 1991). Moreover, the downregulation of serum\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eE\u003csub\u003e2\u003c/sub\u003e levels in the present study may be attributed to the fact that ST is a derivative of DHT which is a non-aromatizable androgen that does not serve as a precursor for the ovarian production of estrogen, consequently resulting in reduced circulating estrogen levels.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIt is inferred that prolonged treatment of ST resulted in aberrant endometrial proliferation, histoarchitectural anomalies, compartment-specific differential immunoexpression of hormone receptors in the endometrium and myometrium, and altered serum hormonal milieu.\u003c/p\u003e\n"},{"header":"Declarations","content":"\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u0026nbsp;\u003c/strong\u003eOne of the authors (LSI) thanks Indian National Science Academy, New Delhi for the INSA Visiting Scientist Award to visit NIRRH, Mumbai. CRS thanks DST, New Delhi for the award of INSPIRE fellowship. PSK thanks KUD for the University Research Scholarship. All experiments were conducted in accordance with the regulations of CPCSEA guidelines and the Institutional Animal Ethical Committee No.639/GO/02/a/CPCSEA of the Karnatak University, Dharwad, Karnataka, India.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnawalt BD (2019) Diagnosis and Management of Anabolic Androgenic Steroid Use. J Clin Endocrinol Metab 104:2490\u0026ndash;2500. https://doi.org/10.1210/jc.2018-01882\u003c/li\u003e\n\u003cli\u003eArmstrong DT, Moon YS, Leung PCK (1976) Uterotrophic effects of testosterone and 5\u0026alpha; dihydrotestosterone in intact and ovariectomized immature female rats. Biol Reprod 15:107-114. https://doi.org/10.1095/biolreprod15.1.107\u003c/li\u003e\n\u003cli\u003eBasaria S, Wahlstrom JT, Dobs AS (2001) Anabolic-Androgenic Steroid Therapy in the Treatment of Chronic Diseases. 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Steroids 165:108572. https://doi.org/10.1016/j.steroids.2020.108752\u003c/li\u003e\n\u003cli\u003eSimitsidellis I, Gibson DA, Cousins FL, Esnal-Zufiaurre A, Saunders PTK (2016) A role for androgens in epithelial proliferation and formation of glands in the mouse uterus. Endocrinol 157:2116-2128. https://doi.org/10.1210/en.2015-2032\u003c/li\u003e\n\u003cli\u003eStouffer, R.L., Hearn, J.P. (1998) Endocrinology of the Transition from Menstrual Cyclicity to Establishment of Pregnancy in Primates. In: Bazer, F.W. (eds) Endocrinology of Pregnancy. Contemporary Endocrinology, vol 9. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-4612-1804-3_2 \u003c/li\u003e\n\u003cli\u003eTakeda H, Chodak G, Mutchnik S, Nakamoto T, Chang C (1990) Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor. 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Expert Opin Investig Drugs 20(1):87-97. https://doi.org/10.1517/13543784.2011.544651\u003c/li\u003e\n\u003cli\u003eYoung KH, Kraeling RR, Bazer FW (1989) Effects of prolactin on conceptus survival and uterine secretory activity in pigs. J Reprod Fertil 86(2):713-722. https://doi.org/10.1530/jrf.0.0860713 \u003c/li\u003e\n\u003cli\u003eZhang J, Bricker L, Wray S, Quenby S (2007) Poor uterine contractility in obese women. BJOG Int J Obstet Gynaecol 114(3):343\u0026ndash;348. https://doi.org/10.1111/j.1471-0528.2006.01233.x\u003c/li\u003e\n\u003cli\u003eZinaman MJ, Cartledge T, Tomai T, Tippett P, Merriam GR (1995) Pulsatile GnRH stimulates normal cyclic ovarian function in amenorrheic lactating postpartum women. J Clin Endocrinol Metab 80: 2088-2093. https://doi.org/10.1210/jcem.80.7.7608260\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Mouse, Estrogen receptorα, Androgen receptor, Prolactin receptor, Serum hormone levels, Stanozolol","lastPublishedDoi":"10.21203/rs.3.rs-7580090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7580090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAbuse of Anabolic-Androgenic Steroids (AAS) by professional and recreational athletes for endurance performance and physique is increasing globally, consequently resulting in secondary pathophysiological effects. The present investigation aims to know the efficacy of one of the AAS, stanozolol, on uterine physiology. A total of 15 female mice were assigned to three experimental groups (n=5). \u0026nbsp;ST was dosed subcutaneously (low-dose, 0.5 mg/kg bwt; high-dose, 5.0 mg/ kg bwt or 1% alcohol-baseline control) for 30 days, and treatment was withdrawn on the 31\u003csup\u003est\u003c/sup\u003e day.\u003c/p\u003e\n\u003cp\u003eMorphometric evaluation of uterus demonstrated aberrant endometrial luminal epithelial proliferation, marked by irregular branching with papillary formations. Prolonged administration of ST results in disruption of uterine organization, reflecting its deleterious impact on uterine histoarchitecture.\u003c/p\u003e\n\u003cp\u003eIn high-dose-treated mice, immunoexpression of ERα was downregulated in the endometrial compartment while significantly upregulated in myometrium, concomitant with increased myometrial thickness leading to myometrial hypertrophy. Results indicate the androgenic-to-anabolic efficacy of ST in myometrial compartment. ST, being a derivative of DHT, does not convert into estrogen; instead it may bind to ERα in the myometrium, suggesting a compartment-specific effect of ST on murine uterus. Furthermore, substantial hyperexpression of PRLR in both endometrium and myometrium, indicates a potential stimulatory effect of ST on AR-mediated release of PRL.\u003c/p\u003e\n\u003cp\u003eHigh-dose ST induced a significant increase in circulating T and PRL levels, while decreasing E\u003csub\u003e2\u003c/sub\u003e concentrations, which reflects an altered hormonal milieu.\u003c/p\u003e\n\u003cp\u003eIt is inferred that prolonged treatment of ST resulted in aberrant endometrial proliferation, histoarchitectural anomalies, compartment-specific differential immunoexpression of hormone receptors in the endometrium and myometrium, and altered serum hormonal milieu.\u003c/p\u003e","manuscriptTitle":"Stanozolol, an Anabolic-Androgenic Steroid, Modulates Expression of Receptors and Disrupts Uterine Histoarchitecture in Mus musculus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 11:59:21","doi":"10.21203/rs.3.rs-7580090/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-14T17:26:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-14T15:01:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290157576665496544557282896639611145669","date":"2025-10-07T11:26:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"219307572613166086541717190776397434774","date":"2025-10-03T14:49:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212721605227280902958595094823365447","date":"2025-10-02T06:33:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328956678473079730746257959306800472021","date":"2025-10-02T03:06:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T01:43:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50804651994463676392390150593406933969","date":"2025-09-11T21:43:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-10T14:28:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-10T14:26:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-10T08:30:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Histology","date":"2025-09-10T07:38:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e7b4bd08-b879-4a5d-b398-8134d8341f97","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-01-14T22:08:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 11:59:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7580090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7580090","identity":"rs-7580090","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}