The Role Of Micro-RNA200-c In Implantatıon Of Embryo To Endometriyum

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Serkant UNAL This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6594610/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract The objective of this study was to examine the impact of microRNA-200c on embryo implantation and the expressions of α5β3, LIF, IL-6, CD105 (endogolin), and VEGF, which have a role in implantation. The experimental model comprised 30 female and 4 male rats. The rats were divided into two groups: the first group comprised non-pregnant rats, while the second group comprised pregnant rats. The pregnant rats were then separated into two groups: an experimental pregnat group and a control pregnant group. In the experimental group, rats were administered a microRNA200-c mimic solution (50 µl of 0.9% sodium chloride) to the right uterine horn, while the left horn received an injection of 0.9% sodium chloride. The procedure was performed on day 3 of the pregnancy. The rats in the control group were not administered any treatment. On days 6 and 9 of gestation, the uteri were removed by cervical dislocation, after which the number of embryos was counted. The effects of α5β3, LIF, IL-6, CD105 (Endogolin), IL-6, CD105 (Endogolin) and VEGF proteins in the uterine tissues of the experimental pregnat groups were analysed using immunohistochemistry. MiRNA-200c reduced the number of implanted embryos by suppressing the expression of α5β3 integrin, LIF, IL-6, CD105 (endogolin), and VEGF. Based on these results, it can be concluded that mimic treatment may interfere with the process of implantation and early pregnancy by reducing the expression levels of In addition, integrin α5β3, LIF, IL-6, CD105 (Endogolin), VEGF. miRNA-200c α5β3 LIF IL-6 CD105 (Endogolin) VEGF implantation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Implantation is defined as the process by which the embryo attaches to the epithelium of the endometrium and enters the maternal structure to form the placenta ( 1 ). Implantation takes place in 3 stages. In the first stage, called apposition, the embryo attaches to the surface of the endometrium ( 2 , 3 ). During this process, an increase in adhesion molecules (integrin, selectin, cadherin, Ig superfamily) is observed on the endometrial surface ( 4 ). Stage 2 is called adhesion, and it is during this period that adhesion molecules form tight junctions between the endometrium and the embryo. At the same time, the process of decidualisation of the endometrium begins ( 5 ). Cytokines such as IL-6, LIF and VEGF play a role in the decidualisation process. In the third stage, called invasion, the embryo invades the endometrium. During this process, the components of cell adhesion molecules, basement membrane components and the extracellular matrix are degraded by serine endopeptidases and metalloproteinases( 6 ). Successful implantation of the embryo depends on the regulation of several genetic factors at the transcriptional and post-transcriptional levels. These gene expression regulations prepare the embryo to approach, attach and penetrate the endometrium ( 7 ). As miRNAs have the ability to target a significant number of genes it is possible that they play a role in the regulation of gene expression in the embryo. MicroRNAs (miRNAs) are endogenous non-coding RNAs approximately 22 nucleotides in size ( 8 , 9 ). There are more than 2000 miRNAs in the human genome. MiRNAs regulate target genes in various cells and conditions. Profiles of these molecules are linked to pathologies such as infertility, endometriosis, and preeclampsia. ( 10 – 12 ). Previous studies have shown that the expression of microRNA-200 (MiR-200) family members is low during the receptive period of the uterus ( 13 – 17 ). In a study conducted by Jimenez in 2014 adecrease in the expression levels of miR-200a, miR-200b and miR-200c in endometrial stromal cells was observed during the implantation process compared to before implantation ( 17 ). Liu et al. reported that when mouse uterine horns were injected with miR-200a, the implantation rate and number of implanted embryos decreased ( 11 ). Zheng et al. (2017) found that injection of miR-200c on day 3 of pregnancy in mice inhibited the formation of uterine receptivity by inhibiting α-1,3-fucosylation biosynthesis on glycoprotein CD44 and targeting FUT4, which further inactivates the Wnt/β-catenin signalling pathway ( 18 ). Zheng et al. also analysed the levels of miR-200 family members in the serum of healthy and infertile women and found that miR-200c was the most sensitive and specific marker for infertility ( 18 ). As demonstrated in the extant literature, miR-200c has been shown to reduce embryo implantation, yet information regarding the mechanism of this condition remains extremely limited ( 13 – 18 ). The present study therefore investigated the relationship between miR-200c and the following markers of implantation and early pregnancy: αvβ3 integrin, LIF, IL-6, CD105 and VEGF, using immunohistochemical analysis. MATERIALS AND METHODS Animals Eight-week-old female Wistar albino rats, with an average weight of 150–160 g, were obtained from the Experimental Surgery Application Centre of Pamukkale University. All animal experiments were approved by the Animal Experiments Ethics Committee of Pamukkale University and were conducted in according to the "Guidelines for Experimental Animal Care and Use of the National Institutes of Health" (No. 2018/01). The rats were housed in standard cages at 22°C under a 12-hour light-dark cycle, with free ad libitum access to food and water. Experimental design A total of 30 female rats and 4 male rats were used in the study. Six of the rats were designated as Group 1. Rats in estrus were included in group 1. Pregnancy was established in 24 of them. 4 female rats and 1 male rat were kept together in the cage for 1 night. After 1 day, a smear was taken and if sperm were seen, it was accepted as the 1st day of pregnancy. The pregnant rats were then randomly divided into an experimental group treated with miR-200c (n = 12) and a pregnant control group (n = 12). On the day 3 of gestation (n = 12), 5 µg of miRNA-200c mimic in 50 µl of 0.9% sodium chloride was injected into the right uterine horn and 50 µl of 0.9% sodium chloride was injected into the left uterine horn under anaesthesia ( 18 ). We planned to sacrifice rats on two different days to observe changes in the implantation process. Pregnant rats in the experimental and control groups were sacrificed on days 6 and 9 of gestation. The right and left uterine horns of each group were evaluated as a subgroup. Nine subgroups were formed, including those sacrificed on days 6 and 9, with group 1 (n = 6 in each subgroup). The groups are summarised in Table 1 . Immunohistochemical and histological analyses were performed on uterine tissue samples collected from rats in Group 1, Group 2a and Group 2b. Table 1 Groups Control Non-pregnant rats Grup1 Rats whose right horns were removed on the 6th day after miRNA-200c application to the right horns. Grup2a Rats whose left horns were removed on the 6th day after and 0.9% sodium chloride application to the left horns. Grup2b Rats whose right horns were removed on the 9th day after miRNA-200c application to the right horns. Grup3a Rats whose left horns were removed on the 9th day after and 0.9% sodium chloride application to the left horns Grup3b The right uterine horns were collected from pregnant rats on day 6 (control pregnant). Grup4a The left uterine horns were collected from pregnant rats on day 6 (control pregnant). Grup4b The right uterine horns were collected from pregnant rats on day 9 (control pregnant). Grup5a The left uterine horns were collected from pregnant rats on day 9 (control pregnant). Grup5b The number of embryos in each group is as follows: In order to observe the effect of microRNA-200c treatment on embryo implantation, uterine tissues were collected from rats sacrificed on days 6 and 9 of pregnancy. The number of embryos was then counted. Histological parameters The excised uterine horn was fixed in neutral buffer formaldehyde for a period of 72 hours. The tissues were then washed for 1hour under running water. The tissues were then dehydrated using a series of ethanol concentrations, ranging from 50–100%. Following a 2-hour soak in xylene to clear the tissue sections, the tissues were left in liquid paraffin for a further 2 hours. The final step involved embedding the tissues in paraffin blocks. For histological and immunohistochemical analysis, 5µm thick sections were cut from the paraffin blocks using a Leica RM-2125 rotary microtome (Leica, Wetzlar, Germany) and mounted on lysine-coated glass slides (Marienfeld Laboratory Glassware, Histobond, Marienfeld, Germany). Hematoxylin eosin staining The sectiones were deparaffinised in xylene, rehydrated through graded ethanols, and stained with hematoxylin and eosin (Merck, Germany). They were examined and photographed under an Olympus BX51 microscope. Immunohistochemistry Following deparaffinization, the sections were rehydrated through a descending ethanol series for 2 minutes in each bath. Following this, the sections were rinsed with tap water and then placed in phosphate-buffered saline (PBS) for a period of 10 minutes. The endogenous peroxidase activity was blocked by treatment with 1:9 30% H 2 O 2 :methanol. The slides were then washed with PBS three times. Thereafter, serum blocking solution (Reagent A) was added to the sections for incubation for 10 min at room temperature. Following this, Reagent A was removed and primer antibody was added drop-wise to wet slides. Sections were then subjected to an overnight incubation with relevant primary antibodies. The primary antibodies used were αvβ3 (Bios Lot: 7G11M19), LIF (Bios Lot: 9G23N13), IL-6 (Bios Lot: 6G24M21), CD105 (Bios Lot: 8G22M18) and VEGF (Bios Lot: 9G16M21), all of which were diluted to 1:100. All primary antibodies were diluted with Phosphate Buffered Saline (PBS). Sections were washed three times with PBS for 2 min each, then treated with Broad Spectrum Second Antibody (Reagent B) for 20 min. The sections were then washed three times with PBS for two minutes on each occasion. Thereafter, they were treated with horse radish peroxidase conjugate streptavidin (HRP-SA) (Reagent C) for a period of 10 minutes. Following a further three washes with PBS for two minutes each, the sections were treated with DAB-Plus Substrate Kit (1018723A;Invitrogen) for a period of between three and 10 minutes. The sections were then counterstained with Harris hematoxylin (Merck). The sections were then washed with tap water and dehydrated through 70, 80, 90 and 100% ethanol for 2 min each. Finally, the sections were coverslipped with entellan. They were examined and photographed under an Olympus BX51 microscope. H-SCORE analysis Separate histological scores (H SCORE) were obtained for αvβ3 integrin, LIF, IL-6, CD105 and VEGF positive staining in uterine lumen and gland epithelial cells and stroma cells. The H SCORE was determined as follows: ∑Pi(I + 1). The staining intensity (I) was categorised as follows: 0 = no expression, 1 = mild, 2 = moderate and 3 = intense, and the percentage of stained cells for each intensity (Pi) was determined. Statistical analysis The Mann-Whitney U test and Kruskal-Wallis analysis of variance were used for independent group analysis. H-score results were analysed using Tukey post hoc tests to determine differences between groups. All statistical analyses were performed using IBM SPSS Statistical Version 21. Differences between means were considered significant if P < 0.05 or less. RESULTS Statistical comparison of embryo numbers Stage 1: Determination of the effect of miRNA-200c on the number of embryos compared to control pregnant groups. The effect of miRNA-200c on embryo numbers was evaluated by comparing with the pregnant control groups. The results obtained revealed that there was a statistically significant difference between the number of embryos in group2a-4a (p = 0,015) and group3a-5a (p = 0,041) (Fig. 1A,B). Stage 2: Determination of the effect of %0,9 sodium chloride on embryo number in comparison to control pregnant groups In a second stage, the effect of 0.9% sodium chloride applied to the left uterine horn of rats in the experimental group on the number of embryos was measured in comparison with the pregnant control group. The results of this analysis showed that there was no significant difference between the number of embryos in the right and left uterine horns of groups 2b-4b (p = 0.180) and groups 3b-5b (p = 0.132) (Fig. 1A,B). Stage 3: Determination of whether there is a spontaneous natural difference between the number of embryos in the left and right uterine horns of rats. At this stage of the experiment, an attempt was made to test the probability that the possible difference in the number of embryos was not caused by the injection of miRNA-200c into the rats, but by the fact that the number of embryos distributed in the right and left uterine horns of the rats is naturally unidirectional in all rats. For this purpose, the number of embryos in the right and left uterine horns of group 2a-2b, (p = 0,002) group 3a-3b, (p = 0,026), group 4a-4b (p = 0,180), group5a-5b (p = 0,699) were statistically evaluated. (Fig. 1A, B). A statistically significant difference was detected between the number of embryos in the right and left uterine horns of group2a-2b (p = 0,002), group3a-3b (0,026) rats. (Fig. 1A, B).It was observed that there was no significant difference between the number of embryos in the right and left uterine horns of group 4a-4b (p = 0,180 ), group5a-5b (p = 0.699) (Fig. 1A,B). Stage 4: To determine whether there is a difference between the number of embryos in the groups injected with 0.9% sodium chloride and miRNA-200c and the number of embryos in the control pregnant groups at days 6 and 9 In stage 4, we investigated the impact of mi-RNA200c on embryo implantation over time by measuring the number of embryos in the same directional uterine horns of each experimental and pregnant group on day 6 and 9 of pregnancy (Fig. 1A,B). It was observed that there was no significant difference between the number of embryos in the right uterine horns of the group2a-3a, (p = 0,699) group 4a-5a (p = 0,699). There was no significant difference between the number of embryos in the left uterine horns of groups 2b-3b (p = 0.699) and group 4b-5b (p = 0.485). Histological findigs The endometrium, myometrium and perimetrium layers of the uterine tissue obtained from group 1 were found to be normal structure (Fig. 2A). It was observed that the epithelial tissue surrounding the lumen separated from the stroma and formed cavities in the uterine tissues obtained from group 2a. The presence of haemorrhage areas in the endometrium was also noted. In addition, some areas of the lumen and gland epithelium lost continuity and formed interruptions. However, myometrium and perimetrium were normal.(Fig. 2B). Lumen and gland epithelial cells, myometrium and perimetrium were found to be normal in the uterine tissue obtained from group 2b. Desudalisation of stromal cells was observed in this group (Fig. 2C). Immunohisthochemical findigs The α5β3 response in the luminal epithelial and glandular cells of the uteri from group 1, group 2a and group 2b were similar (p > 0,05). In group 2b, the expression of α5β3 in the stromal cells was higher than in group 1 and group 2a (p < 0.05) (Fig. 3A,B). LIF expression was found to be increased in the luminal glandular epithelium of groups 1 and 2a in comparison to group 2b. In contrast, the level of LIF expression in the stomal/decidual cells of group 2b was higher than in groups 1 and 2a (p < 0.05) (Fig. 4A,B). IL-6 expression in uterine luminal epithelial and glandular cells and stromal/desidual cells of group 2b was significantly higher than that of group 1 and group 2a (p < 0.05). Howewer, a significant increase in IL-6 expression was observed in the glandular epithelium and stromal cells of group 1 when compared with group 2a (p < 0.05) (Fig. 5A, B). In comparison with groups 1 and 2a, CD105 expression was found to be significantly lower in luminal epithelial cells of group 2b. In contrast, a significant increase in CD105 expression was observed in stromal/desidual cells in Group 2b (p 0.05). The expression of CD105 in glandular cells was found to be similar across the groups (p > 0.05) ( Fig. 6A, B). The expression of VEGF (vascular endothelial growth factor) in the luminal epithelium was found to be significantly higher in group 1 than in groups 2a and 2b. Our study demonstrated that there was an elevation in the expression of vascular endothelial growth factor (VEGF) in glandular epithelial cells within groups 1 and 2a when compared with those within group 2b. Nevertheless, a substantial augmentation in VEGF expression was evident in stromal/decidual cells of group 2b when contrasted with groups 1 and 2a (see Fig. 7A, B). DISCUSSION Uterine receptivity is a hallmark event for successful implantation of the embryo. Poor uterine receptivity has been associated with infertility and abortion. In recent years, microRNAs (miRNAs) have been reported to be associated with embryo implantation, embryo development, decidualisation and infertility ( 13 – 19 ). It has been found that the expression of microRNAs differs between receptive and non-receptive endometrium, with Let-7, mir-23, miR-200, miR-30 and Mir-183 being more frequently associated with endometrial receptivity ( 13 , 19 ). Previous studies have shown that injection of the miR-200 family into the horns of the uterus reduces the implantation of embryos ( 11 , 18 ). In our study, similar to previous studies, a significant reduction in the number of embryos was observed in the uterine horn of miR-200c-treated rats compared to sodium chloride-treated pregnant rats and untreated rats, in contrast, no significant difference was observed between the number of embryos in the uterine horn of rats treated with sodium chloride and the number of embryos in untreated rats. The our study was conducted for the purpose of elucidating the mechanism of action of miR-200c on implantation. An evaluation was therefore performed of the relationship between mirRNA200-c and particular molecules implicated in the implantation stages (αVβ3 integrin, LIF, IL-6, CD105 and VEGF), which were analysed by immunohistochemical methods in rats.. It has been demonstrated that a decline in the expression of these molecules, particularly in stromal cells, contributes to the molecular mechanism that underlies this adverse effect. As has been documented, αvβ3 integrin is expressed in the glandular and luminal epithelium of the uterus during the implantation window. A correlation has been demonstrated between its deficiency or absence and infertility ( 20 – 22 ). As demonstrated by numerous studies, the αvβ3 integrin is of significant importance in the process of decidualisation ( 20 , 21 ), It has been reported that this protein acts as a critical receptor for the attachment of the embryo to the uterus ( 21 ). In the our study, the expression of αvβ3 in the luminal epithelium was found to be similar between groups. Nevertheless, a higher reaction was observed in stromal/desidual cells in Group 2b rats when compared to Group 1 and Group 2a. We demonstrated that the administration of microRNA-200c led to a decrease in αvβ3 integrin expression in both glands and stromal cells. LIF plays an important role in the preimplantation development of the embryo, subsequent implantation and early pregnancy ( 23 ). LIF has been demonstrated to be involved in uterine regulation for implantation, blastocyst growth and development, embryo endometrial interaction, embryo implantation, decidualisation, uterine inflammatory response to implanted embryos and trophoblast invasion ( 24 , 25 ). In a study conducted by Margıoula-Siarkou et al. in endometrial biopsy samples taken from infertile and fertile women, LIF expression in epithelial tissue was found to be statistically higher than in infertile women, while no difference was observed in stromal cells ( 26 ). However, in another study by Niknafs et al in 2019, it was reported that the highest LIF expression in endometrial tissue taken from rats on day 4 of pregnancy was observed in tissue taken on day 4 of pregnancy, with localised expression in epithelial tissue and to a lesser extent in stromal cells ( 27 ). In the our study, LIF expression in the lumen and gland epithelium of group 2b endometrium was found to be weak compared to group 2a, whereas expression was found to be intense in stromal/deidual cells. The discrepancy between our findings and those of other studies may be attributable to the fact that our tissues were obtained from rats on the 6th day of gestation, at a stage preceding the decidualisation process. The our study found that administration of the miR-200c decreased LIF expression in stromal cells Interleukin-6 is secreted from glandular and luminal epithelial cells of the endometrium throughout the menstrual cycle, especially during the mid-secretory phase and early stages of pregnancy. The secretion of this protein promotes successful embryo implantation and adequate placentation during the implantation period and early stages of pregnancy. Interlökin-6, menstrüel siklus boyunca, özellikle de orta sekretuar faz ve gebeliğin erken dönemlerinde endometriyumun glandüler ve luminal epitel hücrelerinden salgılanır. Bu proteinin salgılanması, implantasyon dönemi ve gebeliğin erken dönemlerinde başarılı embriyo implantasyonunu ve yeterli plasentasyonu teşvik eder ( 28 , 29 ). The our study found that IL-6 expression in group 2a and group 2b was significantly different between the two groups. In group 2b, IL-6 expression in the lumen, gland and stromal cells was found to be statistically significantly higher than in group 2a. In conclusion, this study demonstrates that microRNA-200c administration suppresses IL-6 expression. CD105 is a type 1 transmembrane glycoprotein that acts as a co-receptor for TGF-. CD105 has been implicated in mechanisms of vasculogenesis and angiogenesis ( 30 ). However, its functional role in uterine receptivity for embryo implantation is still unclear.In a study conducted by Chaddan in 2016, it was reported that CD105 expression was found in epithelial and stromal cells on day 3 of gestation, whereas stromal cells in the implanted region showed strong expression and stromal cells in the non-implanted region showed weak expression in tissue taken on day 6 of gestation. In our study, expression was found to be statistically higher in the endometrial stromal cells in group2b than in group2a. In contrast, expression levels in the luminal epithelium of group2b tissues were lower than those observed in group2a. The results of this study showed that microRNA-200c can inhibit CD105 expression in stromal cells ( 31 ). VEGF is a crucial cytokine for successful embryo implantation. In rodents, the vascular permeability of the uterus increases in the blastocyst regions with the onset of the attachment reaction. This is followed by stromal decidualization and angiogenesis. VEGF has been identified as a mitogen for endothelial cells and an inducer of angiogenesis. It has been reported that VEGF mRNA is expressed in the luminal epithelium in the endometrium on days 1 and 2 of pregnancy, in stromal cells to a lesser extent on day 3 of pregnancy and in luminal epithelial cells, subepithelial cells and stromal region on day 4. Furthermore, the expression of VEGF mRNA has been observed in luminal epithelial and stromal cells immediately surrounding the blastocyst on the 5th day after the first binding reaction ( 31 , 32 ). Our study revealed that the expression of VEGF was found to be slightly weaker in the lumen and glandular epithelium of group 2b uterine tissues compared to group 1 and gryup2a, but very intense in the stromal/decidual cells. WE found that the application of microRNA 200c led to a decrease in the expression of αvβ3, LIF, IL-6, CD105 and VEGF, particularly in stromal cells, and even led to the negative expression of IL-6. Only a few studies to date have shown that high levels of miR-200c inhibit pinopod formation, which is important for endometrial receptivity ( 18 ), and play a role in decidualisation by affecting IL-5 expression in the stroma ( 13 ). This study was the first to investigate the relationship between miR-200c and integrin αvβ3, LIF, IL-6, CD105 and VEGF. Our results suggest that miRNA-200c prevents pregnancy by inhibiting decidualisation. In conclusion, it can be suggested that miRNAs can be used as markers or therapeutic targets in infertility treatments to increase endometrial receptivity and promote pregnancy. Declarations Author Contribution Ayşe AKGÜN and Nazlı ÇİL did the experimentsGülçin ABBAN METE and M. 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Scientific World Journal 2014;2014:201514. doi: 10.1155/2014/201514 . Epub 2014 Jul 24. Margioula-Siarkou C, Prapas Y, Petousis S, et al. LIF And LIFR Expression In The Endometrium Of Fertile And Infertile Women: A Prospective Observational CaseControl Study Mol Med Rep 2016;13(6):4721–8. doi: 10.3892/mmr.2016.5142 Niknafs B, Hesam Shariati MB, Shokrzadeh N. miR223-3p, HAND2, and LIF expression regulated by calcitonin in the ERK1/2-mTOR pathway during the implantation window in the endometrium of mice. Am J ReprodImmunol 2021;85(1):e13333. doi: 10.1111/aji.13333 . Epub 2020 Sep 22. .Jasper MJ, Tremellen KP, Robertson SA. Reduced expression of IL-6 and IL-1α mRNAs in secretory phase endometrium of women with recurrent miscarriage. J Reprod Immunol. 2007;73(1):74–84. doi: 10.1016/j.jri.2006.06.003. Epub 2006 Oct 10. . Lockwood CJ, Yen CF Yen, Basar M et al. Growth Factors, Cytokines, Cell Cycle Molecules Preeclampsia-Related Inflammatory Cytokines Regulate Interleukin-6 Expression in Human Decidual Cells. Am J Pathol 2008;172(6):1571–9.doi: 10.2353/ajpath.2008.070629 . Epub 2008 May 8 Rokhlin OW, Cohen MB,, Kubagawa H, et al. Differential 667 expression of endoglin on fetal and adult hematopoietic cells in human bone marrow. J. Immunol. 1995; 154(9): 4456–4465. Chadchan SB, Kumar V, Maurya VK, et al. Endoglin (CD105) coordinates the process of endometrial receptivity for embryo implantation. Mol Cell Endocrinol 2016;425:69–83. doi: 10.1016/j.mce.2016.01.014 . Epub 2016 Jan 21. Chakraborty I, Das SK. Dey SK. Differential expression of vascular endothelial growth factor and its receptor mRNAs in the mouse uterus around the time of implantation. J Endocrinol 1995;147(2):339–352. doi: 10.1677/joe.0.1470339 . Guo X, Yi H, Li TC, Wang Y, et al. Role of Vascular Endothelial Growth Factor (VEGF) in Human Embryo implantation: Clinical Implantation: Biomolecules 2021;11(2):253. doi: 10.3390/biom11020253 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 13 Jun, 2025 Reviewers agreed at journal 01 Jun, 2025 Reviewers agreed at journal 01 Jun, 2025 Reviews received at journal 21 May, 2025 Reviews received at journal 15 May, 2025 Reviewers agreed at journal 15 May, 2025 Reviewers invited by journal 15 May, 2025 Editor assigned by journal 05 May, 2025 Submission checks completed at journal 05 May, 2025 First submitted to journal 05 May, 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-6594610","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":458107658,"identity":"ed0fa9c4-6280-4b54-bd02-58d47aabda47","order_by":0,"name":"Ayşe AKGÜN","email":"","orcid":"","institution":"Pamukkale University","correspondingAuthor":false,"prefix":"","firstName":"Ayşe","middleName":"","lastName":"AKGÜN","suffix":""},{"id":458107659,"identity":"94529de1-6584-446e-96c9-afa518adb33b","order_by":1,"name":"Gulçin ABBAN METE","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYDACCR44k/EAgwGDHIh14AERWiTAKoFajMGMBOK1MDAkNoBY+LTwz+49+Lkwx6aOn3+NwYEfBXfS54cdfgi0xU5OtwGHJXfOJUvP3JYmITnjjcHBHoNnuRtvpxkAtSQbmx3AYc2NHANp3m2HJQxunDE4zGBwOHfj7ASQlgOJ23Bokb+RY/ybd9t/CXuolnTD2ekf8GoxuJFjBrTlgIQBfw9YS4K8dA5+WwzvnEuz5t2WLDnjBlsB0C+HDTdI5xQcSDDA7Re5272Hb/Nus+Pn7z+88cGPP4fl5Wenb/7wocJODqf34UAiAepUsEoDQspBgB9qqHwDMapHwSgYBaNgJAEAJE5oIMbSJNIAAAAASUVORK5CYII=","orcid":"","institution":"Pamukkale University","correspondingAuthor":true,"prefix":"","firstName":"Gulçin","middleName":"ABBAN","lastName":"METE","suffix":""},{"id":458107660,"identity":"f51b2001-08cb-4249-8e71-89d52360a218","order_by":2,"name":"Nazlı ÇIL","email":"","orcid":"","institution":"Pamukkale University","correspondingAuthor":false,"prefix":"","firstName":"Nazlı","middleName":"","lastName":"ÇIL","suffix":""},{"id":458107661,"identity":"b80d75bb-a62d-4f72-b065-c304f9f3b616","order_by":3,"name":"M. Serkant UNAL","email":"","orcid":"","institution":"Pamukkale University","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"Serkant","lastName":"UNAL","suffix":""}],"badges":[],"createdAt":"2025-05-05 12:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6594610/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6594610/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83078344,"identity":"b801997f-42a7-41ec-9198-64e9e36f0280","added_by":"auto","created_at":"2025-05-19 18:47:22","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":115705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. Number of embryos implanted in the uterus in each group. B. Statistical analysis of total implantation site numbers. The number of embryos was significantly lower in the miRNA application group (* p \u0026lt; 0.05).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6594610/v1/57d88309e12fe720a66eaa5c.jpeg"},{"id":83078347,"identity":"e43b4ab0-ffe5-4d32-aa27-daa8150a902a","added_by":"auto","created_at":"2025-05-19 18:47:22","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":368176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative hematoxylin and eosin (H\u0026amp;E) staining of uterus dokusu in group 1, 2a,2b. A: Group1, B: Group2a, C: Group 2c. Luminal epithelium; (LE), glandular epithelium (GE), stroma (S), myometrium (M), perimetrium (P), separated area between luminal epithelium and stroma (star), and haemorrhage areas (arrow). The hematoxylin and eosin stain. Scale bar: 200 μm, X100\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6594610/v1/fa129c5710b4ffe5221b836b.jpeg"},{"id":83078346,"identity":"5be4c4c7-ef63-4871-b549-0723a0404664","added_by":"auto","created_at":"2025-05-19 18:47:22","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":342939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. α5β3 integrin expression and localisation in uterine tissues in groups 1, 2a and 2b. Luminal epithelium (LE), glandular epithelium (GE), stroma (S), myometrium (M), perimetrium (P) .Haematoxylin and immunopreoxidase Scale bars: 200 μm, 100 μm.X100, 200. \u0026nbsp;B. Semiquantitative scores in the analysis of Integrin α5β3 immunohistochemistry.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6594610/v1/2d1be67077060cb7835104c9.jpeg"},{"id":83078351,"identity":"a2c9d59c-8394-425c-be9c-82a97c3de842","added_by":"auto","created_at":"2025-05-19 18:47:22","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":336113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.LIF expression and localisation in uterine tissues in groups 1, 2a and 2b. Luminal epithelium (LE), glandular epithelium (GE), stroma (S), myometrium (M), perimetrium (P). Haematoxylin and immunopreoxidase Scale bars: 200 μm, 100 μm.X100, 200.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Semiquantitative scores in the analysis of LIF immunohistochemistry\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6594610/v1/f337002b65434e26cf88d466.jpeg"},{"id":83078355,"identity":"7197130e-2e1f-4d41-92fd-b01969dc0618","added_by":"auto","created_at":"2025-05-19 18:47:22","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":341337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. IL-6 expression and localisation in uterine tissues in groups 1, 2a and 2b. Luminal epithelium (LE), glandular epithelium (GE), stroma (S), myometrium (M), perimetrium (P). Haematoxylin and immunopreoxidase Scale bars: 200 μm, 100 μm.X100, 200.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Semiquantitative scores in the analysis of IL-6 immunohistochemistry.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6594610/v1/19f2a628aaea77ba9f638fdd.jpeg"},{"id":83078350,"identity":"a9dc71f0-5507-4eef-a1e3-f29f893dbb5b","added_by":"auto","created_at":"2025-05-19 18:47:22","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":342140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. Integrin α5β3 expression and localisation in uterine tissues in groups 1, 2a and 2b. Luminal epithelium (LE), glandular epithelium (GE), stroma (S), myometrium (M), perimetrium (P). Haematoxylin and immunopreoxidase Scale bars: 200 μm, 100 μm.X100, 200. \u0026nbsp;B. Semiquantitative scores in the analysis of CD105 immunohistochemistry.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6594610/v1/bf535bf232ec7ad269747c36.jpeg"},{"id":83078987,"identity":"71615118-449d-4dfa-a3ff-2bf83b13ce49","added_by":"auto","created_at":"2025-05-19 18:55:22","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":355379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. VEGF expression and localisation in uterine tissues in groups 1, 2a and 2b. Luminal epithelium (LE), glandular epithelium (GE), stroma (S), myometrium (M), perimetrium (P). Haematoxylin and immunopreoxidase Scale bars: 200 μm, 100 μm.X100, 200.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Semiquantitative scores in the analysis of VEGF immunohistochemistry.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6594610/v1/7329e56a2bf56d2acaafd9e6.jpeg"},{"id":83079783,"identity":"7416c438-2477-4e9c-8e00-87231eb89611","added_by":"auto","created_at":"2025-05-19 19:11:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3765081,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6594610/v1/aa05fd77-cd10-4f51-977e-f67f2b18e0e4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Role Of Micro-RNA200-c In Implantatıon Of Embryo To Endometriyum","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eImplantation is defined as the process by which the embryo attaches to the epithelium of the endometrium and enters the maternal structure to form the placenta (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Implantation takes place in 3 stages. In the first stage, called apposition, the embryo attaches to the surface of the endometrium (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). During this process, an increase in adhesion molecules (integrin, selectin, cadherin, Ig superfamily) is observed on the endometrial surface (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Stage 2 is called adhesion, and it is during this period that adhesion molecules form tight junctions between the endometrium and the embryo. At the same time, the process of decidualisation of the endometrium begins (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Cytokines such as IL-6, LIF and VEGF play a role in the decidualisation process. In the third stage, called invasion, the embryo invades the endometrium. During this process, the components of cell adhesion molecules, basement membrane components and the extracellular matrix are degraded by serine endopeptidases and metalloproteinases(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSuccessful implantation of the embryo depends on the regulation of several genetic factors at the transcriptional and post-transcriptional levels. These gene expression regulations prepare the embryo to approach, attach and penetrate the endometrium (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). As miRNAs have the ability to target a significant number of genes it is possible that they play a role in the regulation of gene expression in the embryo.\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) are endogenous non-coding RNAs approximately 22 nucleotides in size (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). There are more than 2000 miRNAs in the human genome. MiRNAs regulate target genes in various cells and conditions. Profiles of these molecules are linked to pathologies such as infertility, endometriosis, and preeclampsia. (\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Previous studies have shown that the expression of microRNA-200 (MiR-200) family members is low during the receptive period of the uterus (\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a study conducted by Jimenez in 2014 adecrease in the expression levels of miR-200a, miR-200b and miR-200c in endometrial stromal cells was observed during the implantation process compared to before implantation (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Liu et al. reported that when mouse uterine horns were injected with miR-200a, the implantation rate and number of implanted embryos decreased (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Zheng et al. (2017) found that injection of miR-200c on day 3 of pregnancy in mice inhibited the formation of uterine receptivity by inhibiting α-1,3-fucosylation biosynthesis on glycoprotein CD44 and targeting FUT4, which further inactivates the Wnt/β-catenin signalling pathway (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Zheng et al. also analysed the levels of miR-200 family members in the serum of healthy and infertile women and found that miR-200c was the most sensitive and specific marker for infertility (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs demonstrated in the extant literature, miR-200c has been shown to reduce embryo implantation, yet information regarding the mechanism of this condition remains extremely limited (\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The present study therefore investigated the relationship between miR-200c and the following markers of implantation and early pregnancy: αvβ3 integrin, LIF, IL-6, CD105 and VEGF, using immunohistochemical analysis.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eEight-week-old female Wistar albino rats, with an average weight of 150\u0026ndash;160 g, were obtained from the Experimental Surgery Application Centre of Pamukkale University. All animal experiments were approved by the Animal Experiments Ethics Committee of Pamukkale University and were conducted in according to the \"Guidelines for Experimental Animal Care and Use of the National Institutes of Health\" (No. 2018/01). The rats were housed in standard cages at 22\u0026deg;C under a 12-hour light-dark cycle, with free ad libitum access to food and water.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eA total of 30 female rats and 4 male rats were used in the study. Six of the rats were designated as Group 1. Rats in estrus were included in group 1. Pregnancy was established in 24 of them. 4 female rats and 1 male rat were kept together in the cage for 1 night. After 1 day, a smear was taken and if sperm were seen, it was accepted as the 1st day of pregnancy. The pregnant rats were then randomly divided into an experimental group treated with miR-200c (n\u0026thinsp;=\u0026thinsp;12) and a pregnant control group (n\u0026thinsp;=\u0026thinsp;12). On the day 3 of gestation (n\u0026thinsp;=\u0026thinsp;12), 5 \u0026micro;g of miRNA-200c mimic in 50 \u0026micro;l of 0.9% sodium chloride was injected into the right uterine horn and 50 \u0026micro;l of 0.9% sodium chloride was injected into the left uterine horn under anaesthesia (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). We planned to sacrifice rats on two different days to observe changes in the implantation process. Pregnant rats in the experimental and control groups were sacrificed on days 6 and 9 of gestation. The right and left uterine horns of each group were evaluated as a subgroup. Nine subgroups were formed, including those sacrificed on days 6 and 9, with group 1 (n\u0026thinsp;=\u0026thinsp;6 in each subgroup). The groups are summarised in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Immunohistochemical and histological analyses were performed on uterine tissue samples collected from rats in Group 1, Group 2a and Group 2b.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl Non-pregnant rats\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrup1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRats whose right horns were removed on the 6th day after miRNA-200c application to the right horns.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrup2a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRats whose left horns were removed on the 6th day after and 0.9% sodium chloride application to the left horns.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrup2b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRats whose right horns were removed on the 9th day after miRNA-200c application to the right horns.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrup3a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRats whose left horns were removed on the 9th day after and 0.9% sodium chloride application to the left horns\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrup3b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThe right uterine horns were collected from pregnant rats on day 6 (control pregnant).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrup4a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThe left uterine horns were collected from pregnant rats on day 6 (control pregnant).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrup4b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThe right uterine horns were collected from pregnant rats on day 9 (control pregnant).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrup5a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThe left uterine horns were collected from pregnant rats on day 9 (control pregnant).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrup5b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eThe number of embryos in each group is as follows:\u003c/h3\u003e\n\u003cp\u003eIn order to observe the effect of microRNA-200c treatment on embryo implantation, uterine tissues were collected from rats sacrificed on days 6 and 9 of pregnancy. The number of embryos was then counted.\u003c/p\u003e\n\u003ch3\u003eHistological parameters\u003c/h3\u003e\n\u003cp\u003eThe excised uterine horn was fixed in neutral buffer formaldehyde for a period of 72 hours. The tissues were then washed for 1hour under running water. The tissues were then dehydrated using a series of ethanol concentrations, ranging from 50\u0026ndash;100%. Following a 2-hour soak in xylene to clear the tissue sections, the tissues were left in liquid paraffin for a further 2 hours. The final step involved embedding the tissues in paraffin blocks. For histological and immunohistochemical analysis, 5\u0026micro;m thick sections were cut from the paraffin blocks using a Leica RM-2125 rotary microtome (Leica, Wetzlar, Germany) and mounted on lysine-coated glass slides (Marienfeld Laboratory Glassware, Histobond, Marienfeld, Germany).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin eosin staining\u003c/h2\u003e \u003cp\u003eThe sectiones were deparaffinised in xylene, rehydrated through graded ethanols, and stained with hematoxylin and eosin (Merck, Germany). They were examined and photographed under an Olympus BX51 microscope.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eFollowing deparaffinization, the sections were rehydrated through a descending ethanol series for 2 minutes in each bath. Following this, the sections were rinsed with tap water and then placed in phosphate-buffered saline (PBS) for a period of 10 minutes. The endogenous peroxidase activity was blocked by treatment with 1:9 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e:methanol. The slides were then washed with PBS three times. Thereafter, serum blocking solution (Reagent A) was added to the sections for incubation for 10 min at room temperature. Following this, Reagent A was removed and primer antibody was added drop-wise to wet slides. Sections were then subjected to an overnight incubation with relevant primary antibodies. The primary antibodies used were αvβ3 (Bios Lot: 7G11M19), LIF (Bios Lot: 9G23N13), IL-6 (Bios Lot: 6G24M21), CD105 (Bios Lot: 8G22M18) and VEGF (Bios Lot: 9G16M21), all of which were diluted to 1:100. All primary antibodies were diluted with Phosphate Buffered Saline (PBS). Sections were washed three times with PBS for 2 min each, then treated with Broad Spectrum Second Antibody (Reagent B) for 20 min. The sections were then washed three times with PBS for two minutes on each occasion. Thereafter, they were treated with horse radish peroxidase conjugate streptavidin (HRP-SA) (Reagent C) for a period of 10 minutes. Following a further three washes with PBS for two minutes each, the sections were treated with DAB-Plus Substrate Kit (1018723A;Invitrogen) for a period of between three and 10 minutes. The sections were then counterstained with Harris hematoxylin (Merck). The sections were then washed with tap water and dehydrated through 70, 80, 90 and 100% ethanol for 2 min each. Finally, the sections were coverslipped with entellan. They were examined and photographed under an Olympus BX51 microscope.\u003c/p\u003e \u003cp\u003eH-SCORE analysis\u003c/p\u003e \u003cp\u003eSeparate histological scores (H SCORE) were obtained for αvβ3 integrin, LIF, IL-6, CD105 and VEGF positive staining in uterine lumen and gland epithelial cells and stroma cells. The H SCORE was determined as follows: \u0026sum;Pi(I\u0026thinsp;+\u0026thinsp;1). The staining intensity (I) was categorised as follows: 0\u0026thinsp;=\u0026thinsp;no expression, 1\u0026thinsp;=\u0026thinsp;mild, 2\u0026thinsp;=\u0026thinsp;moderate and 3\u0026thinsp;=\u0026thinsp;intense, and the percentage of stained cells for each intensity (Pi) was determined.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Mann-Whitney U test and Kruskal-Wallis analysis of variance were used for independent group analysis. H-score results were analysed using Tukey post hoc tests to determine differences between groups. All statistical analyses were performed using IBM SPSS Statistical Version 21. Differences between means were considered significant if P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or less.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eStatistical comparison of embryo numbers\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStage 1: Determination of the effect of miRNA-200c on the number of embryos compared to control pregnant groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of miRNA-200c on embryo numbers was evaluated by comparing with the pregnant control groups. The results obtained revealed that there was a statistically significant difference between the number of embryos in group2a-4a (p\u0026thinsp;=\u0026thinsp;0,015) and group3a-5a (p\u0026thinsp;=\u0026thinsp;0,041) (Fig.\u0026nbsp;1A,B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStage 2: Determination of the effect of %0,9 sodium chloride on embryo number in comparison to control pregnant groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a second stage, the effect of 0.9% sodium chloride applied to the left uterine horn of rats in the experimental group on the number of embryos was measured in comparison with the pregnant control group. The results of this analysis showed that there was no significant difference between the number of embryos in the right and left uterine horns of groups 2b-4b (p\u0026thinsp;=\u0026thinsp;0.180) and groups 3b-5b (p\u0026thinsp;=\u0026thinsp;0.132) (Fig.\u0026nbsp;1A,B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStage 3: Determination of whether there is a spontaneous natural difference between the number of embryos in the left and right uterine horns of rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt this stage of the experiment, an attempt was made to test the probability that the possible difference in the number of embryos was not caused by the injection of miRNA-200c into the rats, but by the fact that the number of embryos distributed in the right and left uterine horns of the rats is naturally unidirectional in all rats. For this purpose, the number of embryos in the right and left uterine horns of group 2a-2b, (p\u0026thinsp;=\u0026thinsp;0,002) group 3a-3b, (p\u0026thinsp;=\u0026thinsp;0,026), group 4a-4b (p\u0026thinsp;=\u0026thinsp;0,180), group5a-5b (p\u0026thinsp;=\u0026thinsp;0,699) were statistically evaluated. (Fig.\u0026nbsp;1A, B).\u003c/p\u003e\n\u003cp\u003eA statistically significant difference was detected between the number of embryos in the right and left uterine horns of group2a-2b (p\u0026thinsp;=\u0026thinsp;0,002), group3a-3b (0,026) rats. (Fig.\u0026nbsp;1A, B).It was observed that there was no significant difference between the number of embryos in the right and left uterine horns of group 4a-4b (p\u0026thinsp;=\u0026thinsp;0,180 ), group5a-5b (p\u0026thinsp;=\u0026thinsp;0.699) (Fig.\u0026nbsp;1A,B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStage 4: To determine whether there is a difference between the number of embryos in the groups injected with 0.9% sodium chloride and miRNA-200c and the number of embryos in the control pregnant groups at days 6 and 9\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn stage 4, we investigated the impact of mi-RNA200c on embryo implantation over time by measuring the number of embryos in the same directional uterine horns of each experimental and pregnant group on day 6 and 9 of pregnancy (Fig.\u0026nbsp;1A,B).\u003c/p\u003e\n\u003cp\u003eIt was observed that there was no significant difference between the number of embryos in the right uterine horns of the group2a-3a, (p\u0026thinsp;=\u0026thinsp;0,699) group 4a-5a (p\u0026thinsp;=\u0026thinsp;0,699).\u003c/p\u003e\n\u003cp\u003eThere was no significant difference between the number of embryos in the left uterine horns of groups 2b-3b (p\u0026thinsp;=\u0026thinsp;0.699) and group 4b-5b (p\u0026thinsp;=\u0026thinsp;0.485).\u003c/p\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eHistological findigs\u003c/h2\u003e\n \u003cp\u003eThe endometrium, myometrium and perimetrium layers of the uterine tissue obtained from group 1 were found to be normal structure (Fig.\u0026nbsp;2A).\u003c/p\u003e\n \u003cp\u003eIt was observed that the epithelial tissue surrounding the lumen separated from the stroma and formed cavities in the uterine tissues obtained from group 2a. The presence of haemorrhage areas in the endometrium was also noted. In addition, some areas of the lumen and gland epithelium lost continuity and formed interruptions. However, myometrium and perimetrium were normal.(Fig.\u0026nbsp;2B).\u003c/p\u003e\n \u003cp\u003eLumen and gland epithelial cells, myometrium and perimetrium were found to be normal in the uterine tissue obtained from group 2b. Desudalisation of stromal cells was observed in this group (Fig.\u0026nbsp;2C).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eImmunohisthochemical findigs\u003c/h2\u003e\n \u003cp\u003eThe \u0026alpha;5\u0026beta;3 response in the luminal epithelial and glandular cells of the uteri from group 1, group 2a and group 2b were similar (p\u0026thinsp;\u0026gt;\u0026thinsp;0,05). In group 2b, the expression of \u0026alpha;5\u0026beta;3 in the stromal cells was higher than in group 1 and group 2a (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;3A,B).\u003c/p\u003e\n \u003cp\u003eLIF expression was found to be increased in the luminal glandular epithelium of groups 1 and 2a in comparison to group 2b. In contrast, the level of LIF expression in the stomal/decidual cells of group 2b was higher than in groups 1 and 2a (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. 4A,B).\u003c/p\u003e\n \u003cp\u003eIL-6 expression in uterine luminal epithelial and glandular cells and stromal/desidual cells of group 2b was significantly higher than that of group 1 and group 2a (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Howewer, a significant increase in IL-6 expression was observed in the glandular epithelium and stromal cells of group 1 when compared with group 2a (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. 5A, B).\u003c/p\u003e\n \u003cp\u003eIn comparison with groups 1 and 2a, CD105 expression was found to be significantly lower in luminal epithelial cells of group 2b. In contrast, a significant increase in CD105 expression was observed in stromal/desidual cells in Group 2b (p 0.05). The expression of CD105 in glandular cells was found to be similar across the groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) ( Fig.\u0026nbsp;6A, B).\u003c/p\u003e\n \u003cp\u003eThe expression of VEGF (vascular endothelial growth factor) in the luminal epithelium was found to be significantly higher in group 1 than in groups 2a and 2b. Our study demonstrated that there was an elevation in the expression of vascular endothelial growth factor (VEGF) in glandular epithelial cells within groups 1 and 2a when compared with those within group 2b. Nevertheless, a substantial augmentation in VEGF expression was evident in stromal/decidual cells of group 2b when contrasted with groups 1 and 2a (see Fig. 7A, B).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eUterine receptivity is a hallmark event for successful implantation of the embryo. Poor uterine receptivity has been associated with infertility and abortion. In recent years, microRNAs (miRNAs) have been reported to be associated with embryo implantation, embryo development, decidualisation and infertility (\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17 CR18\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). It has been found that the expression of microRNAs differs between receptive and non-receptive endometrium, with Let-7, mir-23, miR-200, miR-30 and Mir-183 being more frequently associated with endometrial receptivity (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Previous studies have shown that injection of the miR-200 family into the horns of the uterus reduces the implantation of embryos (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, similar to previous studies, a significant reduction in the number of embryos was observed in the uterine horn of miR-200c-treated rats compared to sodium chloride-treated pregnant rats and untreated rats, in contrast, no significant difference was observed between the number of embryos in the uterine horn of rats treated with sodium chloride and the number of embryos in untreated rats.\u003c/p\u003e \u003cp\u003eThe our study was conducted for the purpose of elucidating the mechanism of action of miR-200c on implantation. An evaluation was therefore performed of the relationship between mirRNA200-c and particular molecules implicated in the implantation stages (αVβ3 integrin, LIF, IL-6, CD105 and VEGF), which were analysed by immunohistochemical methods in rats.. It has been demonstrated that a decline in the expression of these molecules, particularly in stromal cells, contributes to the molecular mechanism that underlies this adverse effect.\u003c/p\u003e \u003cp\u003eAs has been documented, αvβ3 integrin is expressed in the glandular and luminal epithelium of the uterus during the implantation window. A correlation has been demonstrated between its deficiency or absence and infertility (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). As demonstrated by numerous studies, the αvβ3 integrin is of significant importance in the process of decidualisation (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), It has been reported that this protein acts as a critical receptor for the attachment of the embryo to the uterus (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In the our study, the expression of αvβ3 in the luminal epithelium was found to be similar between groups. Nevertheless, a higher reaction was observed in stromal/desidual cells in Group 2b rats when compared to Group 1 and Group 2a. We demonstrated that the administration of microRNA-200c led to a decrease in αvβ3 integrin expression in both glands and stromal cells.\u003c/p\u003e \u003cp\u003eLIF plays an important role in the preimplantation development of the embryo, subsequent implantation and early pregnancy (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). LIF has been demonstrated to be involved in uterine regulation for implantation, blastocyst growth and development, embryo endometrial interaction, embryo implantation, decidualisation, uterine inflammatory response to implanted embryos and trophoblast invasion (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In a study conducted by Margıoula-Siarkou et al. in endometrial biopsy samples taken from infertile and fertile women, LIF expression in epithelial tissue was found to be statistically higher than in infertile women, while no difference was observed in stromal cells (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, in another study by Niknafs et al in 2019, it was reported that the highest LIF expression in endometrial tissue taken from rats on day 4 of pregnancy was observed in tissue taken on day 4 of pregnancy, with localised expression in epithelial tissue and to a lesser extent in stromal cells (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In the our study, LIF expression in the lumen and gland epithelium of group 2b endometrium was found to be weak compared to group 2a, whereas expression was found to be intense in stromal/deidual cells. The discrepancy between our findings and those of other studies may be attributable to the fact that our tissues were obtained from rats on the 6th day of gestation, at a stage preceding the decidualisation process. The our study found that administration of the miR-200c decreased LIF expression in stromal cells\u003c/p\u003e \u003cp\u003eInterleukin-6 is secreted from glandular and luminal epithelial cells of the endometrium throughout the menstrual cycle, especially during the mid-secretory phase and early stages of pregnancy. The secretion of this protein promotes successful embryo implantation and adequate placentation during the implantation period and early stages of pregnancy. Interl\u0026ouml;kin-6, menstr\u0026uuml;el siklus boyunca, \u0026ouml;zellikle de orta sekretuar faz ve gebeliğin erken d\u0026ouml;nemlerinde endometriyumun gland\u0026uuml;ler ve luminal epitel h\u0026uuml;crelerinden salgılanır. Bu proteinin salgılanması, implantasyon d\u0026ouml;nemi ve gebeliğin erken d\u0026ouml;nemlerinde başarılı embriyo implantasyonunu ve yeterli plasentasyonu teşvik eder (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The our study found that IL-6 expression in group 2a and group 2b was significantly different between the two groups. In group 2b, IL-6 expression in the lumen, gland and stromal cells was found to be statistically significantly higher than in group 2a. In conclusion, this study demonstrates that microRNA-200c administration suppresses IL-6 expression.\u003c/p\u003e \u003cp\u003eCD105 is a type 1 transmembrane glycoprotein that acts as a co-receptor for TGF-. CD105 has been implicated in mechanisms of vasculogenesis and angiogenesis (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). However, its functional role in uterine receptivity for embryo implantation is still unclear.In a study conducted by Chaddan in 2016, it was reported that CD105 expression was found in epithelial and stromal cells on day 3 of gestation, whereas stromal cells in the implanted region showed strong expression and stromal cells in the non-implanted region showed weak expression in tissue taken on day 6 of gestation. In our study, expression was found to be statistically higher in the endometrial stromal cells in group2b than in group2a. In contrast, expression levels in the luminal epithelium of group2b tissues were lower than those observed in group2a. The results of this study showed that microRNA-200c can inhibit CD105 expression in stromal cells (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVEGF is a crucial cytokine for successful embryo implantation. In rodents, the vascular permeability of the uterus increases in the blastocyst regions with the onset of the attachment reaction. This is followed by stromal decidualization and angiogenesis. VEGF has been identified as a mitogen for endothelial cells and an inducer of angiogenesis. It has been reported that VEGF mRNA is expressed in the luminal epithelium in the endometrium on days 1 and 2 of pregnancy, in stromal cells to a lesser extent on day 3 of pregnancy and in luminal epithelial cells, subepithelial cells and stromal region on day 4. Furthermore, the expression of VEGF mRNA has been observed in luminal epithelial and stromal cells immediately surrounding the blastocyst on the 5th day after the first binding reaction (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Our study revealed that the expression of VEGF was found to be slightly weaker in the lumen and glandular epithelium of group 2b uterine tissues compared to group 1 and gryup2a, but very intense in the stromal/decidual cells.\u003c/p\u003e \u003cp\u003eWE found that the application of microRNA 200c led to a decrease in the expression of αvβ3, LIF, IL-6, CD105 and VEGF, particularly in stromal cells, and even led to the negative expression of IL-6.\u003c/p\u003e \u003cp\u003eOnly a few studies to date have shown that high levels of miR-200c inhibit pinopod formation, which is important for endometrial receptivity (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), and play a role in decidualisation by affecting IL-5 expression in the stroma (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study was the first to investigate the relationship between miR-200c and integrin αvβ3, LIF, IL-6, CD105 and VEGF. Our results suggest that miRNA-200c prevents pregnancy by inhibiting decidualisation.\u003c/p\u003e \u003cp\u003eIn conclusion, it can be suggested that miRNAs can be used as markers or therapeutic targets in infertility treatments to increase endometrial receptivity and promote pregnancy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAyşe AKG\u0026Uuml;N and Nazlı \u0026Ccedil;İL did the experimentsG\u0026uuml;l\u0026ccedil;in ABBAN METE and M. Serkant UNAL wrote main manuscript text G\u0026uuml;l\u0026ccedil;in ABBAN METE prepared all the figures. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKim SM and Kim JS. 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Role of Vascular Endothelial Growth Factor (VEGF) in Human Embryo implantation: Clinical Implantation: Biomolecules 2021;11(2):253. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biom11020253\u003c/span\u003e\u003cspan address=\"10.3390/biom11020253\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bratislava-medical-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bratislava Medical Journal](https://link.springer.com/journal/44411)","snPcode":"44411","submissionUrl":"https://submission.springernature.com/new-submission/44411/3","title":"Bratislava Medical Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"miRNA-200c, α5β3, LIF, IL-6, CD105 (Endogolin), VEGF implantation","lastPublishedDoi":"10.21203/rs.3.rs-6594610/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6594610/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this study was to examine the impact of microRNA-200c on embryo implantation and the expressions of α5β3, LIF, IL-6, CD105 (endogolin), and VEGF, which have a role in implantation.\u003c/p\u003e \u003cp\u003eThe experimental model comprised 30 female and 4 male rats. The rats were divided into two groups: the first group comprised non-pregnant rats, while the second group comprised pregnant rats. The pregnant rats were then separated into two groups: an experimental pregnat group and a control pregnant group. In the experimental group, rats were administered a microRNA200-c mimic solution (50 \u0026micro;l of 0.9% sodium chloride) to the right uterine horn, while the left horn received an injection of 0.9% sodium chloride. The procedure was performed on day 3 of the pregnancy. The rats in the control group were not administered any treatment. On days 6 and 9 of gestation, the uteri were removed by cervical dislocation, after which the number of embryos was counted. The effects of α5β3, LIF, IL-6, CD105 (Endogolin), IL-6, CD105 (Endogolin) and VEGF proteins in the uterine tissues of the experimental pregnat groups were analysed using immunohistochemistry.\u003c/p\u003e \u003cp\u003eMiRNA-200c reduced the number of implanted embryos by suppressing the expression of α5β3 integrin, LIF, IL-6, CD105 (endogolin), and VEGF.\u003c/p\u003e \u003cp\u003eBased on these results, it can be concluded that mimic treatment may interfere with the process of implantation and early pregnancy by reducing the expression levels of In addition, integrin α5β3, LIF, IL-6, CD105 (Endogolin), VEGF.\u003c/p\u003e","manuscriptTitle":"The Role Of Micro-RNA200-c In Implantatıon Of Embryo To Endometriyum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 18:47:18","doi":"10.21203/rs.3.rs-6594610/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-13T06:42:02+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"45776371358632494101637213651326388070","date":"2025-06-01T12:31:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277558349646387524314341258100638560986","date":"2025-06-01T07:30:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-21T18:54:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-15T19:42:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99504223626501523806751634146320315469","date":"2025-05-15T07:13:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-15T07:01:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-05T13:57:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-05T13:54:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bratislava Medical Journal","date":"2025-05-05T12:43:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bratislava-medical-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bratislava Medical Journal](https://link.springer.com/journal/44411)","snPcode":"44411","submissionUrl":"https://submission.springernature.com/new-submission/44411/3","title":"Bratislava Medical Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7425c34a-fe2f-4c86-b7f9-6c0350876593","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T05:53:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-19 18:47:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6594610","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6594610","identity":"rs-6594610","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}