Serum Reproductive Hormone Profiles and Endometrial Expression of miR-16-5p, VEGF Gene, and Protein, and CD31-Positive Cells following Progesterone and EGCG Administration.

According to the ELISA findings, the EGCG group exhibited the lowest progesterone levels. Therefore, the administration of EGCG reduced serum progesterone concentrations, and this reduction was statistically significant compared to all other groups ( P < 0.05). The highest progesterone levels were observed in the progesterone group, with statistically significant differences when compared to the EGCG ( P < 0.05) and progesterone + EGCG ( P < 0.05) groups [ Figure 2a ]. Serum concentrations of progesterone (a) and estradiol (b), Endometrial expression of miR-16-5p (c), and VEGE gene (d). Letters a, b, and c denote statistically significant differences compared with the control (Co) group, progesterone (Pro) group, and EGCG group, respectively. Data are presented as mean ± SD, with statistical significance set at P < 0.05 As illustrated in Figure 2b , the ELISA results demonstrated that administration of EGCG or progesterone significantly reduced estradiol levels in the serum of mice compared to the control group ( P < 0.05), with the lowest estradiol concentrations observed in the progesterone + EGCG group. A significant difference was noted between the progesterone group and the progesterone + EGCG group ( P 0.05), nor between the progesterone + EGCG group and the EGCG group ( P > 0.05). IHC results demonstrated that the lowest VEGF protein belonged to the EGCG group, which was significantly different from that of the control group ( P < 0.05). The progesterone group showed maximum protein level and in comparison with the control group, it was statistically different ( P < 0.05). VEGF protein level was lower in the progesterone + EGCG group compared with the control group. It was statistically significant ( P < 0.05). In western blot results, the EGCG group showed lower protein levels compared to the progesterone group ( P < 0.05) and control group ( P < 0.05). The western blot analysis also indicated that the VEGF protein concentration was significantly higher in the progesterone group than in the control group ( P 0.05). In both the IHC and western blot results, a statistically significant difference was observed between the progesterone + EGCG group and the progesterone group ( P < 0.05). Similarly, a significant difference was observed between the progesterone + EGCG group and the EGCG group ( P < 0.05). Overall, the IHC and western blot revealed that EGCG decreased VEGF protein while progesterone elevated the expression of VEGF protein, Figure 3 . VEGF protein expression in the endometrium. Immunohistochemical images (a1) and western blot bands (b1) depicting VEGF protein expression. Quantitative analysis of VEGF levels in IHC (a2) and western blot (b2). Letters a, b, and c denote statistically significant differences compared with the control (Co) group, progesterone (Pro/P) group, and EGCG (e) group, respectively. Data are presented as mean ± SD, with statistical significance defined as P < 0.05 As shown in Figure 2c , the real-time PCR results demonstrated that miR-16-5p was upregulated in the progesterone group compared to the control group ( P < 0.05). The EGCG group showed the lowest miR-16-5p levels, which were statistically significant compared to both the control and progesterone + EGCG groups ( P 0.05). However, in the progesterone + EGCG group, miR-16-5p levels were significantly reduced compared to the progesterone group ( P < 0.05) and enhanced compared to the EGCG group ( P < 0.05). According to the real-time PCR results, the lowest and highest VEGF gene levels belonged to the EGCG group and progesterone group but it was not statistically significant compared to the control groups ( P > 0.05). In general, the statistical analysis indicated that no significant difference was found in the comparisons between the groups, Figure 2d . Immunohistochemistry for CD31, a marker for endothelial cells, indicated that the number of CD31-positive cells was significantly higher in the progesterone group compared to the control group ( P 0.05). The EGCG group exhibited the lowest number of CD31-positive cells. This reduction was statistically significant compared to the control group ( P < 0.05), the progesterone group ( P < 0.05), and the progesterone + EGCG group ( P < 0.05), as shown in Figure 4 . Endothelial cell density in the endometrium. (a) Immunohistochemical images showing endothelial cells. (b) Quantitative analysis of endothelial cell density. Letters a, b, and c denote statistically significant differences compared to the control group, progesterone group, and EGCG group, respectively. Data are presented as mean ± SD, with statistical significance established at P < 0.05

The present study suggests that alterations in the natural hormone profile induced by progesterone and EGCG, an anti-angiogenic factor, may influence the endometrial expression of miR-16-5p, leading to post-transcriptional targeting of the VEGF gene. So during the implantation window, this angiomiR may affect VEGF protein expression, promoting endothelial cell proliferation and affecting endometrial receptivity, although additional distinct biological pathways could also be involved and further research is needed to fully understand the underlying processes and confirm these findings. The current research received approval from the Ethics Committee of Isfahan University of Medical Sciences (Isfahan, Iran), with reference number IR.MUI.MED.REC.1400.606. All procedures involving animals were carried out in full compliance with the established guidelines for the care and use of laboratory animals. Data and materials are available upon request by contacting the corresponding author ([email protected]). There are no conflicts of interest.

AngiomiRs, a subclass of microRNAs, exert post-transcriptional regulation on genes associated with angiogenesis, either cell-autonomously or non-cell-autonomously. Considering the significance of angiogenesis in the receptivity of the endometrium during the implantation window, angiomiRs may stand out as potentially vital determinants in female fertility.[ 9 11 ] In this study, the alteration in natural hormone profiles, including progesterone and estradiol, following the administration of exogenous progesterone, was found to upregulate the endometrial expression of miR-16-5p when compared to the control group. In contrast, the EGCG group exhibited a decrease in miR-16-5p expression. It has been demonstrated that microRNA expression can be altered by cyclical changes in hormone profiles across the menstrual cycle.[ 34 ] Progesterone can induce upregulation of several mature miRNAs in mouse endometrial epithelial cells. These include mmu-let-7c-5p, mmu-let-7f-5p, mmu-let-7b-5p, mmu-let-7a-5p, mmu-miR-29a-3p, mmu-miR-1a-3p, mmu-miR-21a-5p, mmu-let-7d-5p, mmu-let-7e-5p, mmu-let-7g-5p, mmu-miR-192-5p, and mmu-miR-320-3p.[ 35 ] The association between miR-200a/miR-141 and miR-205 with hormone receptor status in endometrial cancer has also been reported. The dysregulated expression of miR-22, known as anti-implantation miRNA, and its target genes Tiam1 (T-lymphoma invasion and metastasis 1) and Race1 (Rac family small GTPase 1) has been associated with a reduction in the progesterone/estradiol (P/E2) ratio in patients with recurrent implantation failure.[ 36 37 ] Treatment of endometrial adenocarcinoma (Ishikawa) cell line with progesterone increased relative expression levels of miR-340-5p, miR-42-3p, and miR-671-5p. Progesterone also boosts miR-133a and reduces miR-200 expression in the endometrium.[ 38 39 40 ] Previous research strongly indicates that EGCG has the ability to modulate the expression of various microRNAs. Notable changes in the expression levels of microRNAs, including hsa-miR-125a-3p, hsa-miR-15b-3p, hsa-miR-548av-3p, hsa-miR-125a-3p, hsa-miR-500a-3p, hsa-miR-7706, and hsa-miR-15b-3p, have been observed in non-small-cell lung cancer cells following treatment with EGCG.[ 41 ] Green tea and EGCG can affect the expression of let-7b. EGCG has also been shown to upregulate the expression of miR-210, which plays a crucial role in regulating AKT, MAP kinases, and the cell cycle.[ 29 42 ] It appears that no study has investigated the effect of EGCG on the endometrial expression of miR-16-5p while Previous investigations have elucidated the expression patterns of miR-16-5p following ovulation stimulation and progesterone administration. However, these studies did not determine the serum levels of reproductive hormones.[ 32 43 ] In the current study, the regimen involving EGCG led to decreased concentrations of progesterone and estradiol. The precise mechanism by which EGCG affects the expression of these hormones has not yet been explored. We also demonstrated that compared to the control group, direct supplementation of progesterone resulted in a notable increase in serum progesterone concentration. Concurrently, the levels of estradiol exhibited a decline in the P group. It suggests that progesterone may have a negative feedback effect on estrogen levels. As shown in Figure 1b , researchers have demonstrated that estrogen and progesterone play a role in regulating miRNA production. miRNAs are initially transcribed by RNA polymerase II, resulting in the formation of a precursor molecule known as primary miRNA (pri-miRNA). In the nucleus, the RNase III enzyme Drosha, along with its partner DGCR8, processes the pri-miRNA by cleaving it, producing a shorter precursor miRNA (pre-miRNA).[ 1 2 ] Research has revealed that elevated estrogen receptor levels are linked to increased expression of both Drosha and DGCR8. However, other studies indicate that estrogen alone enhances DGCR8 expression without influencing Drosha. In contrast, progesterone appears to have minimal or no significant effect on the expression of either Drosha or DGCR8.[ 44 45 46 ] Exportin-5 transports pre-miRNA to the cytoplasm, where it is processed by Dicer into double-stranded miRNA. Estrogen and progesterone have been suggested to enhance the expression of both Exportin-5 and Dicer-1.[ 3 4 45 46 ] The miRNA duplex is transported to the RNA-induced silencing complex (RISC), where it is processed into a mature, single-stranded miRNA enabling it to play a crucial role in regulating gene expression.[ 47 ] EGCG has not been thoroughly studied to elucidate the potential mechanisms by which it reduces the expression of angiomirs or miRNAs in general. This effect may be associated with its influence on hormone levels. However, additional research is needed. In this study, compared to the control group, the Progesterone group showed a significant increase in VEGF protein levels, as determined by Western blot and IHC. Conversely, a notable decrease in VEGF protein levels was observed in the EGCG group. VEGF is expressed in the endometrial luminal and glandular epithelium as well as stromal cells.[ 48 ] Studies have indicated that progesterone regulates endometrial receptivity via VEGF and other growth factors, and nuclear progesterone receptors in endometrial cells trigger VEGF production.[ 49 50 ] EGCG influences endometrial angiogenesis-related factors such as growth factors VEGF and VEGFR2.[ 51 ] Furthermore, EGCG can target specific receptors, including the insulin-like growth factor-1 (IGF-1) receptor in the stroma and luminal and glandular endometrial epithelium.[ 52 53 ] It also targets the FAS receptor (CD95) in stromal cells.[ 54 55 ] It can also inhibit the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptors (VEGFRs).[ 56 57 58 ] Interference with these receptors by EGCG could lead to decreased VEGF levels in the endometrium. The exact molecular mechanism underlying the alteration in VEGF protein expression and CD31-positive cell density following the administration of progesterone and EGCG is not completely clear. It is suggested that microRNAs may play a crucial role in this process. It has been reported that VEGF is a direct target for miR-16-5p ( http://mirtarbase.mbc.nctu.edu.tw/php/index.php ). In the canonical and original model of miRNA function, the RISC typically binds to the 3′ untranslated region (UTR) of target mRNAs, initiating translational repression but recent studies have also revealed that miRNAs can interact with other regions of the target mRNAs, such as 5′ UTR and coding sequence (CDS) region. Both negative and positive regulatory effects of miR-16-5p on VEGF protein expression have been shown.[ 32 43 59 ] Additionally, the competing endogenous RNA (ceRNA) hypothesis may be involved in these results. lncRNAs, that are target for miR-16-5p, may act as ceRNAs and compete with mRNA of other targets for binding to miR-16-5p.[ 60 ] However, more studies are required to reveal the role ceRNA and lncRNA-miRNA–mRNA network in the context of endometrium. Furthermore, Wnt, Bcl2, Jagged1, and Fgf2 are associated with the VEGF protein-related pathways. Studies have revealed that they are also targets for miR-16-5p, suggesting an indirect regulation of VEGF protein expression. Additionally, alterations in other miRNAs may have impacted VEGF protein, it is indicated that during non-natural cycles, the expression of certain miRNAs targeting VEGF, including miR-34-5p, miR-423-5p, miR-34b, miR-503, miR-520g, miR-369-3p, and miR-186, is altered,[ 61 62 ] so further investigation is necessary. The present study reveals that, unlike the VEGF protein, the expression of the VEGF gene did not show statistically significant differences across the various groups examined. The mRNA abundance of a gene does not serve as a reliable indicator of its protein expression level. Tian et al .[ 63 ] investigated protein and mRNA levels in multipotent EML cells and MPRO cells, finding that 35% of genes showed significant protein changes without mRNA alterations, and 1% exhibited discordant mRNA-protein expression patterns. The results of the Schwanhäusser et al .[ 64 ] study also demonstrates that proteins were, on average, 900 times more abundant than their corresponding mRNAs and So, diverse regulatory mechanisms operated at post-transcriptional and translational levels. Therefore, it appears that VEGF is regulated post-translationally, by microRNAs. In the present study, the maximum and minimum levels of endothelial cell density, miR-16-5p, VEGF protein, and gene expression belonged to the progesterone group and EGCG group, respectively, that were statistically significant compared with the control group. VEGF and VEGF receptors are important for endothelial cell survival, proliferation, apoptosis, and vascular permeability. These are also essential for endometrial receptivity by promoting angiogenesis and vascular permeability.[ 48 65 ] Studies revealed that the activation of nuclear progesterone receptors induced endothelial cell proliferation. It also modulates junctional proteins such as CDH5, PECAM-1, and CLDN5, which play a crucial role in facilitating endothelial cell proliferation and migration. Conversely, during the implantation window, EGCG can regulate the expression of genes associated with apoptosis and autophagy and inhibit the proliferation and migration of endometrial endothelial cells. It is also demonstrated that altered levels of steroid hormones could potentially alter molecular patterns in the endometrium, impacting uterine receptivity. This alteration might involve changes in miRNA profiles and VEGF protein expression.[ 50 66 67 ]

Forty mature female and twenty adult male mice (NMRI, 10–12 weeks, 25–40 g) were housed in a home. Throughout the study, these mice were maintained under standard conditions, which included a constant room temperature of 22°C, a 12-h light and 12-hour dark cycle, and unrestricted access to food and water. The mice were randomly divided into four groups: 1) Control group (CO): No intervention was made. 2) Luteal phase support or progesterone group (P): The mice were given progesterone (Aburaihan Co-Iran) at a dose of 1 mg/mouse IP daily for 3 days, starting 72 h after day 0.[ 32 ] 3) EGCG group (E): The mice were treated with EGCG (Sigma-Aldrich, E4143) at a dose of 5 mg/kg IP at 0, 24, 48, and 72 h.[ 26 ] 4) progesterone + EGCG (PE): The mice were given progesterone at a dose of 1 mg/mouse IP daily for 3 days, starting 72 h after day 0. Additionally, they received EGCG at a dose of 5 mg/kg IP at 0, 24, 48, and 72 h0 after day 0 [ Figure 1a ]. (a) Overview of the experimental design. A summary of the different experimental groups, the specific drug injection protocols used for each group, and the methods employed for assessing various markers. (b) Estrogen and progesterone effects on microRNA synthesis. EP5, exportin-5; TFs, transcription factors; ceRNA, competing endogenous RNA; CDS, coding sequence; GTP, guanosine triphosphate; Ran: small GTPase Ran As shown in Figure 1a , in each experimental group, a pair of two female mice and one male mouse underwent overnight mating within a designated cage. The confirmation of successful mating relied on the detection of sperm in a vaginal smear the subsequent morning, which was subsequently designated as the first day of pregnancy. Just before implantation, the mice were humanely sacrificed under anesthesia, blood collection was performed via cardiac puncture, and then approximately one-third of the middle uterine horns were excised for further analysis. The right uterine horns were meticulously fixed in 10% neutral buffered formalin to facilitate immunohistochemistry (IHC) investigations. In contrast, the left uterine horn samples were rapidly immersed in liquid nitrogen to preserve their molecular integrity for subsequent western blot and real-time PCR analyses.[ 32 ] All analyses were conducted under blind conditions. Blood was centrifuged for 10 min at 4,000 rpm and the serum was separated. Serum estradiol and progesterone were measured using an ELISA kit (Monobind –USA) with a sensitivity of 8.2 pg/mL and 0.1 ng/mL, respectively. All protocols for hormone assay were done based on the kit’s instructions.[ 33 ] IHC for VEGF protein Immunohistochemical staining was utilized to determine the level of VEGF protein expression and quantify the density of CD31-positive cells in the endometrium. Uterine samples were fixed in a solution of 10% natural buffered formalin, followed by dehydration in ethanol, embedding in liquid paraffin, and sectioning into 4-μm thick slices. Subsequently, these sections were deparaffinized using xylene and rehydrated through descending concentration gradients of alcohols. Antigen retrieval was achieved through boiling in Tris-buffered saline at pH 9. To block endogenous peroxidase activity, the slides were immersed in 3% hydrogen peroxide. Nonspecific binding was prevented by incubating the sections in a solution of 5% bovine serum albumin at room temperature. The next step involved an overnight incubation of the sections with a mouse monoclonal anti-VEGF primary antibody (Diagnostic Biosystems: PDM165) at 4°C. Afterward, the samples were treated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (Protaqs: 300155400) in accordance with the manufacturer’s guidelines. To visualize the results, the sections were counterstained with Harris’ hematoxylin. Subsequently, images were captured using a light microscope (Olympus Corporation 40×) and analyzed using ImageJ software (NIH, MD, USA). Parameters were adjusted manually within the software, and all evaluators were blinded to maintain objectivity.[ 26 ] IHC for CD31 For identifying endothelial cells, CD31 was employed as a marker in a similar immunohistochemical procedure. The mouse monoclonal anti-CD31 antibody (Zytomed system: BMS044) was applied following the aforementioned methodology.[ 26 ] The quantification of VEGF protein levels in the endometrium was carried out utilizing the western blot technique. After protein extraction with radioimmunoprecipitation assay (RIPA) buffer, protein concentration was determined using a BCA assay (Thermo Fisher Scientific, USA). Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and blocked with 5% skim milk. Membranes were incubated overnight with an anti-VEGF antibody (Biorbyte -Orb191500), followed by horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam- AB6721). Results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam-181602), and protein bands were visualized with ECL western blotting detection system from GE Amersham, UK, and their densitometry was subsequently analyzed using the ImageJ software developed by NIH, MD, USA.[ 32 ] The quantification of miR-16-5p and VEGF gene expression was carried out through real-time PCR analysis. Total RNA extraction from the endometrium tissues was conducted in accordance with the TRIzol Reagent instructions from Rojetechnology Co-Iran. The RNA purity was subsequently assessed using the NanoDrop 2000 system from Thermo Scientific (USA). Subsequently, cDNA was synthesized using the RT-PCR Pre-Mix Kit sourced from Bio Fact-Korea. For real-time PCR, the Real-Time PCR Master Mix Kit (Bio Fact- Korea) was used along with specific primers [ Table 1 ]. Data analysis was performed using the 2 −ΔΔCt method. Primer sequences for real-time PCR In this study, statistical analyses were conducted using SPSS version 29 (IBM, New York, NY, USA). A one-way ANOVA test was employed to evaluate differences between the groups, followed by Tukey’s post hoc test for multiple comparisons. All data were presented as the mean ± standard deviation (SD), and a P value of < 0.05 was considered statistically significant.