{"paper_id":"35a7a3f3-82ad-4672-abc5-b0e011907723","body_text":"Int. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3045 \nInternational Journal of Biological Sciences \n2025; 21(7): 3045-3060. doi: 10.7150/ijbs.98653 \nResearch Paper \nThe Therapeutic Potential of EGCG and Pro-EGCG in \nMitigating Ovarian Hyperstimulation Syndrome: \nUnraveling the Modulatory Mechanism through the \nVEGF Pathway  \nSijia Wang1,2#, Lanlan Fang2#, Luping Cong1, Qiongqiong Jia2,3, Waner Wu1, Mingpeng Zhao1, Tinchiu Li1, \nJacqueline Pui Wah Chung1, Ka Kei Fung1, Vivian Ching Man Lam1, Yingpu Sun2, Jung-Chien Cheng2, \nDavid Yiu Leung Chan1 \n1. Assisted Reproductive Technology Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, The Chinese University of Hong Kong, Hong \nKong SAR, 999077, China. \n2. Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, \nZhengzhou, 450003, China. \n3. The Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada. \n#These authors contributed equally to this work.  \n Corresponding authors: David Yiu Leung CHAN, Assisted Reproductive Technology Unit, Department of Obstetrics and Gynaecology, Faculty of Medicine, \nThe Chinese University of Hong Kong, Hong Kong SAR, 999077, China; E-mail: drdcyl16@cuhk.edu.hk. Jung-Chien CHENG, Center for Reproductive Medicine, \nHenan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450003, China ; E-mail: \njungchien.cheng@gmail.com. Yingpu SUN, Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated \nHospital of Zhengzhou University, Zhengzhou, 450003, China; E-mail: syp2008@vip.sina.com. \n© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (http s://creativecommons.org/licenses/by/4.0/). \nSee https://ivyspring.com/terms for full terms and conditions. \nReceived: 2024.05.20; Accepted: 2025.03.14; Published: 2025.04.22 \nAbstract \nOvarian hyperstimulation syndrome (OHSS) is a severe complication of controlled ovarian \nhyperstimulation (COH) during in vitro fertilization (IVF) treatment, characterized by increased capillary \npermeability. Vascular endothelial growth factor (VEGF) is a key mediator in OHSS, with serum VEGF \nlevels correlating with its severity. In this study, we investigated the therapeutic potential of \n(-)-epigallocatechin-3-gallate (EGCG) and its derivative, Pro -EGCG, in mitigating OHSS. Using both in \nvitro and in vivo models, including primary human granulosa-lutein cells, the human granulosa-like tumor \nKGN cell line, and a rat OHSS model induced with pregnant mare serum gonadotropin, we found that \nEGCG and Pro -EGCG significantly reduced OHSS progression. This was supported by histological \nanalyses, reductions in ovarian weight, and decreased VEGF expression at both transcriptomic and \nproteomic levels. Mechanistic studies revealed that EGCG and Pro-EGCG inhibit TGF-β-induced VEGF \nproduction through suppression of the TGF-β/Smad and PKA-CREB signaling pathways. RNA sequencing \nfurther validated the downregulation of VEGF expression following treatment. These findings highlight \nthe potential of EGCG as a novel adjuvant therapy for managing OHSS, providing a mechanistic basis for \nits clinical application. \nKeywords: OHSS, EGCG, VEGF pathway, OHSS animal model, RNA-Seq \nIntroduction \nOvarian Hyperstimulation Syndrome (OHSS) is \na severe complication associated with assisted \nreproductive technologies (ARTs), particularly in in \nvitro fertilization (IVF) procedures1. It is characterized \nby excessive ovarian enlargement, often accompanied \nby the formation of multiple ovarian cysts  and \nincreased vascular permeability. This leads to fluid \nextravasation into the abdominal and pleural cavities \n2, resulting in significant morbidity and, in severe \ncases, life -threatening complications. The severity of \n \nIvyspring  \nInternational Publisher \n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3046 \nOHSS varies widely, with symptoms ranging from \nmild to severe. The reported incidence rate ranges \nfrom 0.5% to 5%, depending on individual conditions \nand the treatment protocols employed\n3. Common \nclinical symptoms include abdominal pain, bloating, \nand respiratory distress. Severe cases may present \nwith ascites, pleural effusions, and electrolyte \nimbalances4. Although the precise etiology of OHSS \nremains unclear, it is primarily attributed to the \nstimulation of multiple ovarian follicles by exogenous \ngonadotropins. This overstimulation triggers \nexcessive vascular endothelial growth factor (VEGF) \nrelease, which increases capillary permeability5. \nVEGF plays a critical role in angiogenesis and \nvascular permeability 6,7. Elevated VEGF levels, \nmainly originating from hyperstimulated ovaries, \nincrease vascular permeability, causing fluid leakage \ninto the peritoneal and pleural cavities. This leads to \ncharacteristic OHSS symptoms such as abdominal \ndistension, ascites, and respiratory distress\n8. VEGF \nand its receptors are expressed in granulosa cells of \npreovulatory follicles and granulosa-lutein cells of the \ncorpus luteum 9-11. Notably, VEGF levels in follicular \nfluid surpass those in serum and correlate with OHSS \nseverity12,13. Previous studies have shown that human \nchorionic gonadotropin (hCG) increases VEGF \nexpression in human granulosa cells and elevates \nserum VEGF concentrations\n14-17. Significantly, both \nanimal models and human studies have highlighted \nthe therapeutic potential of targeting VEGF or its \nreceptors to prevent OHSS development18,19. \nEpigallocatechin gallate (EGCG), a major \nbioactive polyphenol found in green tea, has garnered \nsignificant attention due to its diverse biological \neffects and potential therapeutic applications in \nfemale reproductive diseases, including polycystic \novary syndrome (PCOS), endometriosis, and uterine \nfibroids\n20,21. EGCG is known for its antioxidant, \nanti-inflammatory, and anti-cancer properties, as well \nas its ability to modulate cell signaling pathways \ninvolved in cell proliferation, apoptosis, and \nangiogenesis \n22. Studies have demonstrated that \nEGCG inhibits VEGF and its receptor expression in \npathological processes such as gastric, ovarian, and \nbreast cancers \n23-26. EGCG octaacetate (Pro -EGCG ), a \nmodified form of  EGCG,  enhances its stability and \nbioavailability in vivo  \n27. However, there is limited \nresearch on the effects of EGCG on women \nexperiencing OHSS during IVF treatment, and the \nspecific mechanisms through which EGCG exerts its \neffects on OHSS remain unclear. In this study, we \ninvestigated the effects of EGCG on OHSS and its \nunderlying molecular mechanisms using in vitro  and \nin vivo models.  \nOur findings show that EGCG or its prodrug, \nPro-EGCG, attenuates OHSS development in a rat \nmodel. In KGN cells and primary human \ngranulosa-lutein (hGL) cells, both EGCG and \nPro-EGCG significantly reduce VEGF and VEGFR -2 \nexpression. Furthermore, EGCG inhibits \nTGF-β-induced VEGF production by suppressing the \ncanonical SMAD signaling pathway. Additionally, \nEGCG downregulates VEGF expression via the \n67-kDa laminin receptor -mediated PKA -CREB \npathway. These findings provide new insights into \nthe regulatory mechanisms underlying pathological \nangiogenesis in OHSS and suggest that EGCG and its \nderivatives could serve as potential therapeutic agents \nfor managing this condition. \nMaterials and methods \nCell cultures and reagents \nThe KGN cell line, a human granulosa -like \ntumor cell line, possesses functional \nfollicle-stimulating hormone receptors, and it was \nobtained from the laboratory of Prof. Weiyi Chen by \ncourtesy at the Chinese University of Hong Kong. \nPrimary human granulosa -lutein (hGL) cells were \nisolated via density centrifugation from follicular \nfluid collected from women undergoing oocyte \nretrieval at the IVF laboratory of the Prince of Wales \nHospital, Hong Kong. Both KGN and hGL cells were \ncultured in DMEM/F12 medium (Gibco) medium \nsupplemented with 10% charcoal/dextran- treated \nfetal bovine serum (FBS) (Gibco), along with \n100 U/mL penicillin and 100  μg/mL streptomycin \n(Gibco). The cell cultures were maintained at 37  °C in \na humidified atmosphere containing 5% CO2. \nRecombinant human TGF -β1 was procured from \nR&D Systems. The pregnant mare serum \ngonadotropin (PMSG) was obtained from Solarbio. \nAntibodies against caspase -3 and the 67 -kDa laminin \nreceptor were purchased from Abcam, VEGF \nantibody was acquired from Thermo Fisher Scientific, \nAntibodies against phospho -CREB, CREB, SMAD2, \nphospho-SMAD2, SMAD3, and phospho- SMAD3 \nwere obtained from Cell Signaling Technology. \nDetailed information regarding the antibodies used \nfor western blot analysis in this study is provided in \nSupplemental Table 1.  \nHuman follicular fluid collection \nThe studies involving clinical samples received \napproval from the Joint Chinese University of Hong \nKong - New Territories East Cluster Clinical Research \nEthics Committee (CREC Ref No: 2018.533) for the \ncollection of human follicular fluid and the primary \ngranulosa cells. Infertile women meeting specific \ncriteria were enrolled in the study after providing \n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3047 \nwritten informed consent between September 2021 \nand December 2022. Detailed information about the \novarian stimulation protocols is provided in a \nprevious study\n28. The inclusion criteria were as \nfollows: women aged 20 -35 years with a BMI of \n19-24.9, regular menstrual cycles, tubal factor \ninfertility or male factor infertility, and no \ncomplications such as diabetes or abnormal thyroid \nfunction. Exclusion criteria included polycystic \novarian syndrome (PCOS), endometriosis, diminished \novarian reserve, chromosomal abnormalities, or \nhydrosalpinx. Follicular fluid was collected during \noocyte retrieval, and hGL cells were isolated and \npurified using density centrifugation. \nCell intervention \nKGN and hGL cells were cultured in 6 -well \nplates (1 × 10 ⁶ cells/well) or 12 -well plates (5 × 10 ⁵ \ncells/well). When the cells reached approximately \n80% confluence, they were treated with various \nconcentrations of EGCG (Sigma -Aldrich, #E4143) or \nPro-EGCG (Abcam, #ab145182) or dimethyl sulfoxide \n(DMSO) (Sigma -Aldrich). DMSO was used as the \nvehicle control, as both EGCG and Pro- EGCG were \ndissolved in DMSO. To maintain consistency, the \ncontrol group received the same volume of DMSO as \nthe experimental groups. After treatment, cells were \nharvested for protein and RNA extraction. To \ninvestigate the effects of EGCG on the function of \nTGF-β, KGN cells were pre- treated with EGCG ( 10 \nµM, 25 µM ) for 24 h, followed by treatment with \nrecombinant human TGF-β. \nCell viability and proliferation assays \nKGN and hGL  cells were seeded into 12 -well \nplates at a density of 5 × 10⁴ cells per well or 96 -well \nplates at a density of 1 × 10 ⁴ cells per well. For \ncytotoxicity analysis, cells were allowed to reach \napproximately 80% confluence before being treated \nwith varying concentrations of EGCG or Pro-EGCG in \nserum-free F12/DMEM medium for specified \ndurations. For proliferation assays, treatments with \nvarying concentrations of EGCG or Pro -EGCG were \ninitiated immediately after seeding in F12/DMEM \nmedium supplemented with 10% FBS and continued \nfor the designated time points. For treatments \nexceeding 24 h, the culture medium was replenished \ndaily with fresh drugs at the same concentrations. \nCell viability and proliferation were assessed \nusing the MTT assay and cell counting methods. For \nthe MTT assay, a solution of MTT in sterile PBS (5 \nmg/mL) was prepared and stored at 4 °C, protected \nfrom light. A 10 µL aliquot of this MTT solution was \nadded to each well of a 96 -well plate, followed by \nincubation at 37 °C in the dark for 4 hours. After \nincubation, 100 µL of DMSO was added to each well \nto dissolve the formazan crystals. Absorbance at 570 \nnm was measured using a spectrophotometric \nmicroplate reader. For cell counting, images were \ncaptured using a Leica microscope for analysis and \ndocumentation. Live and dead cells were counted in \nsix random fields of view per well. The results were \nused to evaluate cell proliferation and viability under \nthe specified treatment conditions. \nWestern blotting analysis \nCells were harvested and lysed using RIPA lysis \nbuffer (Cell Signaling Technology) containing 1% \nprotease inhibitors (Boster) on ice. After \ncentrifugation, the supernatant was carefully \ncollected, and protein concentration was determined \nusing a BCA assay kit (Bio -Rad). Equal amounts of \nprotein were mixed with 5X SDS protein loading \nbuffer (Solarbio) and heated at 100°C for 10 min. \nProteins were separated by SDS -PAGE (Bio -Rad \nLaboratories) and transferred onto PVDF membranes \n(Bio-Rad Laboratories). Membranes were blocked for \n1 h in 5% non- fat dry milk dissolved in Tris -buffered \nsaline (TBS). After blocking, the membranes were \nincubated overnight at 4°C with primary antibodies \ndiluted in 5% non- fat milk -TBS. Following primary \nantibody incubation, membranes were washed five \ntimes with TBS containing 0.1% Tween-20 (TBST). The \nmembranes were then incubated with \nHRP-conjugated secondary antibodies for 1 h at room \ntemperature. Immunoreactive bands were visualized \nusing an enhanced chemiluminescent substrate \n(Bio-Rad Laboratories), and imaging was performed \nusing a ChemiDoc MP Imager (Bio-Rad Laboratories). \nReverse transcription-quantitative real-time \nPCR (RT-qPCR) \nTotal RNA was isolated and purified from cells \ncollected at the designated time points using the \nRNeasy Mini Kit (Qiagen) or TRIzol reagent \n(Invitrogen), following the manufacturer's \ninstructions. A 50 ng aliquot of RNA was reverse \ntranscribed into first -strand complementary DNA \n(cDNA) using the cDNA Synthesis Kit (Takara Bio). \nEach 20 μL qPCR reaction contained 1X TB Green \nPremix Ex Taq (Takara Bio), 20 ng of cDNA, and 0.8 \nμM of the appropriate primer pairs. RT -qPCR was \nperformed using an Applied Biosystems 7500 Fast \nReal-Time PCR System with a 96-well optical reaction \nplate. The primer sequences used are listed in \nSupplementary Table 1. Assay specificity was \nvalidated by performing a melting curve analysis and \nelectrophoresis of the PCR products on an agarose gel. \nAll RT -qPCR experiments were carried out in \ntriplicate. Negative controls included water and RNA \n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3048 \nsamples without reverse transcription. Relative \nmRNA quantification was calculated using the \ncomparative Ct method, with GAPDH as the \nreference gene, applying the 2\n–∆∆Ct formula. \nSmall interfering RNA (siRNA) transfection \nTo downregulate endogenous mRNA \nexpression, cells were transfected with 50 nM \nOn-TARGETplus SMARTpool siRNAs targeting \nspecific genes (Dharmacon). Transfection was \nperformed using Lipofectamine RNAiMAX \n(Invitrogen), following the manufacturer’s \ninstructions. A siCONTROL Non -TARGETING pool \nsiRNA (Dharmacon) was used as a control. After \ntransfection, cells were incubated for 48 hours before \nproceeding with the experimental procedures. The \nefficacy of gene knockdown was assessed by western \nblot analysis. \nRat OHSS model \nFemale Sprague -Dawley (SD) rats, aged three \nweeks and weighing between 32 -36 g, were obtained \nfrom the Laboratory Animal Services Center at The \nChinese University of Hong Kong for the study. The \nrats were housed in a controlled environment with \nfree access to food and water. Ethical approval for the \nanimal experiments was granted by The Chinese \nUniversity of Hong Kong Animal Experimentation \nEthics Committee.  \nA total of 32 female SD rats were randomly \nassigned to four experimental groups (n = 8 per \ngroup) using a computer -generated randomization \nsequence to minimize selection bias. The groups \nincluded a control group and three OHSS model \ngroups. OHSS was induced based on a modified \nversion of a previously established protocol to \nenhance reproducibility\n29. Specifically, 23 -day-old SD \nrats received intraperitoneal injections of pregnant \nmare serum gonadotropin (PMSG) at 10 IU/day for \nfour consecutive days. On the fifth day, human \nchorionic gonadotropin (hCG) was administered \nintraperitoneally at 10 IU to trigger ovulation. The \ncontrol group received a single injection of PMSG (7 \nIU) on the third day, followed by hCG (10 IU) 48 \nhours later. \nTo assess the therapeutic effects of EGCG and \nPro-EGCG, rats in the OHSS model groups were \ntreated with either vehicle control (DMSO) or \nEGCG/Pro-EGCG (10 mg/kg, i.p. ) from day 4 to day \n6. On the seventh day, all animals were euthanized \nunder anesthesia, and peripheral blood, ascitic fluid, \nand ovarian tissue samples were collected for further \nanalysis. Investigators responsible for sample \ncollection and data analysis were blinded to the group \nallocations to minimize potential bias. Body weights \nwere recorded every two days throughout the \nexperiment to monitor the health status of the \nanimals.   \nHistological staining and \nimmunohistochemistry (IHC) analysis \nOvarian tissue samples were obtained from \ndistinct cohorts of both control and OHSS rats. The \novarian tissues were fixed in 4% paraformaldehyde \nsolution (Sigma) and then embedded in paraffin. The \nembedded tissues were sectioned into 4 -μm-thick \nslices. These sections underwent a series of \npreparatory steps, including deparaffinization in \nxylene, rehydration through an ethanol gradient, \nantigen retrieval in citrate buffer (pH 9.0) by \nmicrowave treatment, and blocking of endogenous \nperoxidase activity with 3% hydrogen peroxide. Next, \nthe tissue sections were incubated overnight at 4°C \nwith anti-rat VEGF antibody at a 1:100 dilution. Prior \nto antibody incubation, a blocking step was \nperformed at room temperature for 20 min using 5% \nnormal goat serum. After washing with PBS, the \ntissue sections were visualized using the DAB \nSubstrate Kit (Dako, Denmark) and counterstained \nwith hematoxylin. Images were acquired using a \nmicroscope and specialized software. The staining \nintensity was evaluated semi-quantitatively using the \nH-score method, which takes into account both the \nintensity of staining and the proportion of positively \nstained cells. The H -score was calculated using the \nformula: H-score = Σpi(i+1), where ‘i’ represents the \nstaining intensity (1 = weak; 2 = moderate; 3 = strong), \nand ‘pi’ denotes the percentage of cells exhibiting each \nintensity (ranging from 0% to 100%). To ensure \nobjectivity, two independent researchers, blinded to \nthe experimental groups, performed the staining \nintensity assessment.   \nAssessment of vascular permeability using \nEvans Blue dye \nVascular permeability was assessed in rats via \nintravenous injection of Evans Blue dye \n(Sigma-Aldrich, #E2129)  following a standardized \nprotocol. A 5% Evans Blue dye solution was prepared \nby dissolving 20 mg of dye in 1 mL of sterile saline \nand filtering it through a 0.22- μm syringe filter to \nremove particulates. Rats were restrained, and the tail \nwas sterilized with 75% ethanol before injection. \nUsing a 30 G needle attached to a 1 mL syringe, 100 μL \nof the prepared dye solution was administered into \nthe tail vein. The dye was allowed to circulate for 30 \nmin to ensure uniform vascular distribution. After the \nincubation period, the rats were euthanized by \nintraperitoneal injection of a ketamine -xylazine \nmixture, following guidelines approved by the \n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3049 \nAnimal Experimentation Ethics Committee. To collect \nperitoneal fluid, 3 mL of sterile saline was injected \ninto the abdominal cavity and allowed to rest for five \nmin. A 1 mL aliquot of the peritoneal fluid was then \ncollected, transferred to a 1.5 mL microcentrifuge \ntube, and mixed with 200 μL of 0.1 M NaOH to \nprecipitate proteins. The samples were centrifuged at \n2000 rpm for 10 minutes at 4°C, and the supernatant \nwas transferred to a fresh tube. The absorbance of the \nsupernatant was measured at 620 nm using a \nmicroplate reader. A standard curve was generated \nfrom known concentrations of Evans Blue dye to \nquantify the dye content in each sample. Vascular \npermeability was calculated and compared between \nexperimental groups to evaluate differences in \nvascular leakage associated with OHSS.  \nMeasurement of rat serum VEGF: \nEnzyme-linked immunosorbent assay \nThe concentration of VEGF in rat serum samples \nwas determined using an enzyme -linked \nimmunosorbent assay (ELISA), following the \nmanufacturer’s instructions. The Rat VEGF ELISA kit \nwas purchased from R&D Systems (#RRV00). \nStatistical analysis \nData are presented as the mean ± SEM from at \nleast three independent experiments. Statistical \nanalyses were performed using Prism 8 software. For \ncomparisons between two groups, a t -test was used. \nFor comparisons among multiple groups, a one -way \nanalysis of variance (ANOVA) was performed, \nfollowed by Tukey's multiple comparison test. The \nassumptions were checked by assessing normality \nusing the Shapiro -Wilk test. Effect sizes were \nmeasured using Cohen’s d for t -tests and R² for \nANOVA.  A p -value of less than 0.05 was considered \nstatistically significant. \nResults \nEGCG inhibits VEGF expression \nIt has been documented that, in physiological \nconditions, the serum concentration of EGCG remains \nbelow 1µM\n30,31. However, with EGCG \nsupplementation, serum levels can elevate to as high \nas 7 µM\n32. To evaluate the effect of EGCG on Vascular \nEndothelial Growth Factor (VEGF), KGN cells were \ntreated with EGCG at concentrations ranging from 1 \nto 50 µM. As shown in Figure 1A, treatment with \nvarying EGCG concentrations for 24 hours did not \nsignificantly alter cell morphology. Cytotoxicity \nassays demonstrated that the inhibitory effect of \nEGCG on KGN cell proliferation depends on both \nconcentration and duration. Similarly, the cytotoxic \neffect of EGCG on KGN cells showed a dose - and \ntime-dependent pattern (Figure 1B). Lower \nconcentrations of EGCG appeared to confer resistance \nto cytotoxic effects, which may be attributed to \nantioxidant properties. Cell counting provided \nadditional validation, showing results consistent with \nthose obtained from the MTT assay (Figure 1C). These \nfindings suggest that at lower concentrations and \nshorter exposure times, EGCG within physiological \nlimits does not induce cytotoxic effects in KGN cells. \nCaspase-3, a critical executioner caspase, plays a key \nrole in the final stages of apoptosis, mediating DNA \nfragmentation, protein degradation, and membrane \nblebbing\n33. To confirm the apoptotic effect of EGCG in \nKGN cells, western blot analysis was performed to \nassess caspase-3 levels, with etoposide included as a \npositive control. As shown in Figure 1D, only \ntreatment with 50 µM EGCG significantly increased \ncaspase-3 expression, indicating apoptosis induction. \nWestern blot analysis revealed that EGCG at \nconcentrations of 1 and 5 µM had no effect on VEGF \nprotein levels. In contrast, treatment with 10 µM, 25 \nµM, and 50 µM significantly downregulated VEGF \nprotein expression after 24 hours (Figure 1E). These \nfindings were supported by RT -qPCR results, which \nshowed consistent inhibition of VEGF mRNA levels \nin KGN cells (Figure 1F). Previous research has \nhighlighted the critical role of VEGF and its receptor \nVEGFR-2 in OHSS -related angiogenesis\n19. \nInterestingly, EGCG reduced VEGFR -2 mRNA levels \nat concentrations of 5 µM and higher following 24 \nhours of treatment (Figure 1F). To investigate the \nmechanisms underlying the effect of EGCG on VEGF \nexpression, experiments were conducted using 25 µM \nEGCG. Western blot analysis confirmed significant \ninhibition of VEGF protein expression after 24 hours \n(Figure 1G). Additionally, RT -qPCR results showed \nthat EGCG reduced VEGF and VEGFR -2 mRNA \nlevels at both 12- and 24-hour time points (Figure 1H). \n67LR-mediated PKA-CREB activation is \nrequired for the EGCG-reduced VEGF \nexpression \nTo investigate the molecular mechanism \nunderlying EGCG-induced downregulation of VEGF \nexpression, it is crucial to determine whether EGCG \nexerts its regulatory effects through direct \nintracellular entry or by interacting with a membrane \nreceptor. The 67 -kDa laminin receptor (67LR) has \nbeen well -documented as a membrane receptor for \nEGCG, mediating its physiological functions\n34. \nPrevious studies have shown that 67LR is expressed \nin KGN cells and plays a critical role in \nEGCG-induced activation of the PKA-CREB signaling \npathway, which promotes StAR expression and \n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3050 \nprogesterone production in both KGN and hGL \ncells35. To evaluate the involvement of 67LR in \nEGCG-mediated VEGF suppression, specific siRNA \ntargeting 67LR (si-67LR) was used to knockdown the \nendogenous 67LR mRNA levels in KGN cells. \nKnockdown of 67LR partially mitigated the inhibitory \neffects of EGCG on VEGF protein levels (Figure 2A). \nThe protein kinase A (PKA)/cAMP -responsive \nelement-binding protein (CREB) signaling cascade is a \nwell-established pathway mediating VEGF \nexpression downstream of 67LR. To examine the \ninfluence of EGCG on CREB activation, \nphosphorylated and total CREB levels were assessed \nby western blot after EGCG treatment. EGCG \ntreatment increased CREB phosphorylation, with \nmaximal activation observed at 30 min (Figure 2B). \n \n \nFigure 1. EGCG inhibits VEGF expression. (A) KGN cells were treated with vehicle control (DMSO) or various concentrations of EGCG (1 µM, 5 µM, 10 µM, 25 µM, and \n50 µM) for 24 h, and cellular morphology was observed microscopically.  (B, C) Cell viability (B) and proliferation (C) were evaluated via MTT assay and cell counting, \n\n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3051 \nrespectively, following treatment with DMSO or EGCG at the indicated concentrations for 24, 48, and 72 h. (D) Western blot analysis of caspase-3 protein levels in KGN cells \ntreated with DMSO or EGCG (5 µM, 10 µM, 25 µM, and 50 µM) for 24 h. Etoposide (50 µM and 100 µM) was included as a positive control. (E, F) VEGF protein expression (E) \nand VEGF/VEGFR-2 mRNA levels (F) were determined by western blot and RT-qPCR, respectively, in KGN cells treated with DMSO or EGCG at the specified concentrations \nfor 24 h. (G, H) Time-course experiments were conducted to assess VEGF protein expression (G) and VEGF/VEGFR-2 mRNA levels (H). KGN cells were treated with DMSO \nor 25 µM EGCG for 3, 6, 12, and 24 h (G) or for 1, 3, 6, 12, and 24 h (H). Results are presented as the mean ± SEM from at le ast three independent experiments. Significant \ndifferences are denoted by asterisks (*p < 0.05, **p < 0.01). \n \nIn cells treated with si -67LR, EGCG- induced \nCREB activation was attenuated (Figure 2C). To \nfurther investigate the role of the CREB pathway in \nEGCG-mediated VEGF suppression, the PKA \ninhibitor H89 was employed. Pre- treatment of KGN \ncells with H89 abolished the suppressive effects of \nEGCG on VEGF protein levels (Figure 2D). To confirm \nthe necessity of CREB and rule out potential off-target \neffects of pharmacological inhibitors, CREB \nexpression was downregulated using specific siRNA \n(si-CREB). Knockdown of CREB inhibited the \nsuppressive effect of EGCG on VEGF protein levels \n(Figure 2E). Finally, to confirm the role of PKA in the \nCREB pathway, KGN cells were pre-treated with H89, \nwhich significantly blocked the phosphorylation of \nCREB, as shown by western blot analysis (Figure 2F). \nThese results collectively highlight the requirement of \n67LR-mediated PKA -CREB activation for \nEGCG-induced downregulation of VEGF expression \nin KGN cells. \nEGCG inhibits VEGF expression via TGF-β \nsignaling pathways  \nTGF-β, a multifunctional cytokine involved in \nvarious physiological and pathological processes, \nincluding angiogenesis and tissue remodeling, plays a \nkey role in stimulating VEGF expression and secretion \nin human granulosa -lutein cells via the classical \nSMAD signaling pathway\n36. This stimulation \nenhances angiogenesis and vascular permeability, \nsuggesting a potential contribution to OHSS. The \nclassical SMAD pathway is initiated when TGF -β \nligands bind to cell surface receptors, such as the \nTGF-β type 2 receptor (TβRII). This interaction \ntriggers the phosphorylation of receptor -regulated \nSMADs (R-SMADs), including SMAD2 and SMAD3. \nThe phosphorylated R -SMADs form complexes with \nSMAD4 and translocate into the nucleus, where they \ninteract with transcriptional partners to regulate gene \nexpression\n37. EGCG has been shown to inhibit TGF -β \nsignaling by directly interacting with TβRII 38. This \ninteraction plays a crucial role in modulating TGF -β \nsignaling pathways and has potential therapeutic \nimplications for managing OHSS. Molecular docking \nanalysis, illustrated in Figure 3A, demonstrates the \npotential binding position of EGCG with TβRII. \nPrevious research confirmed that treating KGN cells \nwith TGF-β for 3 hours significantly increased VEGF \nexpression\n36. However, pre- treatment with varying \nconcentrations of EGCG prior to TGF -β exposure \nsignificantly reduced VEGF expression at both the \nmRNA and protein levels (Figure 3B). This \nsuppression highlights the effectiveness of EGCG in \ncounteracting the TGF -β-induced increase in VEGF. \nThe activation of TGF -β involves phosphorylation of \nSMAD2 and SMAD3, which subsequently bind to the \ncommon mediator SMAD4. Western blot analysis \nconfirmed the phosphorylation and activation of \nSMAD2 (Figure 3C) and SMAD3 (Figure 3D) \nfollowing TGF-β treatment. Pre-treatment with EGCG \nsignificantly reduced the levels of phosphorylated \nSMAD2 and SMAD3, demonstrating its inhibitory \neffect on their activation. To further explore the role of \nthe classical SMAD signaling pathway in the \nreduction of TGF -β-induced VEGF expression by \nEGCG, endogenous SMAD4 expression was silenced \nusing specific siRNA (si -SMAD4). Silencing SMAD4 \nattenuated the ability of EGCG to suppress VEGF \nexpression, confirming the involvement of SMAD4 in \nthis regulatory mechanism. \nEGCG and Pro-EGCG exhibit similar cytotoxic \neffects and effectively reduce the expression of \nVEGF and VEGFR-2 \nEGCG is known to exhibit poor bioavailability \ndue to low absorption, rapid metabolism, and \nreduced stability. To address these limitations, a \nmodified variant, Pro -EGCG, has been developed, \noffering improved stability, bioavailability, and \nbiological activity in vivo . Pro -EGCG has \ndemonstrated significant inhibitory effects on the \ndevelopment, growth, and angiogenesis of \nexperimental endometriosis in mice. In this study, the \neffects of EGCG and Pro -EGCG on KGN cell \nproliferation were assessed using the MTT assay at \nvarious concentrations and time points (Figure 4A). \nEGCG effectively suppressed KGN cell proliferation, \nparticularly at higher concentrations and longer \ndurations. Notably, Pro -EGCG exhibited an even \nstronger proliferation-suppressive effect compared to \nEGCG. The cytotoxicity of EGCG and Pro -EGCG on \nKGN cells was also evaluated using the MTT assay \n(Figure 4B). EGCG showed cytotoxic effects starting at \n25 µM after 48 hours. In contrast, Pro -EGCG \ndemonstrated significant cytotoxicity at lower \nconcentrations and shorter durations, highlighting its \ngreater potency.  \n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3052 \n \nFigure 2. 67LR -mediated PKA-CREB activation is required for EGCG -induced reduction in VEGF expression. (A)  KGN cells were transfected with 50 nM \ncontrol siRNA (si-Ctrl) or 67LR siRNA (si -67LR) for 48 h, followed by treatment with 25 µM EGCG for 24 h. Protein levels of VEGF, 67 -kDa laminin receptor (67LR), and \nα-Tubulin were analyzed by western blot. (B) Phosphorylated and total CREB protein levels were assessed via western blot in KGN cells treated with 25 µM EGCG for 30 or \n60 min. (C) KGN cells were transfected with 50 nM si-Ctrl or si-67LR for 48 h and then exposed to 25 µM EGCG for 30 min. Protein levels of phosphorylated and total CREB, \nalong with 67LR, were examined by western blot. (D) KGN cells were pre-treated with vehicle control (DMSO) or 1 µM H89 for 1 h before treatment with 25 µM EGCG for \n24 h. Protein levels of VEGF and α-Tubulin were measured via western blot. (E) KGN cells were transfected with 50 nM si-Ctrl or CREB siRNA (si-CREB) for 48 h, followed by \ntreatment with 25 µM EGCG for 24 h. Western blot analysis was performed to evaluate protein levels of VEGF, CREB, and α-Tubulin. (F) Phosphorylated and total CREB protein \nlevels were evaluated by western blot in KGN cells pre-treated with DMSO or 1 µM H89 for 1 h, followed by treatment with 25 µM EGCG for 30 min. Results are presented \nas the mean ± SEM from at least three independent experiments. Significant changes are indicated by asterisks (*p < 0.05, **p < 0.01). \n\n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3053 \n \nFigure 3. EGCG inhibits VEGF expression via TGF-β  signaling pathways. (A) Molecular docking analysis demonstrates the interaction between EGCG and TGF-β \nreceptor type II (TβRII). (B) KGN cells were pre-treated with vehicle control (DMSO), 10 µM EGCG, or 25 µM EGCG for 24 h, followed by stimulation with 5 ng/mL TGF-β \nfor 3 h. VEGF and α-Tubulin protein levels were analyzed by western blot (left panel). VEGF mRNA levels were determined by western blot and RT-qPCR (right panel). (C) KGN \ncells were pre-treated with DMSO or 25 µM EGCG for 24 h, then treated with 5 ng/mL TGF-β for 30 min. Western blot analysis was performed to assess phosphorylated and \ntotal SMAD2 protein levels, with α-Tubulin serving as a loading control. (D) Phosphorylated and total SMAD3 protein levels were also analyzed under the same conditions using \nwestern blot. (E) KGN cells were transfected with 50 nM control siRNA (si-Ctrl) or SMAD4 siRNA (si-SMAD4) for 48 h, followed by exposure to 25 µM EGCG for 30 min. \nProtein levels of VEGF, SMAD4, and α-Tubulin were evaluated by western blot. Results are presented as the mean ± SEM of at least three independent experiments. Significant \ndifferences are indicated by asterisks (*p < 0.05, **p < 0.01). \n \nThe regulatory effects of Pro -EGCG on VEGF \nand VEGFR-2 expression in KGN cells were examined \nin a concentration- dependent manner over 24 hours \n(Figure 4C). Compared to the inhibitory effects of \nEGCG on VEGF and VEGFR-2 (Figure 1F), Pro-EGCG \nexhibited greater efficiency, suppressing VEGF and \nVEGFR-2 RNA expression at lower concentrations. \nThe time-dependent effects of EGCG and Pro -EGCG \non VEGF expression were also evaluated at the same \nconcentration (Figure 4D). Both compounds inhibited \nVEGF expression at all time points; however, EGCG \nreached its maximum effect at 48 hours, while \nPro-EGCG achieved similar results within 24 hours. \nBoth compounds suppressed VEGFR -2 expression \nacross all time points in a time -dependent manner. \nFurther analysis was conducted on primary human \ngranulosa cells treated with DMSO, EGCG, or \nPro-EGCG. These treatments significantly reduced \nVEGF and VEGFR -2 expression at both the RNA \n(Figure 4E) and protein levels (Figure 4F). \nEGCG or Pro-EGCG alleviate OHSS in rats \nTo further explore the modulatory effects of \nEGCG and Pro -EGCG on OHSS pathogenesis, we \ndeveloped an OHSS rat model and evaluated these \ncompounds as potential treatments. Consistent with \nprevious findings, induction of OHSS led to \nsignificant ovarian enlargement and increased \novarian weight\n39,40. Treatment with EGCG or \nPro-EGCG effectively alleviated the severity of these \nsymptoms (Figures 5A -C). Histological analysis \nconfirmed a higher number of corpora lutea in OHSS \nrats compared to the control group, consistent with \nearlier studies40. Treatment with EGCG or Pro -EGCG \nsignificantly reduced the number of corpora lutea in \nOHSS rats (Figures 5D, 5E).  \n\n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3054 \n \nFigure 4.  EGCG and Pro -EGCG exhibit similar cytotoxic effects and effectively reduce VEGF and VEGFR -2 expression. (A) KGN cells were treated with \nvehicle control (DMSO) or varying concentrations of EGCG (upper panel) or Pro-EGCG (lower panel) for 0, 24, 48, and 72 h. Cell viability was assessed using the MTT assay. \n(B) Cytotoxicity was further evaluated under the same conditions as in (A) using the MTT assay. (C) KGN cells were treated with DMSO or different concentrations of \nPro-EGCG for 24 h, and VEGF and VEGFR-2 mRNA levels were quantified by RT-qPCR. (D) KGN cells were exposed to DMSO, 10 µM EGCG, or 10 µM Pro-EGCG for 24, \n48, and 72 h. VEGF and VEGFR-2 mRNA levels were analyzed by RT-qPCR. (E) Primary hGL cells were treated with DMSO, 10 µM EGCG, or 10 µM Pro-EGCG for 24 h, and \nVEGF and VEGFR-2 mRNA levels were determined by RT-qPCR. (F) Protein levels of VEGF in primary hGL cells were examined by western blot after treatment with DMSO, \n10 µM EGCG, or 10 µM Pro-EGCG. Results are presented as the mean ± SEM from at least three independent experiments. Significant differences are denoted by asterisks (*p \n< 0.05, **p < 0.01). \n\n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3055 \n \nFigure 5. EGCG and Pro-EGCG alleviate OHSS development in rats. (A) Representative images of ovaries from each experimental group. (B, C) Ovarian weight (B) \nand ovarian weight normalized to body weight (C) were measured after euthanasia. (D)  Representative H&E -stained ovarian sections. Images were captured at 400× \nmagnification; scale bars represent 50 μm. (E) Percentage of corpora lutea in ovarian sections was quantified (n = 4 per group). (F) Representative IHC staining of ovarian tissues \nfor VEGF, with images captured at 400× magnification; scale bars represent 50 μm. (G) Quantitative analysis of VEGF protein expression in ovarian tissues based on IHC staining. \nVEGF expression was evaluated as positive staining intensity. (H-J) mRNA levels of VEGF (H), VEGFR-2 (I), and TGF-β (J) in rat ovaries were quantified by RT-qPCR. (K) Levels \nof Evans Blue dye in peritoneal fluid were measured at OD620 nm across different groups. (L) Serum VEGF protein levels were quantified using ELISA. Results are presented as \nthe mean ± SEM from at least three independent experiments. Significant differences are indicated by asterisks (*p < 0.05, **p < 0.01). \n\n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3056 \nImmunohistochemistry revealed a marked \nupregulation of VEGF expression in granulosa cells, \nluteal cells, and stromal cells within the ovaries of \nOHSS rats. Administration of EGCG or Pro -EGCG \nsignificantly attenuated VEGF expression in these \novarian tissues (Figures 5F, 5G). RT -qPCR analysis \nfurther demonstrated that VEGF mRNA levels were \nelevated in the ovaries of OHSS rats. This upregula -\ntion was significantly suppressed by treatment with \nEGCG or Pro -EGCG (Figure 5H). Similarly, mRNA \nlevels of VEGFR -2 and TGF -β were significantly \nincreased in OHSS rat ovaries but were effectively \nreduced following treatment with EGCG or Pro - \nEGCG (Figures 5I, 5J). To validate the effects of EGCG \nand Pro-EGCG on vascular permeability in the OHSS \nmodel, Evans Blue dye was used. Vascular \npermeability was significantly elevated in OHSS rats \ncompared to controls. Treatment with EGCG or \nPro-EGCG significantly reduced this increase in \nvascular permeability (Figure 5K). Lastly, VEGF levels \nin rat serum were quantified using an ELISA assay. \nSerum VEGF levels were significantly higher in OHSS \nrats, consistent with the pathogenesis of the condition \n(Figure 5L). Importantly, administration of EGCG or \nPro-EGCG led to a marked reduction in serum VEGF \nlevels. \nRNA sequencing results in KGN cells Treated \nwith EGCG or Pro-EGCG \nTo validate the transcriptomic changes in KGN \ncells following treatment with EGCG and Pro-EGCG, \nwe subjected these KGN cells treated by EGCG or \nPro-EGCG to RNA-Seq analysis. The top 200 upregu -\nlated and downregulated genes were represented in \nFigure 6A. The Venn diagram illustrates that 11,258 \ngenes were co -expressed across different groups \n(Figure 6B). The Principal Component Analysis (PCA) \nresults of the samples highlight the robustness and \nreliability of the observed differences in gene \nexpression among the control and treated groups \n(Figure. 6C). The statistics of the number of \ndifferential genes (including up -regulation and \ndown-regulation) for each compare group and the \nthreshold for screening are shown in Figure 6D. \nComparing the EGCG- treated group to the control \ngroup, the results revealed that 627 genes were \nup-regulated, while 964 genes were down -regulated. \nIn the case of the Pro- EGCG-treated group compared \nto the control group, 1062 genes were up -regulated, \nwhile 1416 genes were down -regulated. These \nfindings provide insights into the specific gene \nexpression alterations induced by EGCG and \nPro-EGCG treatments in relation to the control group. \nThe volcano plot depicting the comparison of gene \nexpression levels between EGCG -treated and control \ncells was highlighted the expression of VEGFa (Figure \n6E), same in volcano plot depicting the comparison of \ngene expression levels between Pro -EGCG treated \nand control cells (Figure 6F). GO, an acronym for \nGene Ontology, represents a prominent bioinforma -\ntics framework aimed at standardizing the delineation \nof gene attributes across diverse species. Comprising \nthree primary categories, namely cellular component, \nmolecular function, and biological process, GO facili -\ntates a comprehensive understanding of gene func -\ntionalities. Following a rigorous GO enrichment ana -\nlysis, the top 30 GO Terms, deemed most statistically \nsignificant, were meticulously curated for presenta -\ntion and analysis (Figure 6E, 6G). The RNA -seq ana-\nlysis provides further confirmation that both EGCG \nand Pro -EGCG treatments significantly reduce the \nexpression of VEGF, corroborating our earlier study. \nDiscussion \nWhile the occurrence of severe OHSS is \nrelatively infrequent, it persists as a substantial and \nconcerning complication associated with in vitro  \nfertilization procedures. The incident of OHSS is \nheavily monitored by all local ART regulatory bodies. \nThe pathophysiology of OHSS, although extensively \nstudied, remains enigmatic, resulting in predomi -\nnantly empirical and anticipatory clinical manage -\nment approaches. A range of strategies has been \ndeployed to preclude the onset of OHSS, including \nreducing gonadotropin dosages, employing GnRH \nantagonists for ovulation triggering, implementing \ncryopreservation techniques, considering cycle \ncancellation, and others\n41-43. Nonetheless, none of \nthese approaches have proven entirely effective in \nproviding comprehensive protection against the \ndevelopment of OHSS. Consequently, the pursuit of \nan optimal pharmaceutical intervention to proactively \nforestall this potentially life -threatening complication \nremains a paramount challenge within the domain of \nassisted reproductive technologies. Recognizing the \ncentral involvement of VEGF in OHSS pathogenesis, \ninterventions aimed at VEGF modulation have been \nharnessed as a preventive measure against this \nsyndrome\n19. To the best of our knowledge, this is the \nfirst study to demonstrate the therapeutic potential of \nEGCG and its derivative, Pro -EGCG, in alleviating \nOHSS, providing novel evidence of their efficacy in \nboth in vitro and in vivo models. Our findings reveal \nthat EGCG treatment suppresses VEGF production by \ninhibiting the TGF-β/Smad and PKA-CREB signaling \npathways, highlighting a previously unrecognized \nmechanism. These results establish EGCG as a \npromising adjuvant therapy for OHSS, offering a \nstrong mechanistic foundation for its potential clinical \napplication. \n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3057 \n \nFigure 6. RNA-sequencing results in KGN cells treated with EGCG and Pro-EGCG. (A) Heatmap displaying the top 200 most upregulated and downregulated genes. \n(B) Venn diagram illustrating co-expression of genes across different treatment groups. (C) Principal component analysis (PCA) of all samples to visualize variance and clustering. \n(D) Bar chart showing the number of differentially expressed genes (DEGs) in each group comparison.  (E) Volcano plot depicting gene expression differences between \nEGCG-treated and control cells. (F) Volcano plot showing gene expression differences between Pro-EGCG-treated and control cells. (G) GO (Gene Ontology) enrichment \nanalysis scatter plot for EGCG -treated versus control cells, highlighting GO terms with significant enrichment (padj < 0.05). (H) GO enrichment analysis scatter plot for \nPro-EGCG-treated versus control cells, showing significantly enriched GO terms (padj < 0.05). \n\n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3058 \nThis study delved into the therapeutic potential \nof EGCG and its modified form, Pro -EGCG, as agents \nfor OHSS treatment. EGCG is a naturally occurring \ncompound found in green tea and has a \nwell-established safety profile, making it a promising \ncandidate for therapeutic use. Its accessibility and \ncost-effectiveness contribute to its appeal as a \npotential treatment. Investigations revealed that \nEGCG exerts inhibitory effects on key factors such as \nVEGF and its receptor VEGFR-2, which are implicated \nin the pathogenesis of OHSS. This suggests that \nEGCG may help mitigate the angiogenic and vascular \npermeability processes responsible for OHSS \ndevelopment. In essence, the multifaceted benefits of \nEGCG, including safety, affordability, and capacity to \ntarget key factors in OHSS pathophysiology, \nunderscore its potential as a valuable therapeutic \napproach for addressing this complex syndrome. \nA pivotal aspect of the study involved \ndeciphering the mechanisms through which EGCG \nexerts an inhibitory effect on VEGF expression. \nFindings indicated that EGCG interacts with the 67LR \nand subsequently triggers the protein kinase A \n(PKA)/cAMP-responsive element -binding protein \n(CREB) signaling pathway, resulting in the \nsuppression of VEGF expression. While previous \nresearch suggested that TGF -β stimulation can \nenhance VEGF expression through the classical \nSMAD signaling pathway, this study, for the first \ntime, shed light on the specific molecular events \nthrough which EGCG inhibits VEGF in KGN cells and \nprimary human granulosa cells. The interaction \nbetween TGF -β and EGCG was investigated to \nunderstand how TGF -β might be involved in EGCG \neffects. TGF-β is a multifunctional cytokine known to \nparticipate in various physiological and pathological \nprocesses, including angiogenesis and tissue \nremodeling. Previous studies confirmed that EGCG \nbinds to TGF -β type II receptor (TβRII), effectively \nhindering TGF-β actions by disrupting its interaction \nwith TβRII in MRC -5 cells\n38. Many studies also \ninvestigated EGCG inhibitory effects on TGF -β \nthrough SMAD or ERK1/2 signaling pathways 44,45.  \nThis study establishes a novel link between \nEGCG capability to inhibit VEGF expression and the \nclassical SMAD signaling pathway associated with \nTGF-β. The results suggest that EGCG capacity to \ninteract with TGF -β receptors, particularly TβRII, \nenables it to interfere with the activation of SMAD2 \nand SMAD3. This interference disrupts their \ntranslocation into the nucleus, where they typically \nengage with transcriptional partners to regulate gene \nexpression. Consequently, this interference results in \ndownstream effects on the expression of genes \npertinent to angiogenesis, such as VEGF and its \nreceptor VEGFR -2, both of which are pivotal in the \ncontext of OHSS pathogenesis. This elucidation of the \nprecise mechanisms underpinning VEGF modulation \nprovides a foundational understanding of the \npotential therapeutic effects of EGCG in OHSS. \nPro-EGCG demonstrated robust proteasome \ninhibition and induction of cell death in cancer cells\n46. \nIts potency surpassed that of EGCG in inhibiting \nproliferation, transforming activity, and inducing \napoptosis in various human cancer cell types, \nincluding breast, prostate, leukemic, and simian virus \n40-transformed cells\n47. Consistent with previous \nresearch, this study revealed that Pro-EGCG exhibited \nsuperior efficacy compared to EGCG in inhibiting \nproliferation and inducing cytotoxicity in KGN cells. \nThese findings further support the notion that \nPro-EGCG possesses potent cell death -inducing \nproperties and outperforms EGCG in terms of \nefficiency. Additionally, this study confirmed that \nPro-EGCG effectively inhibited the expression of \nVEGF and its receptor in KGN cells. Notably, the \ninhibitory effects on VEGF and its receptor were \nobserved at lower concentrations compared to EGCG, \nproviding further evidence of its enhanced efficiency. \nPrevious studies demonstrated that Pro -EGCG and \nEGCG do not affect ovarian follicles in mice ovaries, \nsuggesting that this natural anti -angiogenic agent \nmay not disrupt normal ovulation\n27. Consequently, \nPro-EGCG emerges as a stable and potent green tea \npolyphenol with the potential to serve as a novel \nanti-angiogenic agent for OHSS. \nThe time -dependent inhibitory effects of EGCG \nand Pro-EGCG on VEGF and VEGFR -2 expression in \nKGN cells are noteworthy. The differential optimal \ntime points for each compound indicate that their \nmechanisms of action may involve distinct pathways \nand kinetics. Therefore, further investigations are \nwarranted to elucidate the specific mechanisms of \naction for Pro- EGCG in the context of OHSS. \nAdditionally, due to the limitations imposed by the \nsample size in this study, additional experiments are \nneeded to confirm the effects of Pro-EGCG in primary \nhGL cells. Interestingly, the study revealed that \nPro-EGCG, despite its enhanced stability and \nbioavailability, did not exhibit a significantly stronger \ntherapeutic effect in the OHSS animal model when \ncompared to EGCG. This unexpected finding raises \nimportant questions about the nuanced interactions of \nthese compounds with the complex biological \npathways implicated in OHSS. Potential explanations \nfor this observation could include differences in \npharmacokinetics, tissue distribution, or specific \ninteractions with the OHSS pathogenic cascade. \nFurther research in these areas will provide a more \ncomprehensive understanding of the therapeutic \n\nInt. J. Biol. Sci. 2025, Vol. 21 \n \n \nhttps://www.ijbs.com \n3059 \npotential and underlying mechanisms of Pro-EGCG in \nthe management of OHSS. \nDespite their potential, EGCG and Pro -EGCG \nhave several limitations. The poor bioavailability and \nrapid metabolism of EGCG may restrict its clinical \nutility27,48, while the long -term safety and \npharmacokinetics of Pro- EGCG remain insufficiently \nexplored. Both compounds exhibit dose -dependent \ncytotoxicity and broad-spectrum activity on signaling \npathways, including PI3K/AKT and MAPK, which \nraises concerns about potential off -target effects 49. \nAdditionally, translating findings from animal \nmodels to human applications presents challenges \ndue to interspecies differences in drug metabolism \nand ovarian physiology. Future research should focus \non conducting clinical trials to assess the safety, \nefficacy, and pharmacodynamics of Pro- EGCG in \nhuman populations. \nIn conclusion, our study highlights the \ntherapeutic potential of EGCG for mitigating OHSS \nand elucidates its mechanisms of action, particularly \nits modulation of VEGF expression. While Pro -EGCG \noffers improved stability and potency, its similar \nefficacy to EGCG in OHSS warrants further \ninvestigation. These findings contribute to the \ngrowing understanding of OHSS pathogenesis and \noffer a foundation for refining therapeutic strategies \nin assisted reproductive technologies. \nSupplementary Material \nSupplementary tables.  \nhttps://www.ijbs.com/v21p3045s1.pdf \nAcknowledgements \nFunding \nWe would like to thank the Hong Kong OG \nTrust Fund (Ref 6906795 and 6904985) and the Hong \nKong government for their support through the Hong \nKong Research Matching Grant (RMG01 -8601386) for \nthis basic clinical research. This work was also \nsupported by operating grants from the National \nNatural Science Foundation of China (32070848, \n32371169 to Lanlan Fang, and 32170868 to Jung-Chien \nCheng). Special thanks to our IVF team members for \ntheir invaluable support in advancing innovative \nresearch. \nAuthor contributions \nDLYC, JCC, and YPS conceived and supervised \nthe study. SW and LLF wrote the original draft of the \nmanuscript. TL, JPWC, and DLYC revised the \nmanuscript. 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