{"paper_id":"a68ac281-a80a-4e86-ae79-a3aaa570d006","body_text":"Vol.:(0123456789)\nReproductive Sciences (2025) 32:2375–2387 \nhttps://doi.org/10.1007/s43032-025-01913-8\nREPRODUCTIVE BIOLOGY: ORIGINAL ARTICLE\nEffect of Nifedipine in Preventing Ovarian Hyperstimulation Syndrome \nthrough TRPC1 Ion Channel Inhibition\nEmel Kocal1  · Remzi Atilgan1  · Şehmus Pala1  · Melike Aslan2  · Tuncay Kuloğlu2  · Nevin Ilhan3  · \nEbru Etem Önalan4  · Serhat Hançer2  · Gizem Kaymaz Bircan4 \nReceived: 4 January 2025 / Accepted: 9 June 2025 / Published online: 18 June 2025 \n© The Author(s) 2025\nAbstract\nOvarian hyperstimulation syndrome (OHSS) is a life-threatening complication that usually develops as a result of triggering \novulation with human chorionic gonadotropin (hCG) after gonadotropin treatment, and in whose pathophysiology vascular \nendothelial growth factor (VEGF) and inflammatory mediators play a role. Nifedipine, used especially in the treatment of \nhypertension, is a calcium channel blocker. Nifedipine also has anti-inflammatory effects via transient receptor potential \ncanonical (TRPC1) ion channel inhibition. VEGF also regulates the angiogenic process through TRPC channels. In our study, \nwe investigated the potential of nifedipine to prevent OHSS due to its TRPC1 blocking effect and anti-inflammatory effects. \nA total of 28 rats were randomly divided into four equal groups. Group (G) 1 control group (n = 7). Rats in G2 (n = 7) were \nadministered 30 IU pregnant mare serum gonadotropin for 4 days and OHSS was induced by administering 30 IU hCG on the \nfifth day. Rats in G3 (n = 7) were induced to have OHSS and were given 100 μg/kg oral cabergoline, while rats in G4 (n = 7) \nwere induced to have OHSS and were given 20 mg/kg intraperitoneal nifedipine. On the fifth day, all rats were decapitated \nand VEGF, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and hypoxia-inducible factor (HIF)-1α levels were \nmeasured in their serum and tissues. TRPC1 gene expression and immunohistochemical analysis were performed in ovarian \ntissue. We showed that nifedipine inhibited VEGF and some inflammatory factor levels more than cabergoline. We showed \nthat nifedipine may achieve these effects through TRPC1 blockade and suppression of inflammatory factors.\nKeywords OHSS · Rat · Nifedipine · TRPC1 · VEGF\nIntroduction\nFluid leakage into the third space as a result of increased \ncapillary permeability is the hallmark of OHSS [1 ], a \ncomplication that typically arises as a result of infertility \ntherapies and occasionally has a potentially fatal potential. \nHistopathological alterations brought on by OHSS in the \novaries include increased luteal follicle cysts, necrosis and \nneovascularization, edema, and ovarian enlargement [2 ]. \nThe American Society for Reproductive Medicine (ASRM) \nclassifies OHSS into three categories: mild, moderate, or \nsevere, based on the presence of OHSS symptoms. Moderate \nto severe OHSS occurs in approximately 1–5% of in vitro \nfertilization (IVF) cycles with an incidence of up to 20% \nin high-risk patients [3, 4]. It has been reported that 11,562 \ncases were hospitalized due to OHSS in the United States \nfrom 2002 to 2011, and approximately 4.4% of these cases \nexperienced life-threatening complications [5 ].\nResearch has demonstrated a connection between OHSS \nand elevated vascular permeability as a result of proinflam-\nmatory cytokine production brought on by hCG-induced \novulation [6 ]. Furthermore, it has been demonstrated that \nvascular endothelial growth factor (VEGF) and a variety \nof angiogenic substances and cytokines contribute to the \ndevelopment of OHSS [7]. VEGFs are produced by granu-\nlosa cells as a result of gonadotropin stimulation, and their \nproduction increases significantly after hCG administration. \n * Remzi Atilgan \n remzi_atilgan@hotmail.com\n1 Department of Obstetrics and Gynecology, Firat University \nSchool of Medicine, Elazig, Turkey\n2 Department of Histology and Embriology, Firat University \nSchool of Medicine, Elazig, Turkey\n3 Department of Biochemistry, University School of Medicine, \nElazig, Turkey\n4 Department of Medical Biology, Firat University School \nof Medicine, Elazig, Turkey\n\n2376 Reproductive Sciences (2025) 32:2375–2387\nIn addition, other systemic and local vasoactive substances, \nincluding interleukin (IL)−2, IL-6, IL-8, IL-10, IL-18, \nangiotensin II, histamine, prolactin, prostaglandins, insulin-\nlike growth factor (IGF) 1, and transforming growth fac-\ntor (TGF) b, are also directly and indirectly involved in the \npathogenesis of OHSS [4, 8–10]. Hypoxia-induced factor-1 \nalpha (HIF-1α) is an essential transcription factor and plays \na critical role in almost all processes of wound healing and \nvascular remodeling [11, 12]. It has been reported that over-\nexpression of HIF-1α can activate the VEGF/AKT/mTOR \nsignaling pathway by increasing VEGF expression and lead \nto increased p-AKT and p-mTOR levels [13]. According to \nsome research, reducing vascular permeability—typically by \ninhibiting VEGF secretion—can stop the development and \nprogression of OHSS [1, 14, 15]. In this work, we examined \nhow the ancient medication nifedipine affected OHSS by \ninhibiting VEGF secretion via the transient receptor poten-\ntial canonical (TRPC1) ion channel. Through pro-angiogenic \nmolecules like VEGF and basic fibroblast growth factor, \nendothelial TRPC channels regulate the angiogenic process \nby delivering calcium ions [16]. Through VEGF and basic \nfibroblast growth factor, among other factors, TRPC1 has \nbeen demonstrated to control angiogenesis [17]. Nifedipine \nis a vasoselective calcium channel blocker that inhibits volt-\nage-dependent L-type calcium channels in cells and stops \ncalcium ions from entering cells. It is particularly used to \ntreat hypertension. Oral administration of nifedipine results \nin nearly total absorption. Nifedipine's main adverse effects \ninclude headache, palpitations, burning and redness in the \nface and legs, and ankle edema from skin vascular dilata-\ntion [18–20].\nMaterials and Methods\nThe Fırat University Experimental Animals Research Center \nconducted this randomized controlled experimental study in \ncompliance with ethical guidelines after receiving approval \nfrom the Fırat University, Faculty of Medicine Experimental \nAnimals Ethics Committee on February 27, 2023. The study \nused 28 female Spraque-Dawley rats that weighed 45–60 g \nand were 22–23 days old. FÜDAM supplied and provided \naccommodation for the animals. The rats were housed in \nspecially designed cages with city water and pellet meal, \nmaintained at room temperature between 22 and 25 °C, and \nexposed to 12 h of light (7:00–19:00) and 12 h of darkness \n(19:00–7:00). The bottoms of the cages were cleaned daily.\nAccording to the principles of laboratory animal care, \nall research animals were cared for in accordance with the \nguidelines for the care and use of animals that our insti-\ntutions have approved (NIH Guide for the Care and Use \nof Laboratory Animals, Institute of Laboratory Animal \nResources, National Research Council, Washington, D.C.). \nTwenty-two-day-old female rats (weighing 45–50 g) were \nrandomly divided into 4 groups.\nGroup (G) 1 (control, n = 7) = group given 0.1 ml saline \nintraperitoneally (ip) for five consecutive days (between days \n22 and 26).\nG2 (OHSS group, n  = 7) = 30 IU pregnant mare serum \ngonadotropin (PMSG) (Folligon®-Intervet; Schering-\nPlough Animal Health, Pune, India) was given subcutane-\nously for 4 consecutive days and 30 IU hCG (Chorulon®-\nIntervet; Schering-Plough Animal Health Boxmeer, The \nNetherlands) was given on the fifth day to induce severe \nOHSS (Severe OHSS protocol).\nG3 (n = 7) = Severe OHSS induced and given 100 μg/\nkg cabergoline [(Dostinex – Pharmacia and Upjohn SpA, \nMilan, Italy) dissolved in 5% glucosal] orally on the day of \nhCG administration [21].\nG4 (n = 7) = Severe OHSS induced and given 20 mg/\nkg intraperitoneal nifedipine (Adalat – 20 Tablets, Bayer \nHealthcare, New Zealand) in saline solution on the day of \nhCG administration [22].\nOn the 27th day, the rats were given 80 mg/kg ketamine \n(Ketalar, Eczacıbaşı, İstanbul, Turkey) and 20 mg/kg xyla-\nzine (Rompun Vet, Bayer AB, İstanbul, Turkey) to induce \nanesthesia and open their abdomens. Following their com-\nplete removal, the ovaries were promptly weighed on a pre-\ncision scale, and their respective weights were noted. Next, \n3–4 cc of blood were extracted from each rat's right ventricle \nand placed in gel biochemistry tubes. High-dose anesthetic \nwas used to put the rats to sleep after the blood collection \nprocedure. Up until the day of the trial, right ovarian tissue \nwas kept at −80 °C for genetic and biochemical analysis. \nFor immunohistochemistry analysis, 10% formaldehyde was \nused to fix the left ovary tissue, which was then embedded \nin paraffin blocks. The consort of the experimental study is \nshown in Fig.  1.\nImmunohistochemical Examination\nSections from paraffin blocks that were 4–6 μm thick were \nput on polylysine slides. For antigen retrieval, deparaffinized \ntissues were boiled in a citrate buffer solution at a pH of 6 \nfor 15 min in a 750 W microwave oven after passing through \na graded alcohol series. The tissues were boiled and then \nallowed to cool for about 20 min at room temperature. To \nstop endogenous peroxidase activity, they were incubated \nwith hydrogen peroxide block solution (Hydrogen Peroxide \nBlock, TA-125-HP, Lab Vision Corporation, USA) for five \nminutes after being cleaned for 3 × 5 min with PBS (Phos-\nphate Buffered Saline, P4417, Sigma-Aldrich, USA). After \n5 min of applying Ultra V Block (TA-125-UB, Lab Vision \nCorporation, USA) solution to prevent background stain-\ning, tissues were incubated with a 1/200 diluted TRPC1, \nfor 60 min at room temperature in a humid environment. \n\n2377Reproductive Sciences (2025) 32:2375–2387 \nFollowing the primary antibody application, the tissues \nunderwent 3 × 5 min PBS washes before being incubated for \n30 min at room temperature in a humid environment with the \nsecondary antibody (biotinylated Goat Anti-Polyvalent (anti-\nmouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, \nUSA). Tissues were incubated with Streptavidin Peroxidase \nFig. 1  The consort of the experimental study\n\n2378 Reproductive Sciences (2025) 32:2375–2387\n(TS-125-HR, Lab Vision Corporation, USA) for 30 min at \nroom temperature in a humid environment after the second-\nary antibody was applied. They were then rinsed with PBS \nfor 3 × 5 min each time. Once the image signal was collected \nunder a light microscope, the tissues were simultaneously \nwashed with PBS and 3-amino-9-ethylcarbazole (AEC) \nSubstrate + AEC Chromogen (AEC Substrate, TA-015 and \nHAS, AEC Chromogen, TA-002-HAC, Lab Vision Corpora-\ntion, USA). The tissues were washed with PBS and distilled \nwater, counterstained with Mayer's hematoxylin, and then \ncoated with the proper covering solution (Large Volume \nVision Mount, TA-125-UG, Lab Vision Corporation, USA). \nUnder a Leica DM500 microscope, the created preparations \nwere inspected, assessed, and captured on camera (Leica \nDFC295).\nHistoscore was created based on the prevalence (0.1: \n< 25%, 0.4: 26–50%, 0.6: 51–75%, 0.9: 76–100%) and sever-\nity (0: none, + 0.5: very little, + 1: little, + 2: moderate, + 3: \nsevere) of immunoreactivity in staining. Histoscore = preva-\nlence x severity [6]. The explanation scheme about immuno-\nhistochemistry examination is shown in Fig.  2.\nQuantitative Real Time Polymerase Chain Reaction \n(qRT‑PCR)\nVariations in the levels of TRPC1 gene mRNA expression \nin ovarian tissues were found using the qRT-PCR technique. \nA tissue sample weighing 30–40 mg was isolated using Tri-\nzolTM Reagent (Cat no. 15596018, Thermo Fischer Sci-\nentific). Thirty microliters of diethylpyrocarbonate (DEPC) \nwater were used to dissolve the resulting RNA pellet. A nan-\nodrop device (BioSpec-nano, Shimadzu) was used to meas-\nure the quantities of RNA. The lowest RNA value read was \naccepted as the standard value to guarantee equal RNA lev-\nels in the complementary DNA (cDNA) synthesis stage. A \ntotal volume of 10 μL was used for cDNA synthesis, which \nincluded 5 μL of RNA sample, 1 μL of 10 × RT Buffer, 0.5 \ndNTP mix (2.5 mM), 1 μL of Random Hexamer (50 μM), \n0.5 μL of Reverse Transcriptase, 0.25 μL of RNase Inhibitor, \nand 1.75 μL of Nuclease-free H2O.\nAfter placing in the thermal cycler, the samples were \nmaintained at 25 °C for 10 min, 37 °C for 120 min, 85 °C \nfor 5 min, and 4 °C for the final temperature. The samples \nwere placed in the thermal cycler and kept in the device \nfor 10 min at 25 °C, 120 min at 37 °C, 5 min at 85 °C and \nthe final temperature was 4 °C. cDNAs obtained by reverse \ntranscription were used to identify rat-specific β-actin \n(housekeeping or control gene) with the names Rn-β-actin \nforward 5ˈ-AGC CAT GTA CGT AGC CAT CC-3ˈ and 5ˈ-TCG \nGAA CCG CTC ATT GCC G-3ˈ [23] and 5ˈ -TTC CAA AGA \nGCA GAA GGA CTG-3ˈ and 5ˈ-AGG TGC CAA TGA ACG \nAGT G-3ˈ primers for TRPC1 were amplified by qRT-PCR \nin triplicate [24]. One microliter of cDNA sample, 5 µL of \n2 X Magic SYBR Mix (Procomcure Biotech GmbH), 0.5 \nµL of Forward Primer (Sentegen), 0.5 µL of Reverse Primer \n(Sentegen), and 3 µL of Nuclease-free H2O were added to \neach well to generate the qRT-PCR combination. The plate \nwas then covered with optical adhesive film.\nUsing the Applied Biosystems 7500 RT-PCR system, it \nwas amplified at 95 °C for 5 min, 95 °C for 10 s, 58 °C for \n30 s, 72 °C for 30 s, and 72 °C for 2 min. Differences in gene \nexpression resulting from qRT-PCR were calculated using \nthe 2-∆∆CT technique.\nBiochemical Study\nSerum was isolated from blood samples that were collected \nin plain tubes and centrifuged for 10 min at + 4 °C at 4000 \nrpm. Eppendorf tubes were used to separate the serum sam-\nples, which were then kept at −80 °C until the day of analy-\nsis. Samples removed from −80 0C were rapidly thawed at \n25 ℃ in a shaking water bath (Köttermann labortechnik, \ntype 3047, Germany) and biochemical analyses were per -\nformed. After removing the entire ovarian tissue, the tis-\nsues (1:9; w:v) were placed in tubes with 0.01 M phosphate \nbuffer (PBS; pH 7.4) and homogenized for 3 min at 4 °C \nand 16,000 rpm. The resulting homogenates were separated \nfrom the supernatants after being centrifuged at 5000xg for \n15 min at + 4ºC. The amount of protein in the supernatant \nwas measured by measuring the blue complex produced \nby proteins at 650 nm using Folin-Phenol reagent in alka-\nline medium. Using the Enzyme-Linked Immuno Sorbent \nAssay (ELISA), the levels of VEGF, TNF-α, IL-1β, IL-6, \nand HIF-1α were assessed in serum and supernatants. \nValues per milligram of protein were computed from the \nsupernatant results. VEGF, HIF-1α, TNF-α, IL-1β, and \nIL-6 levels in tissue and serum were examined using the \nappropriate ELISA kit protocols. The EPOCH 2 microplate \nreader (BioTek Instrument, Inc., USA) was used to spectro-\nphotometrically read absorbances at 450 nm. The relevant \nbiochemical parameter's unit was used to present the results. \nTable  1 lists all of the biochemically examined parameters \nalong with the manufacturer, country of origin, catalogue \nnumber, kit measurement range, and kit sensitivity.\nStatistical Analysis\nAll of the data collected for this study were statistically eval-\nuated using the SPSS 22.0 package. Numerical data were \nexpressed as median (minimum—maximum). To determine \nif the variables were normally distributed, the Shapiro–Wilk \ntest was employed. For general comparisons between more \nthan two groups, the Kruskal Wallis test was employed. Fol-\nlowing Kruskal Wallis, the post-hoc Dunn test was employed \nto compare the two groups. The level of statistical signifi-\ncance was set at P < 0.05.\n\n2379Reproductive Sciences (2025) 32:2375–2387 \nFig. 2  Explanation scheme about immunohistochemistry examination\n\n2380 Reproductive Sciences (2025) 32:2375–2387\nResults\nTRPC1 Immunoreactivity\nIn comparison to G1, TRPC1 immunoreactivity was statis-\ntically significantly higher in G2 ( p = 0.010) and G3 (p  = \n0.023). Comparing G1 and G4, there was no statistically sig-\nnificant difference in TRPC1 immunoreactivity (p = 0.998). \nTRPC1 immunoreactivity in G3 did not change statistically \nsignificantly from that in G2 (p  = 0.996); however, it did \ndecrease statistically significantly in G4 (p  = 0.002). How-\never, when comparing G4 to G3, there was a statistically sig-\nnificant reduction in TRPC1 immunoreactivity (p = 0.006), \n(Table  2), (Fig.  3).\nGenetic Findings\nThe administration of cabergoline did not reverse the signifi-\ncant decrease in TRPC1 gene expression that was observed \nas a result of OHSS exposure (G1 vs. G2; p = 0.009), while \nthe administration of nifedipine significantly increased \nTRPC1 expression in comparison to G2 and G3 (p  = 0.007 \nand p = 0.008). Figure  4 displays the qRT-PCR fold change \ngraph. These findings demonstrate that whereas nifedi -\npine administration corrected the decline in TRPC1 levels \nbrought on by OHSS, cabergoline administration did not.\nBiochemical Findings\nSerum VEGF Levels Serum VEGF levels in G2 were found \nto be statistically significantly higher than those in G1 (p \n< 0.007). Serum VEGF levels were shown to be statistically \nsignificantly lower in G3 (p  = 0.011) and G4 (p  = 0.008) \nthan in G2. Nevertheless, there was no noticeable statisti-\ncally significant difference between G3 and G4 (p = 0.935), \n(Table  3).\nTissue VEGF Levels Tissue VEGF levels in G2 were statis-\ntically significantly higher than those in G1 (p  = 0.003). \nIn comparison to G2, tissue VEGF levels were statistically \nsignificantly lower in the G3 (p = 0.046) and G4 (p = 0.01) \ngroups. G3 and G4 did not, however, differ statistically sig-\nnificantly (p = 0.616), (Table  4).\nSerum HIF‑1 α Levels When comparing G2, G3, and G4 to \nG1, no statistically significant difference was found (p  = \n0.597), (Table  3).\nTissue HIF‑1 α Levels When comparing G2, G3, and G4 to \nG1, no statistically significant difference was found (p  = \n0.164), (Table  4).\nSerum IL‑1β Levels Serum IL-1β levels in G2 were found \nto be statistically significantly higher than those in G1 (p  < \n0.001). When comparing G3 to G2, there was no statistically \nsignificant change in IL-1β levels (p = 0.307), whereas G4 \nshowed a statistically significant reduction (p = 0.021). Nev-\nertheless, there was no discernible statistically significant \nchange between G3 and G4 (p = 0.997), (Table  3).\nTissue IL‑1β Levels Tissue IL-1β levels in G2 were found to be \nstatistically significantly higher than those in G1 (p = 0.043). \nThere was no statistically significant difference in tissue IL-1β \nlevels between G3 (p = 0.239) and G4 (p = 0.859) compared \nto G2. Nevertheless, there was no noticeable statistically sig-\nnificant difference between G3 and G4 (p = 0.998), (Table 4).\nSerum IL6 Levels  Serum IL6 levels in G2 were found to \nbe statistically significantly higher than those in G1 (p  = \n0.003). There was no statistically significant change in IL6 \nlevels in G3 (p = 0.384) as compared to G2, but there was \na statistically significant decrease in G4 (p  = 0.002). Nev -\nertheless, there was no observable statistically significant \ndifference between G3 and G4 (p = 0.547), (Table  3).\nTissue IL6 Levels Tissue IL6 levels in G2 did not differ statisti-\ncally significantly from those in G1 (p = 0.928). In comparison \nto G2, IL6 levels were statistically significantly lower in G3 \n(p = 0.021) and G4 (p = 0.044). G3 and G4 did not, however, \ndiffer statistically significantly (p = 0.996), (Table 4).\nTable 1  Country, company, catalog number, kit measurement range and kit sensitivity of the Enzyme-Linked Immunosorbent Assay (ELISA) \nkits used in the study\nVEGF  vascular endothelial growth factor, TNF − α  tumor necrosis factor − alpha, IL − 1β  interleukin − 1 beta, IL-6  interleukin − 6, HIF \n− 1α hypoxia-inducible factor − 1 alpha\nParameters Company and country Catalog number Measuring range Sensitivity\nVEGF Sunred Biotechnology Company, Shanghai, China 201–11–0660 11–3000 ng/L 10,127 ng/L\nHIF-1 α ELK Biotechnology, Wuhan, China ELK1604 0,16–10 ng/mL 0,056 ng/mL\nTNF − α ELK Biotech. Biotechnology Company, Wuhan, China ELK1396 15.63–1000 pg/mL 6.1 pg/mL\nIL 1-β Sunred Biotechnology Company, Shanghai, China 201–11–0120 25–8000 pg/L 20,118 pg/L\nIL-6 Sunred Biotechnology Company, Shanghai, China 201–11–0136 2–600 pg/mL 1,822 pg/mL\n\n2381Reproductive Sciences (2025) 32:2375–2387 \nSerum TNF‑α Levels Comparing G2 to G1, there was a statisti-\ncally significant increase in serum TNF-α levels (p = 0.002). \nThere was a statistically significant decrease in serum TNF-α \nlevels in G4 (p = 0.003), but no statistically significant change \nin G3 compared to G2 (p = 0.093). G3 and G4 did not, how-\never, differ statistically significantly (p = 0.994), (Table 3).\nTissue TNF‑α Levels Tissue TNF-α levels in G2 did not dif-\nfer statistically significantly from those in G1 (p  = 0.928). \nTNF-α levels in G3 were statistically significantly lower than \nthose in G2 (p  = 0.007). There was no statistically signifi-\ncant difference between G4 and G3 (p = 0.985) or between \nG4 and G2 (p = 0.131), (Table  4).\nFig. 3  TRPC1, VEGF, HIF-1α, IL-1β, IL-6 and TNF-α immunohistochemical staining. Immunoreactive staining in all groups is shown with \nblack arrows\n\n2382 Reproductive Sciences (2025) 32:2375–2387\nDiscussion\nIn this study, we examined how nifedipine affects the sup-\npression of VEGF and proinflammatory cytokines that are \ncrucial to the pathophysiology of OHSS, including HIF-1 α, \nIL-1β, IL-6, and TNF-α. Our research revealed that nifedi-\npine, like cabergoline, lowers VEGF levels in ovarian tis-\nsue and serum. We also demonstrated that nifedipine lowers \ncytokine levels. We demonstrated that nifedipine can like-\nwise accomplish these effects by blocking the TRPC1 ion \nchannel. The effectiveness of the old medication nifedipine \nin OHSS is being examined for the first time in our study.\nNumerous investigations have demonstrated that VEGF \nproduction is decreased when TRPC1 is inhibited [16, 17, \n25]. Given that TRPC1 from the TRPC family may be more \nclosely associated with VEGF, which is crucial to the patho-\nphysiology of OHSS, we thought it suitable to investigate its \nactivity in our work. It has been demonstrated that nifedi-\npine inhibits the expression of inflammatory proteins such \nas IL-1β, IL-6, and TNF-α, as well as TRPM-7, TRPC-1, \nTRPC-3, and TRPC-6. This lowers VEGF expression and \neliminates skin redness and edema [25]. As a result of our \ninvestigation, we demonstrated that TRPC1 immunoreactiv-\nity rises in OHSS. It was shown that nifedipine dramatically \ndecreased TRPC1 immunoreactivity, whereas cabergoline \nwas unable to suppress it. However, a significant rise in \nTRPC1 gene expression was observed in comparison to the \ncabergoline group, whereas a significant decrease in gene \nexpression was observed in the nifedipine group when com-\npared to the control group. This rise can be explained by \nthe fact that gene expression has increased to make up for \nthe tissue's decreased TRPC1 immunoreactivity. By binding \nto VEGF2 receptors on endothelial cells, increased VEGF \nbrought on by hCG injection promotes angiogenesis and \ncontributes to the pathogenesis of OHSS by raising cyto-\nsolic  Ca2+ concentration and vascular permeability [9 , 26]. \nIn our study, the conditions of TRPC1 inhibition on OHSS \nwere examined macroscopically. However, the direct effect \nof nifedipine on TRPC1 could not be clearly explained by \nour present findings. A decrease in signal transducer and \nactivator of transcription 3 (STAT3) and NF-κB p65 phos-\nphorylation was reported in TRPC1 siRNA transfected \nlung epithelial cell line (MLE-12) cells. In contrast, STAT3 \nand NF-κB phosphorylation were shown to be increased in \nTRPC1 overexpressing MLE-12 cells compared with the \ncontrol group. Additionally, loss of TRPC1 has been shown \nto cause decreased secretion of IL-6, TNF-α, IL-4, IL-10 and \nIL-1β. These findings support that TRPC1 deficiency may \nexhibit impaired proinflammatory responses via NF-κB and \nSTAT3. It has been suggested that when TRPC1 is inhibited, \nthe cell cycle is arrested in the S phase, and when TRPC1 is \noverexpressed, the G2 and M phases are acceleratedThese \nresults suggest that TRPC1 may affect the cell cycle through \nactivation of the STAT3/NF-κB pathway [27]. In our study, \nnifedipine may have also shown its TRPC1 inhibition effect \non the cell cycle. Further studies should be designed. Rats \nin our study were given hCG to induce ovulation [26]. It \nhas been demonstrated that endothelial cells expressing \nTRPC1 enhance VEGF-induced  Ca2+ influx [28]. High \ncalcium permeability, nonselective cationic channels are \npresent in the TRPC1 protein. Many human tissues and cell \ntypes express these channels in large quantities [29]. Drugs \nto treat cancer, epilepsy, pain, arthritis, and heart conditions \nmay therefore be developed with these ion channels as pro-\nspective targets [30]. Şanlı et al. [31] found that elevated \nTable 2  TRPC1 TRPC1 \nimmunoreactivity histoscore in \nthe ovarian tissue (Values   are \ngiven as median, minimum–\nmaximum)\n* Kruskal–Wallis\na  Compared with G1\nb  Compared with G2\nc  Compared with G3, (p < 0.05)\nGroups TRPC1 immu-\nnoreactivity \nhistoscore\nG1 0,40 (0,30–0,45)\nG2 1,20 (0,90–2,70)a\nG3 1,20 (0,80–1,80)a\nG4 0,40 (0,20–0,60)bc\nP*  < 0,001\nFig. 4  TRPC1 gene expression was shown to be significantly \ndecreased in OHSS (G1) (p=0.009), cabergalin application (G3) \ncould not correct this decrease (comparing G1 and G3; p=0.008), but \nnifedipine application (G4) significantly increased TRPC1 expres-\nsion compared to G2 and G3 (p=0.007 and p=0.008). Data are \nexpressed as mean ± SEM. Significant difference compared to the \nG1 at ap<0.05, compared to G2 at bp<0.05 and compared to G3 at \ncp<0.05. Abbreviations: TRPC1: transient receptor potential canoni-\ncal; VEGF: vascular endothelial growth factor; HIF-1 α: hypoxia-\ninducible factor-1 alpha; IL-1β: interleukin-1 beta; IL-6: interleu-\nkin-6; TNF-α: tumor necrosis factor alpha; G1: Control group; G2: \nOHSS group; G3: OHSS group given cabergoline; G4: OHSS group \ngiven nifedipine\n\n2383Reproductive Sciences (2025) 32:2375–2387 \nTRPM2 and CHRM1 [6 ] immunoreactivity in hyperstimu-\nlated rat ovaries may contribute to the pathophysiology of \nOHSS as a cause or effect of congestion and edema in an \nexperimental investigation examining ion channel activities \nin OHSS. Following a single dosage or prolonged caber -\ngoline administration, Atilgan et al. [21] demonstrated a \nsubstantial decrease in VEGF-2 levels in ovarian tissues. \nIn moderate or severe-risk women, cabergoline has been \nshown to lower the risk of OHSS [32]. Our research showed \nthat the OHSS group had much higher blood and tissue \nVEGF levels than the control group, and that cabergoline \nand nifedipine effectively reduced serum and tissue VEGF \nlevels to a comparable degree to the OHSS group. In one \nstudy, nifedipine decreased the mRNA levels of VEGF and \nTRPC-6 in skin tissue, according to RT-PCR analysis [25]. \nAnother study found that VEGF secretion was decreased \nwhen  Ca2+ channels were inhibited with nifedipine (10–5 \nM), whereas VEGF secretion was increased when L-type \nchannels were stimulated [33]. The recent studies'findings \nmight also help to explain why our study's nifedipine treat-\nment reduced the VEGF levels in rats with OHSS. However, \nresearch on nifedipine has revealed that, in addition to its tra-\nditional mode of action, the drug's pleiotropic effects cause \nthe synthesis of VEGF, the most powerful angiogenic factor. \nBy increasing the distance between cells by its vasodilation \naction and promoting endothelial cell migration, nifedipine \nmay also promote microvascular angiogenesis [34, 35]. \nNevertheless, nifedipine's molecular effects have also been \ndemonstrated to decrease cell death in hypoxic cells [18]. \nBy controlling microcirculation, boosting regional blood \nTable 3  Serum VEGF, HIF-1 \nα, IL-1ẞ, IL-6 and TNF-α \nlevels in all groups (Values   are \ngiven as median, minimum–\nmaximum)\nVEGF vascular endothelial growth factor, TNF − α  tumor necrosis factor − alpha, IL − 1β  interleukin − 1 \nbeta, IL-6  interleukin − 1, HIF − 1α  hypoxia-inducible factor − 1 alpha, G1  Control group, G2  OHSS \ngroup, G3 OHSS group given cabergoline, G4 OHSS group given nifedipine\n* Kruskal–Wallis\na  Compared with G1\nb  Compared with G2, (p < 0.05)\nParametre G1 G2 G3 G4 P*\nVEGF (pg/ml) 4,52\n(1,18–7,27)\n55\n(21–90)a\n4,72\n(1,25–8,42)b\n4,18\n(2,24–6,98)b\n0,002\nHIF-1 α (pg/ml) 0,187\n(0,18–3,25)\n0,184\n(0,17–4,72)\n0,217\n(0,18–7,33)\n0,178\n(0,16–8,54)\n0,597\nIL-1ẞ (pg/ml) 328\n(274–902)\n5268\n(1257–9498)a\n1077\n(755–1992)\n1059\n(214–1196)b\n < 0,001\nIL 6\n(pg/ml)\n2,550\n(0,37–59,19)\n87,037\n(83,78–126)a\n69\n(1,42–92)\n2,074\n(0,63–12,14)b\n0,001\nTNF-α (pg/ml) 208\n(159–341)\n516\n(102–425)a\n285\n(196–408)\n201\n(71–392)b\n0,001\nTable 4  Ovarian tissue VEGF, \nHIF-1 α, IL-1ẞ, IL-6 and \nTNF-α levels in all groups \n(Values   are given as median, \nminimum–maximum)\nVEGF vascular endothelial growth factor, TNF − α  tumor necrosis factor − alpha, IL − 1β  interleukin − 1 \nbeta, IL-6  interleukin − 1, HIF − 1α  hypoxia-inducible factor − 1 alpha, G1  Control group, G2  OHSS \ngroup, G3 OHSS group given cabergoline, G4 OHSS group given nifedipine\n* Kruskal–Wallis\na  Compared with G1\nb  Compared with G2, (p < 0.05)\nParameters G1 G2 G3 G4 P*\nVEGF (pg/ml) 2,066\n(0,52–3,60)\n12,871\n(5,81–18,63)a\n2,850\n(1,49–5,78)b\n1,477\n(1,14–3,83)b\n0,06\nHIF-1 α\n(pg/ml)\n0,947\n(0,6–4,76)\n0,53\n(0,4–0,50)\n0,631\n(0,5–1,24)\n0,8\n(0,3–0,9)\n0,164\nIL-1ẞ (pg/ml) 168\n(83–223)\n427\n(273–649)a\n223\n(82–273)\n238\n(148–289)\n0,048\nIL 6\n(pg/ml)\n1,607\n(0,90–2,78)\n4,740\n(3,98–6,04)\n0,789\n(0,53–0,88)b\n0,719\n(0,32–1,14)b\n0,011\nTNF-α (pg/ml) 462\n(359–617)\n937\n(916–1365)\n197\n(129–389)b\n336\n(304–397)\n0,009\n\n2384 Reproductive Sciences (2025) 32:2375–2387\nflow, and enhancing tissue tolerance to ischemia, nifedipine \napplied topically and systemically to ischemic skin flaps in \nrats has been demonstrated to decrease necrotic areas [36, \n37]. Given that nifedipine may reorganize microcircula-\ntion and tissue homeostasis differently, and because the \nincrease in VEGF in OHSS may be mediated by a variety \nof different mediators, the nifedipine group in our study \nhad lower VEGF levels. However, it has been demonstrated \nthat TRPC1 alleviates edema and redness in the foot skin \nand lowers VEGF expression in the skin of chilblain rats \n[25]. In our study, we showed that nifedipine inhibits the \nTRPC1 channel, which reduces inflammation and VEGF \nproduction in OHSS. However, further molecular studies \nare needed to fully elucidate how this inhibition occurs at \nthe molecular level. Nifedipine's suppression of inflam-\nmation and VEGF production via TRPC1 inhibition can \nbe explained by the findings in similar studies. It has been \nshown in vitro that nifedipine can activate TRPC channels, \nespecially TRPC1, leading to Ca2 + influx into myometrial \ncells, but this effect can only occur at high drug concen-\ntrations (> 10 − 6 M). Despite effective downregulation of \ngenes encoding all channels tested, nifedipine-induced Ca2 \n+ influx was shown to be significantly reduced only in cells \nwhere TRPC1 expression was downregulated. Therefore, it \nwas suggested that the rapid increase in intracellular Ca2 \n+ observed in the pregnant human myometrial 1–41 (PHM1-\n41) cells in response to nifedipine may be due to a direct \nblocking effect of nifedipine on TRPC1 [36]. Our findings \nare in line with those of this study. The most significant \nstimulant for VEGF synthesis is hypoxia [38]. HIF-1, which \nattaches to the VEGF gene promoter, is activated to deliver \nthis stimulation [39]. In their experimental investigations, \nPala et al. [40] showed how VEGF contributes to the devel-\nopment of hypoxia in OHSS. Through the transcription of \nseveral hypoxia response genes, HIF-1α controls angiogen-\nesis [41]. In mice suffering from myocardial infarction (MI), \nit has been demonstrated that HIF-1α directly increases the \nexpression of TRPC1. This has been shown to improve heart \nfunction following MI by promoting angiogenesis [17]. \nThere was no discernible difference between the groups in \nour study when serum and tissue HIF-1 α levels were evalu-\nated across all groups. The findings of the aforementioned \nresearch are contrary to these findings. There may not have \nbeen enough hypoxic conditions in the rat ovaries where we \nproduced OHSS, and additional compensatory mechanisms \nmight have been at work.\nA significant rise in systemic inflammatory cytokines \nand vasoactive substances, including VEGF, interleukin-1β, \nIL-6, and TNF-α, in serum, follicular, and ascitic fluid may \nbe the etiology of OHSS [6 , 42]. Moreover, hCG causes \novulation, permits a rise in the number of oocytes, and \nstarts inflammatory secretion in human granulosa cells, all \nof which contribute to the systemic inflammatory response \nduring OHSS [42, 43]. Serum levels of IL-1β, IL-6, and \nTNF-α were higher in our OHSS group than in the control \ngroup, according to our study. We demonstrated that nifedi-\npine markedly decreased TNF-α, IL-1β, and IL-6 levels. \nThere was no difference between the OHSS, cabergoline, \nand nifedipine groups, despite the fact that tissue IL-1β \nlevels were higher in the OHSS group than in the control \ngroup. In contrast to the OHSS group, the nifedipine and \ncabergoline groups had lower levels of IL-6. Comparing \nthe cabergoline and OHSS groups, the cabergoline group's \ntissue TNF-α levels were noticeably lower. There was no \nsignificant difference between the cabergoline and nifedipine \ngroups when compared. Numerous inflammatory responses \nof vascular endothelial cells are regulated by the classical \nimmune inflammatory cytokines TNF-α, IL-1β, and IL-6 \n[44]. By lowering the expression of TRP family proteins, \nnifedipine has been demonstrated to decrease the cytokine \nproduction of IL-1β, IL-6, TNF-α, and VEGF [25]. In our \ninvestigation, we demonstrated that nifedipine had an anti-\ninflammatory impact by significantly lowering the serum \nlevels of inflammatory cytokines, including TNF-α, IL-1β, \nand IL-6, in rats with OHSS. We believe that nifedipine's \nanti-inflammatory action stems from a decrease in TRPC1 \nchannel activation. One impact of nifedipine that may help \nprevent the development of OHSS is its anti-inflammatory \nproperties. In many cell types, TNF-α-induced secretory \nactivities rely on  Ca2+. For instance, it has been demon-\nstrated that nifedipine inhibits the release of IL-6 when it \nblocks the lung's endothelial L-type CaV channels. Nifedi-\npine has been shown to have comparable anti-inflammatory \neffects in organs other than the lung [45]. In human chondro-\ncytes, nifedipine has been demonstrated to suppress oxida-\ntive stress and the release of inflammatory mediators [46]. \nBy showing for the first time the anti-inflammatory benefits \nof nifedipine in a rat OHSS model caused by hCG, our find-\nings advance our understanding in this field. Matrix metallo-\nprotein (MMP)−13, interleukin (IL)−1β, IL-6, tumor necro-\nsis factor (TNF)-α, cyclooxygenase (COX)−2, and inducible \nnitric oxide (NO) have all been demonstrated to be inhibited \nby nifedipine [46]. Another study found that mucus volume \ndecreased when TRPC1 expression was blocked. This has \nbeen linked to lower proinflammatory cytokines such IL-4, \nIL-1β, and TNF-α as well as down-regulated inflammatory \npathways, especially the STAT3/NF-κB signaling pathway \n[27]. Through the TLR4/TRPC1/NF-κB signaling pathway, \nTRPC1 has also been shown to contribute to the inflamma-\ntory response to bacterial infection [47].\nIn guinea pigs with asthma, expression of the TRPC1 chan-\nnel has also been linked to the development of chronic airway \ninflammation [48]. The reduction in inflammatory cytokines \nin the nifedipine group is probably due to the drug's superior \nanti-inflammatory qualities [49]. According to recent research, \nnifedipine's main function in the uterus is thought to be to stop \n\n2385Reproductive Sciences (2025) 32:2375–2387 \ncalcium-dependent muscle contractions, but there is also evi-\ndence that it may have cytoprotective and anti-inflammatory \neffects outside of pregnancy by blocking calcium channels in \nnon-pregnant tissues [48, 50]. Nevertheless, it has been demon-\nstrated that nifedipine has no effect on the release of inflamma-\ntory cytokines or the expression of inflammatory markers from \nhuman myometrial cells and tissues. According to this study, \nthe inhibition of smooth muscle contractions by nifedipine \nwas not mediated by the control of proinflammatory cytokines \n[51]. Some of the variations in serum and tissue inflammatory \ncytokine levels observed in our investigation may be explained \nby the findings of this study. However, our study used serum \nand ovarian tissue, whereas their study used uterine tissue. The \ndifferences between species should also be considered.\nThe limitation of our study include the fact that it was an \nexperimental investigation and that the outcomes of experi-\nments cannot be used to replicate human results, as well as the \nfact that it was carried out in a small population because of \nthe limited number of rats. In our study, the short-term effects \nof nifedipine and cabergoline were investigated, and the long-\nterm effects of both drugs on OHSS could not be evaluated. \nIn particular, the possible side effects of long-term inhibition \nof TRPC1 and the effect on other visible effects were not \ninvestigated, which is one of the limitations of our study. In \nour study, only the severe OHSS model was used. Examin-\ning the mild and moderate OHSS models could reveal how \nthe effects of drugs change in these cases and could be more \nmeaningful for clinical practice. This constitutes another limi-\ntation of our study. Our study's strengths are that it is the first \nto examine the activities of TRPC1 and cytokines in OHSS, \nthe first to examine the effects of cabergoline and nifedipine \non these parameters, and the first to examine the pathophysi-\nology of OHSS from a different angle. These findings may \nserve as models for future research that can close this gap in \nthe literature. In conclusion, nifedipine inhibits the production \nof proinflammatory cytokines such IL-1β, IL-6, and TNF-α, \nas well as VEGF, most likely via blocking TRPC1 channel \nactivation. This lowers the development of OHSS.\nAcknowledgements None.\nFunding Open access funding provided by the Scientific and Techno-\nlogical Research Council of Türkiye (TÜBİTAK). The current study \nwas supported by Fırat University Scientific Research Project Center \nas project number TF.23.22. This experimental study is derived from \nthe master thesis of Dr. Emel Kocal.\nDeclarations \nConflict of interests All authors declare that they have no conflict of \ninterest.\nOpen Access  This article is licensed under a Creative Commons Attri-\nbution 4.0 International License, which permits use, sharing, adapta-\ntion, distribution and reproduction in any medium or format, as long \nas you give appropriate credit to the original author(s) and the source, \nprovide a link to the Creative Commons licence, and indicate if changes \nwere made. The images or other third party material in this article are \nincluded in the article's Creative Commons licence, unless indicated \notherwise in a credit line to the material. If material is not included in \nthe article's Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will \nneed to obtain permission directly from the copyright holder. To view a \ncopy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.\nReferences\n 1. Hortu I, Karadadas E, Ozceltik G, Tavmergen E, TavmergenGoker \nEN, Yigitturk G, Erbas O. Oxytocin and cabergoline alleviate \novarian hyperstimulation syndrome (OHSS) by suppressing vas-\ncular endothelial growth factor (VEGF) in an experimental model. \nArch Gynecol Obstet. 2021;303:1099–108.\n 2. Cheng JC, Fang L, Li Y, Wang S, Li Y, Yan Y, Jia Q, Wu \nZ, Wang Z, Han X, Sun YP. Melatonin stimulates aromatase \nexpression and estradiol production in human granulosa-lutein \ncells: relevance for high serum estradiol levels in patients \nwith ovarian hyperstimulation syndrome. Exp Mol Med. \n2020;52:1341–50.\n 3. Practice Committee of the American Society for Reproductive \nMedicine. Prevention and treatment of moderate and severe \novarian hyperstimulation syndrome: a guideline. Fertil Steril. \n2016;106:1634–1647.\n 4. Nastri CO, Teixeira DM, Moroni RM, Leitão VM, Martins WP. \nOvarian hyperstimulation syndrome: pathophysiology, stag-\ning, prediction and prevention. Ultrasound Obstet Gynecol. \n2015;45:377–93.\n 5. Timmons D, Montrief T, Koyfman A, Long B. Ovarian hyper -\nstimulation syndrome: a review for emergency clinicians. Am J \nEmerg Med. 2019;37:1577–84.\n 6. Şanlı C, Atılgan R, Kuloğlu T, Pala Ş, İlhan N. The investiga-\ntion of cholinergic receptor muscarinic 1 activity in the rat ovary \nwith induced ovarian hyperstimulation. Turk J Obstet Gynecol. \n2023;20:53–8.\n 7. Wang B, Wang J, Liu Y, Wang L, Du M, Zhang Z, Guan Y. \nsRAGE downregulates the VEGF expression in OHSS ovarian \ngranulosa cells. Gynecol Endocrinol. 2021;37:836–40.\n 8. Kwik M, Maxwell E. Pathophysiology, treatment and prevention \nof ovarian hyperstimulation syndrome. Curr Opin Obstet Gynecol. \n2016;28:236–41.\n 9. Gómez R, Soares SR, Busso C, Garcia-Velasco JA, Simón C, Pel-\nlicer A. Physiology and pathology of ovarian hyperstimulation \nsyndrome. Semin Reprod Med. 2010;28:448–57.\n 10. Palumbo A, Ávila J, Naftolin F. The ovarian renin-angiotensin \nsystem (OVRAS): a major factor in ovarian function and disease. \nReprod Sci. 2016;23:1644–55.\n 11. Lerman OZ, Greives MR, Singh SP, Thanik VD, Chang CC, \nSeiser N, Brown DJ, Knobel D, Schneider RJ, Formenti SC, \nSaadeh PB, Levine JP. Low-dose radiation augments vasculogen-\nesis signaling through HIF-1-dependent and -independent SDF-1 \ninduction. Blood. 2010;116:3669–76.\n 12. Zhang Y, Zhang YY, Pan ZW, Li QQ, Sun LH, Li X, Gong MY, \nYang XW, Wang YY, Li HD, Xuan LN, Shao YC, Li MM, Zhang \nMY, Yu Q, Li Z, Zhang XF, Liu DH, Zhu YM, Tan ZY, Zhang \nYY, Liu YQ, Zhang Y, Jiao L, Yang BF. GDF11 promotes wound \nhealing in diabetic mice via stimulating HIF-1ɑ-VEGF/SDF-1ɑ-\nmediated endothelial progenitor cell mobilization and neovascu-\nlarization. Acta Pharmacol Sin. 2023;44:999–1013.\n 13. Song S, Zhang G, Chen X, Zheng J, Liu X, Wang Y, Chen Z, \nWang Y, Song Y, Zhou Q. HIF-1α increases the osteogenic \n\n2386 Reproductive Sciences (2025) 32:2375–2387\ncapacity of ADSCs by coupling angiogenesis and osteogenesis \nvia the HIF-1α/VEGF/AKT/mTOR signaling pathway. J Nano-\nbiotechnology. 2023;21:257.\n 14. Huang J, Mao Y, Li Q, Hong H, Tang N, Kang X, Huang Y, Liu J, \nGong Q, Yao Y, Li L. Kallistatin prevents ovarian hyperstimula-\ntion syndrome by regulating vascular leakage. J Cell Mol Med. \n2022;26:4613–23.\n 15. Zhang J, Huang J, He X, Li N, Miao Y, Li B, Shao X, Wang \nN. Ginkgo biloba extract 761 reduces vascular permeability of \nthe ovary and improves the symptom of ovarian hyperstimulation \nsyndrome in a rat model. Gynecol Endocrinol. 2022;38:318–23.\n 16. Moccia F, Negri S, Shekha M, Faris P, Guerra G. Endothelial \nCa(2+) signaling, angiogenesis and vasculogenesis: just what it \ntakes to make a blood vessel. Int J Mol Sci. 2019;20:3962.\n 17. Wen X, Peng Y, Gao M, Zhu Y, Zhu Y, Yu F, Zhou T, Shao J, \nFeng L, Ma X. Endothelial transient receptor potential canoni-\ncal channel regulates angiogenesis and promotes recovery after \nmyocardial infarction. J Am Heart Assoc. 2022;11:e023678.\n 18. Manohar K, Gupta RK, Gupta P, Saha D, Gare S, Sarkar R, \nMisra A, Giri L. FDA approved L-type channel blocker Nifedi-\npine reduces cell death in hypoxic A549 cells through modula-\ntion of mitochondrial calcium and superoxide generation. Free \nRadic Biol Med. 2021;177:189–200.\n 19. Coccia G, Bortolotti M, Michetti P, Dodero M. Prospective clin-\nical and manometric study comparing pneumatic dilatation and \nsublingual nifedipine in the treatment of oesophageal achalasia. \nGut. 1991;32:604–6.\n 20. Edraki N, Mehdipour AR, Khoshneviszadeh M, Miri R. Dihy -\ndropyridines: evaluation of their current and future pharmaco-\nlogical applications. Drug Discover Today. 2009;14:1058–66.\n 21. Atilgan R, Pala Ş, Yavuzkır Ş, Başpınar M, Yılmaz M, Ilhan N. \nWhat is the impact of short- and long-term supplementation of \neither cabergoline or clarithromycin on resolving rat ovarian \nhyperstimulation syndrome (OHSS) model? J Obstet Gynaecol. \n2019;39:687–94.\n 22. Oraebosi MI, Olurishe TO, Anafi SB, Bisalla M. Diurnal \nefficacy of alpha-lipoic acid/nifedipine/glimepiride combina-\ntion mitigates diabetic neuropathies in rats. Ann Pharm Fr. \n2022;80:291–300.\n 23. Vnukov VV, Gutsenko OI, Milutina NP, Ananyan AA, \nDanilenko AO, Panina SB, Kornienko IV. Influence of SkQ1 \non Expression of Nrf2 transcription factor gene, are-controlled \ngenes of antioxidant enzymes and their activity in rat blood \nleukocytes. Biochemistry (Mosc). 2015;80:586–91.\n 24. Bartoli F, MoradiBachiller S, Antigny F, Bedouet K, Gerbaud \nP, Sabourin J, Benitah JP. Specific upregulation of TRPC1 and \nTRPC5 channels by mineralocorticoid pathway in adult rat ven -\ntricular cardiomyocytes. Cells. 2019;9:47.\n 25. Zhou Y, Yan H, Li T, Xie M, Li X, Zhao C. New use of old \nmedicine: Nifedipine acts on the TRP family and inflammatory \nproteins in the treatment of chilblain. Burns. 2022;48:372–80.\n 26. Brock TA, Dvorak HF, Senger DR. Tumor-secreted vascular \npermeability factor increase cytosolic Ca2_ and von Wille-\nbrand factor release in human endothelial cells. Am J Pathol. \n1991;138:213–21.\n 27. Pu Q, Zhao Y, Sun Y, Huang T, Lin P, Zhou C, Qin S, Singh \nBB, Wu M. TRPC1 intensifies house dust mite-induced airway \nremodeling by facilitating epithelial-to-mesenchymal transition \nand STAT3/NF-κB signaling. FASEB J. 2019;33:1074–85.\n 28. Jho D, Mehta D, Ahmmed G, Gao XP, Tiruppathi C, Broman M, \nMalik AB. Angiopoietin-1 opposes VEGF-induced increase in \nendothelial permeability by inhibiting TRPC1-dependent Ca2 \ninflux. Circ Res. 2005;96:1282–90.\n 29. Rubaiy HN, Ludlow MJ, Bon RS, Beech DJ. Pico145-pow -\nerful new tool for TRPC1/4/5 channels. Channels (Austin). \n2017;11:362–4.\n 30. Minard A, Bauer CC, Wright DJ, Rubaiy HN, Muraki K, Beech \nDJ, Bon RS. Remarkable progress with small-molecule modula-\ntion of TRPC1/4/5 channels: Implications for understanding the \nchannels in health and disease. Cells. 2018;7:52.\n 31. Şanlı C, Atılgan R, Kuloğlu T, Pala Ş, AydınTürk B, Keser HB, \nİlhan N. Transient receptor potential melastatin 2 ion channel \nactivity in ovarian hyperstimulation syndrome physiopathology. \nTurk J Med Sci. 2021;51:787–95.\n 32. Tang H, Mourad SM, Wang A, Zhai SD, Hart RJ. Dopamine \nagonists for preventing ovarian hyperstimulation syndrome. \nCochrane Database Syst Rev. 2021;4:CD008605.\n 33. Rosenthal R, Heimann H, Agostini H, Martin G, Hansen LL, \nStrauss O. Ca2+ channels in retinal pigment epithelial cells \nregulate vascular endothelial growth factor secretion rates in \nhealth and disease. Mol Vis. 2007;13:443–56.\n 34. Miura S, Fujino M, Matsuo Y, Tanigawa H, Saku K. Nifedi-\npine-induced vascular endothelial growth factor secretion from \ncoronary smooth muscle cells promotes endothelial tube for -\nmation via the kinase insert domain-containing receptor/fetal \nliver kinase-1/NO pathway. Hypertens Res: Off J Japanese Soc \nHypertens. 2005;28:147–53.\n 35. Mendonça RJ, Coutinho-Netto J. Aspectos celulares da cicatri-\nzação. An Bras Dermatol. 2009;84:257–62.\n 36. Fukunaga Y, Izawa-Ishizawa Y, Horinouchi Y, Sairyo E, Ikeda \nY, Ishizawa K, Tsuchiya K, Abe Y, Hashimoto I, Tamaki T. \nTopical application of nitrosonifedipine, a novel radical scaven-\nger, ameliorates ischemic skin flap necrosis in a mouse model. \nWound Repair Regen. 2017;25:217–23.\n 37. Yart L, Frieden M, Konig S, Cohen M, Martinez de Tejada \nB. Dual effect of nifedipine on pregnant human myome-\ntrium contractility: Implication of TRPC1. J Cell Physiol. \n2022;237:1980–1991.\n 38. Abulafia O, Sherer DM. Angiogenesis of the ovary. Am J Obstet \nGynecol. 2000;182:240–6.\n 39. Bausero P, Ben-Mahdi M, Mazucatelli J, Bloy C, Perrot-Appla-\nnat M. Vascular endothelial growth factor is modulated in vas-\ncular muscle cells by estradiol, tamoxifen, and hypoxia. Am J \nPhysiol Heart Circ Physiol. 2000;279:H2033–42.\n 40. Pala Ş, Atilgan R, Ozkan ZS, Kavak SB, Ilhan N, Akpolat N, \nSapmaz E. Effect of varying doses of tamoxifen on ovarian his-\ntopathology, serum VEGF, and endothelin 1 levels in ovarian \nhyperstimulation syndrome: an experimental study. Drug Des \nDevel Ther. 2015;9:1761–6.\n 41. Song W, Liang Q, Cai M, Tian Z. HIF-1alpha-induced up-\nregulation of microRNA-126 contributes to the effectiveness \nof exercise training on myocardial angiogenesis in myocardial \ninfarction rats. J Cell Mol Med. 2020;24:12970–9.\n 42. Orvieto R. Controlled ovarian hyperstimulation–an inflamma-\ntory state. J Soc Gynecol Investig. 2004;11(7):424–6. https://  \ndoi. org/ 10. 1016/j. jsgi. 2004. 05. 001. \n 43. Orvieto R, Dratviman-Storobinsky O, Cohen Y. Interleukin-2 \nproduction by cultured human granulosa cells. Am J Reprod \nImmunol. 2015;74:392–7.\n 44. Stappenbeck F, Wang F, Sinha SK, Hui ST, Farahi L, Mukhame-\ndova N, Fleetwood A, Murphy AJ, Sviridov D, Lusis AJ, Par -\nhami F. Anti-inflammatory oxysterol, oxy 210, inhibits athero-\nsclerosis in hyperlipidemic mice and inflammatory responses of \nvascular cells. Cells. 2024;13:1632.\n 45. Zyrianova T, Zou K, Lopez B, Liao A, Gu C, Olcese R, \nSchwingshackl A. Activation of endothelial large conductance \n\n2387Reproductive Sciences (2025) 32:2375–2387 \npotassium channels protects against TNF-α-induced inflamma-\ntion. Int J Mol Sci. 2023;24:4087.\n 46. Yao J, Long H, Zhao J, Zhong G, Li J. Nifedipine inhibits oxi-\ndative stress and ameliorates osteoarthritis by activating the \nnuclear factor erythroid-2-related factor 2 pathway. Life Sci. \n2020;253: 117292.\n 47. Zhou X, Ye Y, Sun Y, Li X, Wang W, Privratsky B, Tan S, \nZhou Z, Huang C, Wei YQ, Birnbaumer L, Singh BB, Wu \nM. Transient receptor potential channel 1 deficiency impairs \nhost defense and proinflammatory responses to bacterial infec-\ntion by regulating protein kinase Cα signaling. Mol Cell Biol. \n2015;35:2729–39.\n 48. Li N, He Y, Li M. C. Role of transient receptor potential canoni-\ncal 1 in airway remodeling and effect of budesonide on its pul-\nmonary expression in asthmatic guinea pigs [in Chinese]. Nan \nFang Yi Ke Da Xue Xue Bao. 2015;35:1374–9.\n 49. Kang M, Ross GR, Akbarali HI. The effect of tyrosine nitration \nof L-type Ca2+ channels on excitation-transcription coupling \nin colonic inflammation. Br J Pharmacol. 2010;159:1226–35.\n 50. Yamagishi S, Nakamura K, Takenaka K, Matsui T, Inoue H. \nPleiotropic effects of nifedipine on atherosclerosis. Curr Pharm \nDes. 2006;12:1543–7.\n 51. Arman BM, Binder NK, de Alwis N, Beard S, Debruin DA, \nHayes A, Tong S, Kaitu’u-Lino TJ, Hannan NJ. Assessment of \nthe tocolytic nifedipine in preclinical primary models of preterm \nbirth. Sci Rep. 2023;13:5646.\nPublisher's Note Springer Nature remains neutral with regard to \njurisdictional claims in published maps and institutional affiliations.","source_license":"CC0","license_restricted":false}