The Study Of The Pathogenesis And Prevention Of Ovarian Hyperstimulation Syndrome(OHSS)

Background:Ovarian hyperstimulation syndrome?OHSS?is an iatrogenic and serious complication after ovarian stimulation.The ovarian enlargement,ascites,high level of17?-estradiol?E₂?and high vascular permeability?VP?are the features of OHSS.High serum E₂ level on the day of human chorionic gonadotropin?hCG?administration is considered as a risk factor for the incidence of OHSS.The main symptom of OHSS is high VP and the most important regulator is vascular endothelium growth factor?VEGF?.VEGF makes local capillaries leaky by binding to and phosphorylating VEGF receptor 2?VEGFR2?in endothelial cells.In addition,VEGF is a VP enhancer whose potency is5000 times stronger than histamine and plays an important role in the incidence of OHSS.Thus,VEGF promotes OHSS onset by regulating nitric oxide?NO?and junction proteins and thereby increasing VP.Zinc finger gene 217?ZNF217?is a potential oncogene which located on chromosome 20q13.2.Previous study showed that ZNF217 and ER?proteins bound to each other in breast cancer cells while ER?positively regulated the expression of VEGF.Besides,ZNF217 promotes the ER?-dependent transcription of the downstream genes by enhancing the recruitment of ER?to its estrogen response elements?ERE?.Thus,ZNF217 may also participate in the regulation of VEGF and OHSS.Moreover,ZNF217 is a candidate gene of polycystic ovary syndrome?PCOS?,regarded as a high risk factor of OHSS onset.Thus,we suppose that ZNF217 is involved in the pathogenesis of OHSS.To date,discontinuing gonadotropin therapy,reducing human chorionic gonadotropin?hCG?usage and intravenous albumin administration are common approaches in the prevention of OHSS.Therefore,OHSS is a potentially lethal condition with an unclear mechanism and it is necessary for us to investigate a new prevention protocol for OHSS.Kisspeptin?Kp?is a kind of polypeptide encoded by the KISS1 gene and its specific receptor is KISS1R?KISS1 receptor?.Kp-10 inhibits VEGF expression in human umbilical vein endothelial cells?HUVECs?through KISS1R while VEGF is an important mediator of OHSS onset.Moreover,Kp-54,another member of the Kp family,could trigger egg maturation in women at high risk of OHSS.Thus,we suggest that Kp/KISS1R system could prevent OHSS by inhibiting VEGF.Aims:1.We aim to explore the distribution as well as the functions of ovarian ZNF217 in the development of OHSS,especially for the synthesis of estrogen and the regulation of vascular permarbility.2.Our study also aim to investigate the function of Kp/KISS1R system in OHSS prevention and the regulation of high E₂ to Kp/KISS1R system.Methods:1.Building of OHSS rat model and collection of the granulosa cells,follicular fluid and serum of OHSS patients.Patients with high level of E₂?serum E₂ level higher than6000 pg/ml on the day of hCG administration?or with more than 25 dominant follicles were identified as at high risk of OHSS in IVF cycles.Follicles whose diameter was larger than 1.4 cm on the ovum retrieval day during IVF cycles were identified as dominant follicles.2.The vascular permeability,ovarian weight,ovarian size and the expression of ovarian VEGF was detectd to evaluate that if OHSS rat models were successful.3.ThelocationofZNF217intheovarieswasdetectedusing immunohistochemistry.4.The concentration of TSP-1 in human follicular fluid and the concentration of estrogen in rat serum were detected using ELISA kits.5.Small interfering?si?RNA knocking down and transfection of vectors with electroporation were used to change the expression of target genes?ZNF217,TSP-1,CD36,CD47,ER??.6.PCR and western blotting were used to detecte the expression of target genes.7.Exogenous Kisspeptin-10 was injected into OHSS rat models to investigte the prevention of Kisspeptin-10 to the symptom of OHSS.Results:1.ZNF217 appeared in the oocytes,granolosa cells and theca cells in the growing follicles of the ovaries.Highest expression level of ZNF217 was observed in the granulosa cells and luteal cells with immunostaining concentrated both in the nucleus and cytoplasm.2.The expression of ovarian ZNF217 was increased in OHSS rats while ovarian TSP-1 decreased.The same results was detected in the OHSS patients.Patients at high risk of OHSS showed an increased expression of ZNF217 in the granulosa cells with a decreased TSP-1 in the follicular fluid.Moreover,high serum estrogen level?E₂?and high ovarian aromatase were also detected both in the OHSS rats and patients.It is suggested that ZNF217 was closely related to OHSS and E₂ concentration.3.ZNF217 mRNA was reduced to less than 30%compared with control group after specific siRNA treatment and the expression of CYP19A1 and CREB1 also significantly decreased both in mRNA and protein levels.Thus,ZNF217 was involved in the regulation of CREB1,the upstream regulator of CYP19A1.The concentration of E₂ in culture medium of KGN cells was also significantly reduced after ZNF217 knock-down.The over-expression of ZNF217 also demonstrated that ZNF217 positively regulated E₂synthesis through CREB1 and aromatase in KGN cells.4.TSP-1 mRNA significantly increased after ZNF217 while it was reduced after the over-expression of ZNF217.Therefore,ZNF217 negatively regulated TSP-1 mRNA in granulosa cells.Moreover,TSP-1 didn't directly regulate the expression of VEGF in HUVECs.However,claudin1,which is the downstream factor of VEGF pathway and regulates VP,significantly decreased after TSP-1 reduction and increased after TSP-1protein treatment?p=0.017?.Moreover,the promotion function of TSP-1 to claudin1 was inhibited after CD36 knock-down.Meanwhile,NO,a well-known VP enhancer,significantly increased after TSP-1 reduction and decreased after TSP-1 protein treatment.After CD47 reduction,the inhibition of TSP-1 protein to NO was reversed.Thus,TSP-1inhibited NO synthesis after binding with CD47.In a word,ZNF217 positively regulated VP through inhibiting TSP-1 and thereby inhibiting claudin1 as well as promoting NO synthesis.5.The significantly higher ovarian weight,abdominal VP,serum E₂ concentration and VEGF abundance in ovaries and lung tissue of the OHSS group demonstrated that our OHSS models were successful.The expression of Kiss1r significantly decreased both in ovaries and lung tissue of OHSS rats.The exogenous Kp-10 injection inhibited the increase of abdominal VP of the OHSS group almost to the level of the control group.Meanwhile,Kp-10 also significantly enhanced the expression of Kiss1r as well as inhibiting VEGF in the ovaries and lung tissue of OHSS models.Thus,Kp/Kiss1r system has an effect on OHSS prevention by inhibiting VEGF expression.6.KISS1R was also reduced in the granulosa cells of patients at high risk of OHSS with no significant change observed in KISS1,which was consistent with the results of OHSS rats.7.Treatment of HUVECs with high E₂ for 48 hours reduced KISS1R mRNA and protein levels in a concentration-independent manner.Meanwhile,high E₂?>5000 pg/ml? also increased VEGF mRNA and NO synthesis in HUVECs,which both represent the increase of VP.Therefore,a high level of E₂ inhibits KISS1R and promotes the increase of VEGF and VP.The reduction of KISS1R after E₂ treatment was partially reversed both in mRNA and protein levels after ER?knock-down,which suggested that ER?participated in the negatively regulation of E₂ to KISS1R.Thus,E₂ suppressed KISS1R and increased VP in HUVECs through the activation of ER?.Conclusion:We clarified that ZNF217 triggered OHSS onset through promoting E₂ synthesis and inhibiting TSP-1,providing a new insight into the pathogenesis of OHSS.Furhtermore,we demonstrated that Kp-10 prevents the increased VP of OHSS by the activation of KISS1R and the inhibition of VEGF,providing a novel possible strategy to prevent OHSS by exogenous Kp-10 injection.