Expression Of Bone Morphogenetic Protein-9 In Granulosa Cells Assess IVF Oocyte Maturation And Embryonic Development

Objective: In the process of in vitro ferti1ization-embryo transfer,the ability to accurately determine oocyte maturation has a profound impact on fertilization,embryo development,pregnancy establishment and fetal development.To raise oocyte quality is of great significance to improve the IVF pregnancy outcome.BMP-9,which mainly exists in the liver,is a member of BMPs,with the induction and maintenance of embryonic neuronal cholinergic differentiation,regulation of glucose and fatty acid metabolism,regulation of body iron dynamic balance and other important functions,of what is now known as the strongest factor to induce bone formation in BMPs.Recent studies have shown that BMP-9 plays an important role on steroidogenesis using rat original granulosa cells.Therefore,we hypothesized that BMP-9 may be present in human ovarian granulosa cells and participate in the regulation of the reproductive system.In this study,we examined the levels of BMP-9 in granulosa cells of IVF-ET patients,and analyzed the relationship between age,body mass index,fertilization rate,high quality embryo rate and egg utilization rate in order to investigate the effect of BMP-9 on egg quality,fertilization and embryo development.Methods:Subjects were recruited through poster announcement from April 2015 to July 2015 at the forth hospital of He Bei medical university Reproductive Medicine.Eighty-five women participated voluntarily with the diagnosis of tubal factor infertility who underwent IVF using long protocol.ALL patients were at ages from 20 to 38,with a normal menstrual cycle;Antral follicle count is greater than 6.The localization of BMP-9 expressed in granulosa cells was tested by Immunocytochemistry method.The expression levels of BMP-9 protein and m RNA were tested by Reverse Transcription-Polymerase Chain Reaction(RT-PCR)and Western Blot.Controlled ovarian hyperstimulation: The treatment options followed a long-term protocol.All women subcutaneously injected Gn RHa 1.4mg in the mid-luteal phase for pituitary suppression.Patients recieved recombinant FSH 150-225 IU for ovarian stimulation after the down-regulation standard was achieved(LH ? 2.5IU/L,FSH ? 3IU/L,E2 ? 30pg/m,EM ? 5mm).The follicular growth and endometrial changes were monitored by transvaginal ultrasound.Levels of LH,E2,and P were monitored if necessary.When two follicles were larger than 20 mm in diameter and the serum level of E2 reached about 200-300pg/ml per follicle,HCG was given to trigger ovalution.After 36 hours,transvaginal ultrasound�guided follicular aspiration was administered.Assessment oocyte maturation and embryo development: Oocyte cumulus complex was evaluated by the metaphase II oocyte morphologic scoring system.After 18 hours IVF,zygote pronuclear morphology was observed followed by scott scoring standard system.The fertilized eggs were continued to culture for 48 hours,and the development of the embryos were examined by the cleavage stage embryo scoring standard.Granulosa cells collection: All the follicular fluid recovered and separated the precipitate by centrifugation.Granulosa cells were obtained by Percoll gradient centrifugation to exclude red blood cells.After PBS washed,the lower layer is granulosa cells.Part of GCs were added to 2 ml Trizol solution and stored at-80? for RT-PCR.Part of the GCs were cryopreserved at-80? used for Western blot.A small amount of GCs were smeared for immunocytochemistry after fixed by 4% paraformaldehyde.Immunocytochemistry method: Gcs were fixed and permeabilized on the slide;endogenous peroxidase and non-specific binding sites were blocked.The rabbit polyclonal antibody against human BMP-9(ABCAM)at a dilution of 1:200 were used for detection,with PBS instead of primary antibody for negative control.Detection followed by the immunocytochemical method.Leica camera systemwere used for image acquisition.RT-PCR technology: Total RNA of granulosa cells was extracted by Trizol method.Reverse transcription was carried out using PROMEGA kit,in accordance with the instructions.Target gene was amplified.?-actin acted as internal control.Products were run at 20g/L agaros gel electrophoresis.Relative expression of BMP-9 m RNA was electrophoresis band luminosity density ratio between the target gene and internal reference PCR products.Western Blot: GCs were splitted by cell lysis solution;protein bands were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE);Transferred to PVDF membrane;5% skim milk powder closed for 1 hours;PVDF membrane was placed in BMP-9 working fluid(1: 1000)at 4? overnight;USing TBST liquid washed the membrane three times;Plus secondary antibody to incubate for 1 hour,then washed the membrane three times.?-actin was the internal reference.We scanned the result using two-color infrared laser scanner and recorded the gray value.The relative amount of BMP-9 protein was the optical density ratio between the target band and internal reference.Statistical analysis: All data were analyzed by SPSS13.0 statistical software.Data were presented as`x � s.The comparisons between various groups were performed by t test.The correlation analysis was carried out using the Person correlation coefficient.A P value of 0.05 was considered as statistically significant.Results: 1 BMP-9 was expressed in the cytoplasm of human granulosa cells,and the expression levels of BMP-9 were different(figure 1).2 The expression of BMP-9m RNA in granulosa cells 2.1 The expression of BMP-9m RNA in granulosa cells do not have linear correlation with age,BMI,basal FSH,basal LH,GN and GN days(Table 1).2.2 The expression of BMP-9m RNA in granulosa cells do not have linear correlation with the number of eggs obtained,M?oocyte number,the number of fertilization,2PN number,fertilization rate,excellent embryo rate and egg utilization rate(Table 2).3 The expression of BMP-9 protein in granulosa cells 3.1 There were differences in the expression levels of BMP-9 protein in granulosa cells(figure 2).3.1 The expression of BMP-9 protein in granulosa cells do not have linear correlation with age,BMI,basal FSH,basal LH,GN and GN days(Table 1).3.2 The expression of BMP-9 protein in granulosa cells do not have linear correlation with the number of eggs obtained,M?oocyte number,the number of fertilization,2PN number,fertilization rate,excellent embryo rate and egg utilization rate(Table 2).4 The correlation coefficient between BMP-9 m RNA expression and protein expression in granulosa cells was 0.220,P = 0.043,and there was a linear correlation between them(figure 3).5 According to the relative expression of BMP-9 m RNA(0.155-0.690),we select the number of 0.403 as the node.All patients are divided into low expression group(n=41)and high expression group(n=44)(Figure 4).There is no significant difference between the two groups of general information(Table 3).High quality embryos and egg utilization in high expression group were significantly higher than those in low expression group(P <0.05)(Table 4).Conclusions:1 BMP-9 m RNA and protein are expressed in human ovarian granulosa cells.BMP-9 exists in the cytoplasm,and may act as an autocrine/paracrine factor involved in the regulation of the reproductive system.2 BMP-9 m RNA expression has linear correlation with the level of protein expression.3 High expression of BMP-9 can increase the rate of high quality embryos and improve the quality of oocytes,indicating that BMP-9 may be regarded as one of the indexes to evaluate the quality of oocytes.