The Expression Of Amyloid Precursor Protein In Ovarian Granulosa Cells And Follicular Fluid And Its Relation To Polycystic Ovary Syndrome

The late20th century trend to delay birth of the first child until the age at which female fecundity or reproductive capacity is lower has increased the incidence of age-related infertility. According to the human biologic clock, female fertility begins declining significantly in the early30s and culminating in the menopause. Female age is considered as an important factor in fertility.The ovary is no exception to the ravages of aging, which occur in every organ of the body. The mechanism of aging in other system may also involved in ovary. Alzheimer’s disease (AD) is a progressing neurodegenerative and age-related disease. Amyloid precursor protein (APP) is a single transmembrane protein, expressed in all cell types and considered as a critical factor in process of AD. APP is metabolized by two distinct antagonist pathways. The major pathway is driven by an a-secretase cleavage, which releases the soluble N-terminal ectodomain (sAPPα). sAPPa has been shown to stimulate the proliferation of many types of cells and act as a growth factor. The other pathway is driven by P-and y-secretase generates the soluble APP beta protein (sAPPβ) and Aβ. Aβ can bind to several types of receptors and active cellular signaling pathway, which induce cell apoptosis. So far, APP and its proteolytic cleavage products had been well investigated nervous system but not been studied in the field of reproductive system.Polycystic Ovary Syndrome (PCOS) is the most common endocrinopathy in women of reproductive age and is characterized by several kinds of serum hormone disorder. Many studies have shown that oxidative stress (OS) increases in PCOS. Oxidative stress represented by elevated serum levels of advanced glycosylated end products (AGEs) and increased expression of their multiple ligand receptor (RAGE) on granulosa cells in PCOS. AGEs are products of non-enzymatic glycation and oxidation of proteins, lipids and nucleic acid, have been found to induce oxidative stress. Ap is one of the ligands for the RAGE, Ap can induce oxidative stress through nuclear factor kB (NF-kB) dependent pathways via RAGE.In this study, we measured APP mRNA and protein expression in granulosa cells from women undergoing IVF treatment, and detected follicular fluid sAPP levels, to determine APP’s possible role in ovarian aging. We characterized APP and RAGE mRNA and protein expression in granulosa cells in PCOS patients and measured sAPP and AGEs levels in follicular fluid, try to analyze possible correlations of APP, AGEs-RAGE and PCOS.Part I The expression of APP in ovarian granulosa cells and follicular fluid in women undergoing IVF treatment [Objective]The aim of this study was to determine whether amyloid precursor protein (APP) is expressed in human granulose cells and to measure the levels of sAPP in follicular fluid from women undergoing IVF, and to correlate them with some characteristics of the patients and of the IVF protocol to investigate their role in ovarian aging. [Materials&Methods]1、One hundred and six women undergoing IVF treatment at the Center for Assisted Reproducion of Nanfang hospital from January2013to January2014were included in this study. For each patient, follicular fluid (FF) sample from a single dominant follicle form each patient was collected during oocyte retrieval.2、All women in the study were divided into two groups on the basis of age: younger group (age<35y, n=50) and older group(age≥35y, n=56). The study was approved by the Ethics Committee and each woman filled a written informed consent before to participate to it.3、For each patient,granulose cells (GC) were separated by Percoll dendity gradient centrifugation. Total RNA was isolated from GC using trizol reagent, APP mRNA levels of GC were measured by real-time qPCR. Western blot was used to measure the expression of APP in GC. sAPPa and sAPPβ levels in FF were determined by using the sAPP enzyme immunoassay ELISA kit.4、All data were analyzed with SPSS16.0statistical program. Quantitative data are presented as mean±SEM. Statistical analysis was made with the unpaired Student’s t-test. Pearson/Spearman analysis was used to examine the relationship between variables. A P value<0.05was considered statistically significant.[Results]1、APP mRNA and protein were expressed in all the GC samples. sAPP were demonstrated in all FF samples.2、The relative expression levels of APP mRNA and protein in GC were significantly higher in older group than younger group (4.56±3.37vs2.98±2.09, P=0.008) and (2.51±1.54vs1.15±0.68, P=0.000), respectively.3、The expression of APP in GC was statistically significant positive related to age(r=0.506, P=0.000), bFSH(r=0.239, P=0.021), total gonadtrophin dosage used (r=0.216, P=0.038) and was significantly negatively related to AFC (r=-0.268, P=0.009), the No. of collected oocytes(r=-0.2339, P=0.025).4、The sAPPβ level of in FF were significantly higer in the older group than younger group (143.82±30.40vs128.70±36.83ng/ml, P=0.024). The older group also had higher level of sAPPa compared with the younger group (172.22±38.02vs162.44±47.23ng/ml), but this difference did not achieve significance (P=0.247). Spearman analysis has shown a significant correlation of sAPPβ levels with age(r=0.258, P=0.008), AFC(r=-0.235, P=0.016), serum E2level on the day of hCG(r=-0.216, P=0.028). And a significantly positive correlation of sAPPa levels with No. of oocytes retrieved (r=0.236, P=0.015).[Conclusions]1、APP was detectable on the mRNA and protein level in GCs. sAPP was demonstrated in all FF samples. Our finding is the first time to prove that APP and sAPP are expressed in GC and FF from women undergoing IVF treatment.2、The expression of APP protein in GC statistically significant positive correlations with age, serum basal FSH level and total gonadtrophin dosage used, suggesting APP may involved in the process of ovarian aging.3、There is a significantly negative correlation of sAPPβ in FF with age and a significantly positive correlation AFC, serum E2level on the day of hCG. FF sAPPa levels has a significantly positive correlation with No. of oocytes retrieved. Part II The expression of APP and AGE-RAGE in ovarian granulosa cells and follicular fluid in women with Polycystic Ovary Syndrome[Objective]The aim of this part is to compare the expression of APP and RAGE in granulose cells and the sAPP and AGEs levels in follicular fliud between PCOS patients and the controls, and to investigate the potential relationship between APP and PCOS.[Materials&Methods]1、Twenty-five patients diagnosed PCOS according to the Rotterdam criteria and thirty patients undergoing IVF because of tubal infertility as controls were included. The study was approved by the Ethics Committee.2、For each patient, after oocyte retrieval, FF were collected, GC were separated by Percoll dendity gradient centrifugation. Total RNA was isolated from GCs using trizol, APP mRNA, RAGE mRNA levels of GC were measured by real-time qPCR. Western blot was used to measure the expression of APP in GCs. sAPP and AGEs levels in FF were measured by ELISA kit.3、All data were analyzed with SPSS16.0statistical program. Quantitative data are presented as mean±SEM. Statistical analysis was made with the unpaired Student’s t-test. Pearson/Spearman analysis was used to examine the relationship between variables. A P value<0.05was considered statistically significant.[Results]1、The women with PCOS had significantly higher antral follicle count (AFC), total testosterone, serum basal LH levels, LH/FSH ratio, number of collected oocytes, and lower total gonadtrophin dosage used than controls.2、The expression of APP mRNA was higher in PCOS group than control group (4.20±2.61vs3.03±2.42, P=0.090). The relative expression level of APP protein in PCOS group was significantly higher than control group (1.63±0.93vs1.08±0.69,P=0.021).3、sAPPβ concentrations were significantly higher in FF of PCOS patients compared with controls (143.96±25.60vs126.31±33.37ng/ml, P=0.035). PCOS women also had higher levels of sAPPa compared with the control group (179.11±31.58vs167.35±36.87ng/ml), but this difference did not achieve significance (P=0.215). The FF sAPPβ level was statistically significant positive related to serum basal LH level(r=0.413, P=0.040).4、The expression of RAGE mRNA was significantly higher in PCOS group than control group(2.16±1.02vs1.07±0.99, P=0.026). AGEs concentrations were higher in FF of PCOS patients compared with controls (1.31±0.56vs1.11±0.66ng/ml) but this difference did not achieve significance(P=0.137).[Conclusions]1、The expression levels of APP mRNA, APP protein in GC and the mean sAPPβ, sAPPa levels in FF were higher in PCOS patients compare with the controls, suggesting APP and sAPP may involved in the process of PCOS.2、PCOS patients have higher expression levels of RAGE mRNA in GC and higher AGEs concentrations in FF than controls.3、To confirm the hypothesis above, further work is needed to clarify the cause and consequences of this observation. More studies are necessary to clarify how APP play a role in ovary.