The Expression Of Amyloid Precursor Protein (APP) And Its Proteolysis Products In Human Ovary And Their Association With Treatment Outcome In Assisted Reproductive Technology (ART)

With the rapid development of reproductive medicine, assisted reproductive technology (Assisted Reproductive Technology, ART) has been widely used in the treatment of infertility, but pregnancy rate in vitro fertilization and embryo transfer (In Vitro Fertilization-Embryo Transplantation, IVF-ET) the effect is far from optimal outcome. Among factors that determine the successful outcome of the IVF-ET, ovarian reserve plays an important role. The ovarian reserve refers to the quantity of follicles and quality follicles in human ovary, and ovarian reserve represents women’s reproductive potential. During ART treatment, patients with lower follicle number will finally have lower number of oocytes retrieved, lower number of embryos. Poor oocyte quality leads to decreased the fertilization rate, embryo dysplasia, lower implantation rate, higher abortion rate, which all reduce the pregnancy rate. Therefore, exploring the mechanism of ovarian reserve and ovarian response is significant for improving the outcome of ART.The amyloid precursor protein (Amyloid Precursor, Protein, APP) and its proteoltsis products including sAPP a, sAPPβ, Aβ40 and Aβ42 in Alzheimer’s disease (Alzheimer Disease AD) has been investigated extensively, and was found to be expressed in granulosa cells and follicular fluid in porcine ovary. However, APP and its proteolysis products has not been investigated in human ovary function. Previous researches have shown that APP is a transmembrane protein involved in a series of cellular events including axon growth, cell proliferation, cell adhesion and cell migration. Appropriate expression level of APP plays a key role in maintaining cognitive function, and higher expression of APP in AD is considered to be able to impair cognitive function. In addition, in the nervous system, there is a certain correlation between the amount of estrogen level and APP content. APP has two kinds of metabolic pathways and produce the main four proteolysis products. One is the amyloid metabolic pathway, producing sAPPβ, Aβ40 and Aβ42, another is non-amyloid metabolic pathway, producing sAPPα. Although the Aβ40 and Aβ42 oligomers were confirmed as major components of amyloid plaques in AD patients, but Aβ40 and Aβ42 monomer is believed to have neuroprotective effect, playing an important role in maintaining neuronal development and function. The characteristics of sAPP a contain protecting neurons against damage, strengthening the recovery of neurons from ischemic injury, protecting cells against excitotoxicity and oxidative stress injury. Features of sAPPβ contain improving the proliferation of neural progenitor cells and promoting human embryonic stem cells’differentiation to neuron.In the present study, we collect granulosa cells samples and follicle fluid samples of IVF patients and analyse the relationship between APP and its proteolysis products and AFC, number of oocytes retrieved, pregnancy rate. We measured the effect of Aβ 40 or APP over-expression on mRNA levels of Follicle Stimulation Hormone Receptor (FSHR), Insulin-like Growth Factor-1(IGF-1), Insulin-like Growth Factor -1 receptor (IGF-1R), P450 aromatase (P450 arom), cholesterol Side-chain Cleavage Cytochromes P450 (P450scc), Steroidogenic Acute Regulatory protein (StAR) by culturing rat ovarian granulosa cells in vitro and explored the relationship between APP and its proteolysis products and treatment outcome in patients undergoing IVF/ICSI.Part 1:The expression of APP and its proteolysis products in human ovary and their association with ovarian reserve and ovarian response[objective]In the present investigation, we used methods of western blot, immunohistochemistry and ELISA to identify the presence of APP and its proteolysis products in human granulosa cells and follicle fluid. Aslo we analyzed the relationship between APP and its proteolysis products and AFC, number of oocytes retrieved. This can provide some initial evidence for further research of APP and its proteolysis products in ovarian function.To detect the expression of APP in ovarian granulosa cells and sAPPα、sAPPβ、 Aβ42、Aβ40 in follicular fluid and to evaluate their relationships to ovarian reserve and ovarian response in controlled ovarian hyperstimulation in patients undergoing IVF-ET cycles. To comprehend APP expression in human ovarian tissue and mouse ovarian tissue, endometrium, skeletal muscle.[Methods]1. Patients undrgoing IVF/ICSI from 2013 June to 2013 September whose ovarian stimulation was facilitated by long protocol were chosen in Center for Reproductive Medicine in Nanfang Hospital, patients with polycystic ovary system (PCOS), endometriosis, ovarian surgery history, serious male factor were excluded. A total of 51 cases were included. Patients’AFC were detected on day 3-5 during their menstral cycle. All patients underwent pretreatment with oral contraceptives pills in the last menstral cycle and pituitary downregulation with Diphereline in the midluteal. After the pituitary was completely downregulated, ovarian stimulation was initiated with gonadotropin. When at least two leading follicles’ diameter reached 18mm or larger, intramuscular HCG was administered. Oocytes were retrieved 36h after HCG sdministration and number of retrieved oocytes was recorded.2. Granulosa cells were isolated from follicular fluid after oocytes retrieved with Ficoll gradient centrifugation method. APP expression in granulosa cells was detected with Western blotting, and semiquantitative analysis was performed. Follicular fluid was obtained from dominant follicles during oocyte retrieval, and levels of sAPPα、 sAPPβ、Aβ42、Aβ40 in follicular fluid were detected by enzyme-linked immunosorbent assay (ELISA). The relationships between level of APP, sAPPα、 sAPPβ、Aβ42、AP40 and AFC and number of oocytes retrieved were compared. Pearson correlation analysis was used when the data were normally distributed, and spearman correlation analysis was used when the data were abnormally distributed.3. The ovarian tissue specimens of surgical removal were collected from the pathology department. APP location in human ovarian tissue was further detected with immnohistochemistry. Simultaneously APP location in mouse ovarian tissue, skeleton muscle and endometrium.[results]:1. Western blotting results demonstrated that APP was expressed ovarian granulosa cells in patients undergoing IVF/ICSI. ELISA results demonstrated that sAPPα、sAPPβ、Aβ42、Aβ40 were present in follicular fluid in patients undergoing IVF/ICSI.2. There is a significant and negative correlation between APP relative level in ovarian granulosa cells and AFC in patients undergoing IVF/ICSI, and the correlation coefficient is -0.448 (P<0.05). There is also a significant and negative correlation between APP relative level in ovarian granulosa cells and number of oocytes retrieved in patients undergoing IVF/ICSI, and the correlation coefficient is -0.467 (P<0.05). The correlation coefficients between levels of sAPPα、sAPPβ、Aβ42、Aβ40 and AFC in follicular fluid in patients undergoing IVF/ICSI were respectively -0.108,-0.269,-0.164,-0.089, but there are no statistical differences (P>0.05). The correlation coefficients between levels of sAPPα、sAPPβ、Aβ42、Aβ40 and number of oocytes retrieved in patients undergoing IVF/ICSI were respectively 0.032,-0.129,0.098,-0.047, but there are no statistical differences (P>0.05).3. Immunohistochemistry results demonstrated that APP was positively expressed in granulosa cells and cumulus cells of human ovarian tissue, and APP was positively expressed in mouse ovarian granulosa cells, oocyte and skeleton muscle and endometrium.[Conclusions]:1. APP is present in human (patients undergoing or not undergoing IVF/ICSI) ovarian granulosa cells, sAPPα、sAPPβ、Aβ42、Aβ40 are present in follicular fluid of patients undergoing IVF/ICSI. The reverse relationship between APP expression in ovarian granulosa cells and AFC and number of oocytes retrieved suggests that APP expression in ovarian granulosa cells are associated with ovarian reserve and ovarian response in patients undergoing IVF/ICSI.2. We have not found that there was a significant correlation between sAPPα、 sAPPβ、Aβ42、Aβ40 and AFC and the number of oocytes retrieved. For the reason that our sample is small, large sample is needed for further investigation.2. The result that expression of APP in mouse granulosa cells, oocyte and endometrium is positive suggests that APP may be associated with mouse fertility.The second part:The association of APP and its proteolysis products with pregnancy outcome in Assisted Reproductive Technology (ART)[Ojective]:To evaluate the relationship between APP expression in granulosa cells and levels of sAPPα、sAPPβ、Aβ42、Aβ40 in follicular fluid and pregnancy outcome in patients undergoing IVF/ICSI.[Mterials and methods]:1. Patients undrgoing IVF/ICSI from 2013 June to 2013 September whose ovarian stimulation was facilitated by long protocol were chosen in Center for Reproductive Medicine in Nanfang Hospital, patients with polycystic ovary system (PCOS), endometriosis, ovarian surgery history, serious male factor were excluded. Granulosa cells were isolated from follicular fluid after oocytes retrieved with Ficoll gradient centrifugation method. APP expression in granulosa cells was detected with Western blotting, and semiquantitative analysis was performed. Follicular fluid was obtained from dominant follicles during oocyte retrieval, and levels of sAPPα、 sAPPβ、Aβ42、Aβ40 in follicular fluid were detected by enzyme-linked immunosorbent assay (ELISA). Patients’parameters including number of two pronucleus、number of total embryos、number of 8 cell embryos with≤5% fragments、implantation rate、cancelled transplantation rate、clinical pregnancy rate was recorded.2. For data analyse,51 patients were first divided into a clinical pregnant group and a non-pregnant group based on their obtaining pregnancy or not and APP level sAPPα、sAPPβ、Aβ42、Aβ40 levels are compared between the two groups. Then 51 patients were divided into three groups according to the percentile of their Aβ40 concentrations:low concentration group (<145.87pg/ml) (n=12), medium concentration group (145.87-270.98 pg/ml) (n=27), high concentration group (>270.98 pg/ml) (n=12). Number of two pronucleus、number of total embryos、 number of 8 cell embryos with≤5% fragments、implantation rate、cancelled transplantation rate、clinical pregnancy rate were compared between the three groups.4. Continuous variables were tested for normal distribution using method of moment. Results are expressed as means and SD. Comparisons between the groups were calculated by t-test or One Way ANOVA if the data were normally distributed and by Wilcoxon Sign Rank test or Kruskal-Wallis test if the data were abnormally distributed. Statistical analysis was performed by SPSS 16.0. Statistical significance was set at P<0.05.[Results]:1. Aβ40 level in clinical pregnant group was significantly higher than in non-pregnant group (228.60+53.21 pg/ml vs.178.90+76.59 pg/ml, P<0.05). But no statistical differences exist between the two groups in APP, sAPPα、sAPPβ、Aβ42 (2.16+2 vs.1.24+1.08,148.99+42.17 ng/ml P=0.105; vs. ng/ml P=0.627; 157.50 +57.50,127.16+35.02 ng/ml vs.127.08+31.34 ng/ml, P=0.993 pg/ml vs.47.51; 58.31+75.33+19.59 pg/ml, P=0.492).2. The pregnancy rates in Aβ40 low、medium、high concentration groups were respectively 16.6%(2/12)、9.2%(16/27)、58.3%(7/12), the pregnancy rates in medium、high concentration groups were significantly lower than low concentration group (P<0.05). The cancelled transplantation rates in Aβ40 low、medium、high concentration groups were respectively 9%(1/11)、0%(0/27)、9%(1/11), the cancelled transplantation rate in medium concentration group was significantly lower than low、high concentration group (P<0.05). The implantation rates in Aβ40 low、 medium、high concentration groups were respectively 21%(4/19)、49%(25/51)、 33%(8/24), the implantation rate in medium concentration group was highest but there is no statistical significance (P>0.05).3. There were no significant differences between the three groups in two pronucleus、number of total embryos、number of 8 cell embryos with≤5% fragments and proportion of number of 8 cell embryos with≤5% fragments to total embryos.[conclusions]:Appropriate Aβ40 levels in follicular fluid may be associated with IVF pregnancy outcome. We have not found that APP, sAPPa, sAPP β, Aβ42 are significantly associated with IVF pregnancy outcome. Considering that the sample is relatively small, further investigation with large sample is needed.The third part:The effect of Aβ40 on the proliferation and molecules involved in steroidogenesis in rat granulosa cells cultured in vitro[Objective]:To study the impact of different concentrations of Aβ40 on the proliferation of rat granulosa cells and the expression of molecules involved in steroidogenesis to clear out that Aβ40 may affect oocyte development by which pathway.[Methods]:21-day female rat ovarian granulosa cells were cultured in vitro. Different concentrations of Aβ40 (Opg/ml、100 pg/ml、200 pg/ml、300 pg/ml、400 pg/ml) were added into the cultured cells and after 24h the proliferative situation of different concentration groups were detected by CCK-8 and mRNA levels of molecules involved in steroidogenesis including FSHR, IGF-1, IGF-1R, P450 arom, P450scc, StAR were measured by RT-PCR.[Results]:1. When compared with control group, Aβ40 concentrations 200、300、400 pg/ml could promote rat ovarian granulosa cells proliferation (P<0.05).200 pg/ml Aβ40 promoted granulosa cells proliferation most (proliferative rate 114.53%), the second was 300 pg/ml Aβ40 (proliferative rate 110.06%),100 pg/ml Aβ40和400 pg/ml promoted granulosa cells proliferation worst (proliferative rates respectively 106.77% and 106.86%).2. When compared with control group (Opg/ml Aβ40), both of 200 pg/mlAβ40 and 400 pg/ml Aβ40 can significantly up-regulate FSHR、StAR、P450arom gene expression (P<0.05); 200 pg/ml Aβ40 (P<0.05) but not 400 pg/ml Aβ40 can significantly up-regulate IGF-1、P450scc gene expression(P>0.05).400 pg/ml Aβ40 can up-regulate IGF1R gene expression (P<0.05), but 200 pg/ml Aβ40 can not up-regulate IGF1R gene expression (P>0.05)[Conclusions]1. Aβ40 may be able to improve oocyte quality to benefit clinical pregnancy by promoting the proliferation ability of granulosa cells.2. Aβ40 may be able to improve oocyte quality to benefit clinical pregnancy by promoting the gene expression of FSHR、IGF-1、IGF-1R、P450 arom、P450scc、 StAR.4. Different concentrations of Aβ40 can regulate these molecules differently. Excessive concentration Aβ40 may have a negative impact on ovarian granulosa cells, indicating that only appropriate concentration of Ap40 is beneficial to granulosa cell, oocyte and pregnancy.The fourth part:The effect of APP over-expression on molecules involved in steroidogenesis in rat granulosa cells cultured in vitro[Objective]To study the impact of APP over-expression on molecules involved in steroidogenesis in ovarian granulosa cells to explore that APP can influence oocyte development by which pathway.[Methods]21-day SD female rats were intraperitoneally injected with PMSG 50IU per rat and after 48h ovarian granulosa cells were collected. The cultured cells were divided into three groups:control group:culture medium were added nothing; empty vector group:culture medium were added empty vector plasmid and transfection reagent Lipo 2000; APP over expression group:culture medium were added APP eukaryotic expression plasmid and transfection reagent Lipo 2000. The expression change of APP is detected by Western blotting to test transfection rate. After APP was over expressed, the mRNA level changes of molecules involved in steroidogenesis including FSHR、IGF-1、IGF-1R、P450 arom、P450scc、StAR and apoptosis molecules Bax、Bcl2 by RT-PCR.[Results]After APP was over expressed:levels of IGF-1 mRNA was inhibited significantly (P<0.05) and declined to one fourth of control group; levels of mRNA of P450arom and P450scc were significantly up-regulated (P<0.05) and increased to 15 times of control group; mRNA levels of StAR increased sinificantly (P<0.05) and rose to 7 times of control group; mRNA levels of IGF-1 R and FSHR increased sinificantly (P<0.05) and rose to about 2 times of control group; mRNA levels of Bcl2 and Bax increased sinificantly (P<0.05) and rose to about 2 times of control group.[Conclusions]Over-expression of APP may be able to influence oocyte development by influencing these molecules involved in steroidogenesis including FSHR, IGF-1、 IGF-1R、P450 arom、P450scc、StAR and apoptosis molecules Bax、Bcl2.