The Research On Body Mass Index、Metabolism Of Glucose And Lipid And The Possible Mechanisms In Patients With Endometriosis Of In Vitro Fertilizition-Embroy Transfer Cycles

Endometriosis (endometriosis, EMS) leads to high incidence of infertility. In vitro fertilization-embryo transfer (in vitro fertilization and embryo transfer, IVF-ET) is an effective technology to help these patients get pregnant. But studies showed that poor embryo quality was obtained on EMS patients receiving IVF-ET treatment. Furthermore, the embryo implantation and pregnancy rates were lower in EMS patients compared to the patients with tubal adhesion or male factor induced infertility. The relevant mechanisms are unclear.Recent findings indicate that patients with EMS have relatively low BMI (body mass index, BMI). Fat tissue is the most important factor for weigh, so EMS may be associated with the pathogenesis of body fat and glucose metabolism abnormalities. In addition, there are many study findings confirm that the expression levels of leptin (a critical factor in fat metabolism released by adipose tissue) were abnormal in serum, follicular fluid and peritoneal fluid. So it is possible that leptin may regulate the reproductive and endocrine systems via some unknown mechanism. Therefore, leptin may play roles in regulating lipid metabolism/body weight and affecting development of fertilized egg, endometrial receptivity and embryo implantation. It is also possible that the growth of endometrial tissue was related to the lipid metabolism disorder and endocrine abnormalities. Both interactions may lead to the EMS patient’s natural progesterone pregnancy and IVF pregnancy rates decline. Currently, there is no conclusion reached in this field due to the lack of literature and studies.In this study, the clinical data of the EMS patients who accepted IVF-ET progesterone therapy were analyzed to understand the correlation between progesterone EMS patient outcomes and BMI. In addition, EMS patient’s blood, follicular fluid and granulosa cells were collected to analyze the following:leptin, blood glucose, lipids, sex hormone levels, granulosa cells leptin mRNA/receptor mRNA expression, phosphorylation of STAT3 and metabolizing enzymes GLUT4 (GLUT -4). This study explored the relationship between leptin levels in patients with EMS and sugar, fat metabolism and progesterone IVF outcomes. Furthermore, the internal mechanism of the disease pathogenesis and treatment of pregnancy progesterone decline were studied. This study provided the basic theory to improve egg quality and reproduction-assisted pregnancy rates.Chapter 1:Relationship between body mass index and outcome of in vitro fertilization and embryo transfer cycle in patients with endometriosis[Background]EMS is the kind of disease that endometrial tissue grows in other parts of the body outside the uterine cavity. EMS starts at the cellular level and its symptoms include pelvic pain and infertility. Although it is a benign disease, the disease is aggressive and recurrent, similar to "malignant" biological behavior. EMS patients suffer severe dysmenorrhea, mental and psychological stress, and enormous economic pressure to the patient and family, which seriously affect the health and quality of life for the patients with EMS. EMS has become a common gynecologic disease. With the use of the diagnostic laparoscope, incidence rate is still rising. In addition, recent studies have found that the EMS patients with lower BMI resulted in poor outcome after accepted the IVF or intracytoplasmic sperm injection (Intracytoplasmic sperm injection, ICSI). There was no previous study make sure of the relationship between EMS and BMI, especially lack of research on progesterone therapy on EMS patients undergoing IVF.[Study Objectives]In order to discover and understand the relationship bwtween the BMI and EMS, In this part of my study we analyzed and compared the clinical data and lab results of patients who have mild EMS, moderate or severe EMS with patients without EMS. Furthermore, we want to study the correlation between BMI and ovarian response, pregnancy rate and miscarriage rate after IVF-ET treatment.[Study Methods]This study was approved by the ethic committee of the hospital and each patient consent was acquired before the patient was enrolled in this study. A total of 261 patient records from January 2011 to September 2012 at Peking University Shenzhen Hospital Reproductive Medicine Department were reviewed. All patients received surgery (laparoscopy or open surgery, excluding pregnancy-related surgery) within two years before they received reproductive assist technique. According to the criteria made by the American Society of Reproductive Medicine, the patients were divided into 3 groups based on their intra-operative pathology findings:1) Control group (no endometriosis):N=157; 2) the experimental group 1:patients with endometriosis of stage Ⅰ-Ⅱ (mild endometriosis):N=57; 3) the experimental Group 2:patients with endometriosis of Stage Ⅲ-Ⅳ(moderate to severe endometriosis):N=47.All patients were treated with long protocol of pituitary down-regulation, continuous use of recombinant follicle stimulating hormone (Gonal-F 75IU/Serono Inc.) from 3rd to 8th day of menstrual cycle. The injection of rFSH was stopped when at least 2 follicles were larger than 18 mm in diameter. Approximately 36 ± 1h after the injection of recombinant human chorionic gonadotropin (Ovidrel 250ug/Serono Inc.), oocytes were retrieved trans-vaginally. Simultaneously,40-60mg progesterone or combined with HCG was injected every day for luteal support. About 48-72h after fertilization (IVF/ICSI),1 to 3 excellent embryos was picked out and transferred. And the following parameters were studied:total dose of gonadotropin (Gn) used by each patient, days of stimulation by Gn, HCG day E2 levels, number of oocytes retrieved, number of embryos transferred, and relationship between the patient’s BMI and clinical outcomes.[Results]1. There was no significant difference between these three groups in age, duration of infertility and infertility type (primary or secondary infertility), P>0.05.2. Comparing emGroup 2 to the control group, Gn usage was increased (39.53 ± 18.33 vs 32.51 ± 13.15 rFSH). There were significant difference (p<0.05) between Group 2 and the control group in HCG day E2 level (1873.12 ± 1313.26 vs 2428.88 ± 1308.18 pg/ml), number of oocytes (8.15±4.68 vs 11.17±6.46), number of embryos transferred (1.98 ± 0.57 vs 2.21 ± 0.46).3. Comparing the experimental Group 1 and the control group, there was no relationship between BMI and pregnancy rate (36.8% vs.38.2%), p>0.05. Comparing Group 2 to the control group and the experimental Group 1, there were significant differences between BMI (19.63 ± 1.95 vs 21.40 ± 3.02 kg/m2; 19.63 ± 1.95 vs 20.27 ± 2.11 kg/m2) and pregnancy rate (19.1 vs 38.2%; 19.1 vs 36.8%).[Conclusions]1. BMI in EMS patients:patients with moderate to severe EMS had lower BMI compared to patients had mild or no EMS. Patient’s BMI decreased as the aggravation of the disease.2. EMS patient’s BMI and outcomes of assisted reproductive technology: patients with moderate to severe EMS had poor IVF/ICSI pregnancy outcomes.The changing trend of BMI of the EMS patient was consistent with IVF clinical outcomes. The BMI and clinical pregnancy rates of patients who had moderate to severe EMS group were lower than in the mild EMS group and the control group, while BMI in mild EMS group and clinical pregnancy rate were not statistically significant from the control group. Therefore, BMI is a factor which influent the outcomes of pregnancy in EMS patients. Therefore, maybe pregnancy rate can be increased by increasing BMI through diet and exercise as to patients with moderate to severe EMS.3. EMS and ovarian response:infertile patients associate with moderate to severe EMS often have poor ovarian response when stimulate by ovulation induction drugs and its reactivity decreased, at the same time we may obtain less number of eggs.Chapter 2:Relationship between leptin levels and lipid metabolism in Infertile patients with endometriosis[Study background]EMS associate infertility seriously affects patient health and quality of life. The pathogenesis is not currently unclear. In our previous study (results in Chapter 1), it was found that the BMI and assisted reproductive technology pregnancy outcomes were lower in the patients with moderate to severe EMS, comparing to the control group. Therefore, the onset and progression of EMS may be associated with glucose and lipid metabolism abnormalities.[Study objectives]In this chapter, we focused on the analysis of serum and follicular fluid levels of the following hormones on the egg retrieval day:glucose, lipid, sex hormones, insulin and leptin. The results were analyzed with patients’ clinical outcomes, etc. abdominal circumferences, hip circumference and BMI. The results demonstrated the roles of leptin and glucose/lipid metabolism in EMS patients.[Study material and methods]1.After hospital ethics committee approval and obtained patient consent, then a total of 21 patient’s records from June 2013 to December 2013 at Peking University Shenzhen Hospital Reproductive Medicine Department were reviewed. All patients received assisted reproductive treatment in no more than 2 years prior IVF, either laparoscopic or open surgery. According to ASRM, the patients were divided into 3 groups::1) Control group (no endometriosis):N=7; 2) the experimental group 1: patients with endometriosis of stage Ⅰ-Ⅱ (mild endometriosis):N=7; 3) the experimental Group 2:patients with endometriosis of Stage Ⅲ-Ⅳ(moderate to severe endometriosis):N=7;2.Ovulation induction treatment:all patients underwent ovulation induction (the same steps as the patients studied in Chapter 1)3. Measurement of Clinical parameters:On the day of oocytes retrieval, the following were measured by assigned staff:weight, height, hip circumstance, waist circumstance. BMI is calculated as BMI=weight (kg)/height (m) 2.4.Measurement of insulin, glucose, lipid and sex hormones in serum and follicular fluid:On the day of egg retrieval, fasting serum and follicular fuid were collected and measured using automatic biochemical analyzer.5.Measurement of leptin in serum and follicular fluid:On day of egg retrieval, fasting serum and follicular fluid specimens were collected. After centrifugation, they were placed in -70 ℃ freezer. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) assay was used to test leptin level in serum and follicular fluid.6.Statistical methods:SPSS 13.0 software was used to analyze data. As to measurement data we use oneway ANOVA. Data were expressed as mean ± standard deviation. LSD method was used when variance was homogeneity among the three groups, and Welch method was used when when variance was not homogeneity among the three groups. P<0.05 was considered statistically significant.Correlation using bivariate correlation analysis was used, P<0.05 was considered statistically significant.[Study results]1. There was no statistical difference when compared the three groups of patients in the following clinical indicators:height, weight, hip circumference, BMI differences (P> 0.05). The control group had higher abdominal circumference compared to the experimental group 1 (88.14 ± 8.75 vs 75.86 ± 7.10cm, P=0.022)2. There was no statistical difference when compared the three groups of patients with serum insulin, glucose, lipids and sex hormones, serum glucose level of difference progesterone, testosterone, triglycerides, high density lipoprotein and low-density lipoprotein (P> 0.05). Insulin and cholesterol levels in the experimental group 1 were lower than the control group. The luteinizing hormone levels in the experimental group 2 were higher than the control group. The serum estradiol concentration in experimental group 2 was lower than that in experimental group 1 and the control group (P<0.05).3. There were no statistical differences comparing follicular fluid glucose, triglycerides, cholesterol, high density lipoprotein, low density lipoprotein and luteinizing hormone level among the three groups (P> 0.05). Insulin levels in the experimental group 1 and the experimental group 2 were lower than the control group (3.21 ± 1.73 vs 7.03 ± 4.50 uIU/ml; 2.95 ± 2.52 vs 7.03 ± 4.50mmol/1, P= 0.046). Compared to the control group, the experimental group 2 had higher testosterone levels in follicular fluid (P<0.05).4. The serum leptin levels were significantly different in the three groups (the experimental group 2> the experimental group 1> control group) (3667.89 ± 495.18 vs 2286.61 ± 304.18 vs 1080.70 ± 546.37 pg/ml) (P<0.01).The leptin levels in the follicular fluid were significantly higher in the experimental group 2, compared to the experimental group 1 and the control group (1906.71 ± 373.56 vs 1102.96 ± 159.67 pg/ml; 1906.71 ± 373.56 vs 874.67 ± 423.64pg/ml, P<0.01).5. The Serum leptin and insulin levels had a negative correlation trend, but not statistically significant (P= 0.092). The follicular fluid leptin and insulin levels were negatively correlated, Pearson correlation coefficient was 0.46 (P= 0.034).[Study conclusion]1. There were no significant differences in the three groups of patients in terms of BMI, hip circumstances and other clinical indicators. However, EMS infertile patietns had lower leptin levels during the IVF cycles (P<0.01), leptin levels increased with EMS progression. Ovulation inducing drugs may induce excessive secretion of leptin in EMS patients, and too much leptin may damage the egg and embryo to some degree. The more severe the disease, the greater the impact on leptin secretion, which in turn affect the quality of eggs and embryos.2. While no differences in the three groups of patients in terms of BMI, hip circumstances and other clinical indicators, the moderate to severe EMS patient group had significantly higher level of follicular fluid leptin.The follicular fluid leptin levels are critical for egg development, so the excessive amount of leptin in EMS patients undergoing IVF treatment in the EMS may cause injury and follicular maturation disorder in granulosa cells, which will lead to decline in the quality of eggs and embryos and affect the EMS women’s embryo implantation and pregnancy rates.3. Follicular fluid leptin and insulin levels were negatively correlated in EMS patients (correlation coefficient r=0.46, P<0.05). The serum leptin and insulin levels were also negatively correlated trend, but not statistically significant. This result may be due to the small sample in this study. Therefore, when EMS infertile patients receive IVF, there was still a leptin-Insulin feedback pathway. The abnormal serum and follicular fluid leptin levels in patients with EMS may lead to excessive suppression of insulin secretion, which may cause glucose and lipid metabolism disorders.4. There was no significant difference in lipid panel between the mild EMS mild group and other groups. Although we found that the lipid levels in the mild EMS patients were normal, larger sample size is needed to reach a conclusion.Chapter 3:Relationship of leptin, its receptor expression and STAT signaling pathway in the granulosa cells of in infertility patients with endometriosis[Study background]The mechanisms about EMS induced infertility were unclear. In addition, the mechanisms of EMS induced infertility were different in different stage EMS induced infertility.The major cause are (1) a change in the pelvic anatomy of gamete transport barriers; (2) ovarian dysfunction; (3) endometrial abnormalities; (4) fertilization anomalies; (5) abnormal immune and inflammatory responses.[Study objectives]To analyze the different mechanism furtherly of how the level of leptin and leptin receptor in EMS patients affect egg quality. It is our focus to understand if there were lipid and glucose metabolism disorders in granulosa cells. In this study, the levels of leptin mRNA and leptin receptor (ob-R) mRNA expression in granulosa cells were studied. The expression of phosphorylation of STAT3 (a key enzyme in glucose metabolism) and glucose transporter protein -4 (gluscose transporter 4, GLUT4) were studied.To analyze the EMS Patients leptin levels and the incidence of abnormal glucose metabolism and further reveal the mechanism of abnormal low implantation rates and low pregnancy rate progesterone treatment in EMS patients.[Study material and methods]1. Study patients:the same as the patients in Chapter 2.2. Ovulation induction treatment:All patients underwent ovulation induction (the same as the patients in Chapter 1).3. Specimen collection:Patient’s follicular fluids were collected on COH day which include the EMS patients and the control group of patients. The follicular fluids in the high quality egg were collected (follicular diameter>=17mm, clear follicular fluid without bloodshed and with oocytes in the fluid) and centrifuged. The granulosa cell layer were extracted and stored in -70℃ freezer.4. Granulosa cells Leptin mRNA and ob-R mRNA levels:Using reverse transcription-polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) assay to detect Leptin mRNA, ob-R mRNA level, compared with the control group.5. Determination of p-STAT3, GLUT4 content in granulosa cells:Protein extracts from sample granulosa cells were used to measure p-STAT3, GLUT4 protein levels, using EMS histone immunoblotting (Western blotting).6. Statistical Methods:Using internal reference as the criteria, the optical density of GLUT 4 and p-STAT3 were respectively averaged. SPSS 13.0 software was used to analyze data. As to measurement data we use oneway ANOVA. Data were expressed as mean ± standard deviation. LSD method was used when variance was homogeneity among the three groups, and Welch method was used when when variance was not homogeneity among the three groups. P<0.05 was considered statistically significant.[Study results]1. The comparison of leptin mRNA and OB-RLmRNA level in ovarian luteinizing granulosa cells:There was expression of leptin mRNA and OB-RLmRNA in all three groups. Using GAPDH as an internal reference and taking the relative integral values, we discovered that there were no significant differences in the expression levels of leptin mRNA and OB-RLmRNA in all three groups (p>0.05).2. Comparison of the levels of p-STAT3 and GLUT4 in the three groups:The levels of p-STAT3 and GLUT4 in the three groups were tested using Western Blotting. All patients in the three groups had expression of p-STAT 3 and GLUT 4. The levels of p-STAT 3 were significantly different among the three groups. The experiment group 2 had highest p-STAT 3 level, followed by the experiment group 1. The control group had the lowest p-STAT 3 level. The levels of GLUT 4 were significantly different among the three groups. The control group had the highest GLUT 4 level compared to the experiment group 1 and the experiment group 2 (p<0.05). There was no difference between the group 1 and the group 2 (p>0.05).[Study conclusion]1. There were no significant differences in mRNA levels of leptin and OB-RLmRNA EMS levels in patients from the three groups, indicating that changes in ovarian granulosa cell function in patients with EMS were not achieved by regulating the level of leptin and its receptor.2. After ovulation treatment in EMS patients, the expression of p-STAT3 was significantly increased compared with the control group. This trend increased along with the progression of EMS. Therefore, elevated levels of p-STAT3 in the granulosa cells of patients with EMS may indicate thatthe downstream of the leptin signal transduction pathway resulted in limited cell function or damage.3. The levels of GLUT 4 were significantly different among the three groups. The control group had the highest GLUT 4 level compared to group 1 and the group 2 (p<0.05). There was no difference between the group 1 and the group 2 (p>0.05). EMS patients’ glucose transportation and uptake ability may decline compared to non-EMS patients. This mechanism may be one of the factors leading to decline in egg quality and maturity obstacles and granulosa cell secretory function in patients with EMS oocytes decline. In addition, the combined results of our second part of the study showed that the EMS patients had the same trend in follicular fluid insulin level and granulosaa cells GLUT.4. The conclusion is proved once again that insulin regulates GLUT4 glucose metabolism by activating or adjustment.