Research On The Effect Of Gonadotropin-releasing Hormone Antagonist In Preventing Chemeotherapy-induced Pof

Background:In recent years, the increased survival rate of cancer patients with chemotherapy drugs due to an increased emphasis on iatrogenic damage. Ovaries are female reproductive system in the most sensitive parts of the chemotherapy, high dose, prolonged chemotherapy can damage tissue, cause ovarian failure, so the young women with menopausal syndrome and signs of premature aging diseases, infertility and premature ovarian failure (Premature overy failure POF), seriously affecting their quality of life and self-confidence. Therefore, how cancer treatment while preserving the ovaries and the reproductive function of women, with huge medical needs, is a clinically important issue worthy of further study. For the ovarian toxicity of chemotherapy drugs, domestic and foreign scholars have done a series of studies for many year, the purpose is to find an effective and safe method to protect the ovaries, so that chemotherapy drugs to minimize the damage to the ovary. The present study focused on the direction of gonadotropin-releasing hormone analogue (Gonadotropin-releasing-Hormone analogues GnRHa) application and organizational aspects of cryopreservation. Tissue cryopreservation - autologous transplantation (CryoPreservation autotransPlantation) there is a technical, safety, ethical risks, the scope is too narrow and clinical application is not possible, so GnRHa become a focus of research.Gonadotropin-releasing hormone (Gonadotropin-releasing-Hormone GnRH) analogues inhibit the female gonadal axis, inhibiting mitosis follicles retain the most primitive stage of follicles. In a "resting" primordial follicles to avoid damage of chemotherapy, enhanced ovarian reserve, which play a protective effect on ovarian function. Gonadotropin-releasing hormone antagonist (Gonadotropin releasing Hormone Antagonist GnRHant) derivatives as GnRH analogues, one of the female gonadal axis has a strong inhibitory effect, and side effects, dosage and administration time of less than GnRH agonists, is the ideal GnRH agonist treatment of alternative medicine. A number of studies abroad that GnRHa on ovarian function has significant protective effect, still does not support some of the chemotherapy of GnRHa on the ovary has a protective effect, that the mechanism of this protection is still not clear. This study was designed to explore the prophylactic use of GnRH antagonists in ovarian tissue structure on chemotherapy, ovarian endocrine function, levels of follicle development, oocyte fertilization, embryo development played a protective effect and mechanism for clinical cancer treatment, bone marrow transplantation and autoimmune disease and high dose of chemotherapy before the ovarian function using GnRH antagonist protection to provide a theoretical basis.Objective:1 Chemotherapy drugs and gonadotropin-releasing hormone antagonist combination chemotherapy on ovarian reserve ability, for the gonadotropin-releasing hormone antagonist to provide the theoretical basis for clinical application;2 Gonadotropin-releasing hormone antagonist by combination chemotherapy on reproductive function and growth and development of offspring, revealed gonadotropin-releasing hormone antagonist in patients on chemotherapy, long-term quality of life;3 Gonadotropin-releasing hormone antagonist on the prevention of chemotherapy damage the role of POF and ovarian granulosa cells associated mitochondrial apoptotic pathway, gonadotropin-releasing hormone antagonist to clarify the protection mechanism;4 Gonadotropin-releasing hormone antagonist on cultured human luteinized granulosa cells function.Methods:1. Construction and evaluation of chemotherapy in animal models of traumatic POFSD sexually mature rats, intraperitoneal injection of different concentrations (10mg/kg, 20mg/kg, 30mg/kg) cyclophosphamide (CTX) 20天. After treatment, said the body weight, ovarian, uterine wet weight; by vaginal smears detect estrous cycle; rat eyeball blood, ELISA method to detect the serum E₂, FSH levels; HE staining, microscope counting the total number of follicles and follicle number; of important organs: ovary, heart, liver, kidneys, lungs for pathology testing.2. Analysis of gonadotropin-releasing hormone antagonist (Cetrorelix) dose - effectSD sexually mature rats, intraperitoneal injection of different concentrations (1ug / d, 3ug / d, 5ug / d) Western song Rick 10 days after stopping eyeball blood, ELISA method to detect serum levels of FSH, LH level; stop drug 5 days and 10 days, twice again eyeball blood, ELISA method to detect serum levels of FSH, LH level.3. Cetrorelix on the impact of chemotherapy on ovarian functionSD sexually mature rats, intraperitoneal injection 5ug / d 10 days after Rick West Music, Cetrorelix 5μg / d and CTX20mg / (kg·d) while ig 20d. 10 days after drug withdrawal and drug withdrawal, respectively, said the body weight, ovarian, uterine wet weight; by vaginal smears detect estrous cycle; rat eyeball blood, ELISA method to detect the serum E₂, FSH levels; HE staining was counting the total number of follicles and follicle number; of ovarian pathology were detected.4. Detection of Cetrorelix chemotherapy on reproductive function and growth and development of offspringSD sexually mature rats, intraperitoneal injection 5ug / d 10 days after Rick West Music, Cetrorelix 5μg / d and CTX20mg / (kg·d) was given intraperitoneally, for 20 days. 10 days after drug withdrawal, according to the male and female rats with a 2:1 ratio of cross cage the next morning for vaginal sperm smear test to detect vaginal plug and sperm for the pregnancy signs found on the same day as the "pregnant" day. Records of pregnant mice in each group G10, 12,14,17,20 body weight changes, and pregnant rats at 20 days of gestation were killed off neck, abdominal examination pregnancy outcomes. Record the number of uterine implantation, microscope counting the number of bilateral ovarian corpus luteum, litter litter size, live births, absorbed fetus and stillbirth; calculated mating, pregnancy, fetal absorption rate, stillbirth rate; live births, fetal rat each record body weight, body length, tail length, sex ratio; check the appearance of fetal mice, Bouins fixed, check whether the abnormal fetal organs, transparent alizarin red bone staining with or without bone deformities.5. Detection of gonadotropin-releasing hormone antagonist on the prevention of chemotherapy damage the role of POF and ovarian granulosa cells associated mitochondrial apoptosis pathwayTransmission electron microscopy detection of ovarian granulosa cell ultrastructure; use of fluorescent probe JC-1 mitochondrial membrane staining granular cells, laser scanning confocal microscope granular cell mitochondrial membrane potential; in situ end labeling (TUNEL) Determination of ovarian granulosa cells apoptosis, apoptotic index; immunoblotting (Western blotting) to detect Cyt-C in mitochondria and cytoplasm of ovarian and spatial distribution of SP immunohistochemistry to detect Cyt-C in the expression of granulosa cytoplasm; while immunohistochemistry method for particle detection of apoptosis indicators, including the apoptosis-related protein BCL-2, BAX, Caspase-3 expression.6. Detection of gonadotropin-releasing hormone antagonist on cultured human luteinized granulosa cells functionCollected in the 2009,10-2009.12 Xijing Hospital assisted reproductive center line in vitro fertilization - embryo transplantation in patients with follicular puncture mature granule cells, and separation, purification, identification and cultivation. Different concentrations of CTX (10,100,1000ng/mL) were incubated with human granulosa cells after 24h, MTT assay cell survival in each group, ELISA assay granule cells induced by E₂ and P₄ level after 12 hours. Western music of different concentrations Rick Cetrorelix (10,100,1000ng / mL) were incubated in the joint 24 hours before chemotherapy, and chemotherapy in 24 hours, MTT cell viability test in each group, ELISA assay granule cells induced by E₂ and P₄ levels.Results:1. Chemotherapy was successfully constructed and stable animal model of traumatic POFAfter screening a series of drug concentrations, the results show that the toxic effects of CTX in the ovary was positively correlated with dosage, which 20mg/kg.d 20 days of continuous use experimental doses of most POF pathology: Ovarian fibrosis, hemorrhage, necrosis, the level significantly reduced the number of follicles, particularly mature follicles and growth of follicles; rat estrous cycle disorder, manifested as persistent diestrus vaginal smears showed a large number of mainly white; low levels of serum E₂, FSH levels rise; the same time heart, liver, kidney, lung pathological changes does not occur in vital organs. Animal models, clinical dosage and time to maintain a minimum safe dose of chemotherapy drugs entirely consistent with the requirements. The model into a model a short time, ovarian histology, endocrine changes associated with human POF very consistent, subject-based experimental research laid the foundation for the next step.2. Analysis of gonadotropin-releasing hormone antagonist (Cetrorelix) dose - effectCetrorelix drug - dose effect analysis showed that the cetrorelix reversible inhibition of the gonadal axis, and was positively correlated with dosage, which 3ug / d, 5ug / d, 10-day continuous use can be completely inhibited by gonadal axis, showing FSH, LH low, after stopping FSH, LH hormone levels rose rapidly, within 10 days may be back to normal, so the Western song Rick 3ug / d, 5ug / d are safe and effective dose.3. Cetrorelix on the impact of chemotherapy on ovarian functionCetrorelix combined with chemotherapy before and chemotherapy, the preservation of the most primordial follicles in ovarian tissue, making the total number of follicles remain basically unchanged, were quiescent follicles from chemotherapy drugs can damage the original follicle to continue after stopping mature; the same time reduce ovarian fibrosis, hemorrhage, necrosis significantly reduce the protection of ovarian reserve; serum sex hormone levels returned to normal; ovarian wet weight and weight gain in rats, rats generally improved.4. Cetrorelix chemotherapy on reproductive function and growth and development of offspringPregnancy after chemotherapy in rats a temporary decline in body weight and pregnancy rate declined, there still births and fetal absorption in fetal and Western song Rick significant improvement after combination therapy after chemotherapy, pregnant rats, body weight, improve pregnancy rates, lower stillbirth rate, birth rate of absorption. Each drug group with bilateral ovarian corpus luteum count the number of uterine implantation, fetal parental live weight, body length, tail length, sex ratio (male%), offspring shape, internal organs and bones is not affected.5. Detection of Cetrorelix POF chemotherapy injury prevention and the role of mitochondrial pathway of apoptosis of ovarian granulosa cells of the correlationThe results suggest that detection of apoptosis: growth after chemotherapy of ovarian follicles and mature follicular cell apoptosis, and apoptosis of granulosa cells of primordial follicles is lower; Cetrorelix primordial follicles after combination therapy was significantly increased, and can significantly reduce Chemotherapy-induced apoptosis of ovarian granulosa cells of primordial follicles. Found by observing the ultrastructure of granulosa cells, granulosa cells after chemotherapy, significant characteristics of apoptosis, the most obvious change significantly reduced the number of mitochondria, swelling, cristae rupture or vacuolization; and Cetrorelix mitochondria were increased after combined treatment. Detection of mitochondrial membrane potential of granule cells: The results showed that rat ovarian granulosa cells after chemotherapy in mitochondrial membrane potential decreased significantly, Rick West music combined potential of mitochondria than normal after the application. Detection of Cyt-C in the cytoplasm of ovarian spatial distribution of mitochondria and found that: chemotherapy, granulosa cell damage mitochondrial structure and membrane potential, and makes the Cyt-C in the cytoplasmic accumulation, stimulate apoptosis cascade reaction, prevention of Rick West Music of applications to effectively prevent the Cyt-C spatial distribution of changes will kill in the earlier stages of apoptosis. BAX, caspase-3, Bcl-2 results suggest that apoptosis-related protein: Cetrorelix after the combination therapy can significantly reverse chemotherapy-induced BAX, caspase-3 in follicular granulosa cells of high expression and low expression of Bcl-2 .6. Gonadotropin-releasing hormone antagonist on cultured human luteinized granulosa cells functionDifferent concentrations of CTX and human granulosa cells incubated in human granulosa cells of FSH-induced survival and E₂ levels increased with increasing concentration of chemotherapy decreased; and Rick co-incubated with different concentrations of opera in the 24 hours before chemotherapy, the results show that human granulosa cells and FSH-induced E₂ levels increased with the concentration Rick opera.Conclusion:1. Experiments with CTX 20mg/kg.d 20 days continuous use experimental doses of chemotherapy was successfully constructed and stable animal model of traumatic POF. The model into a model a short time, ovarian histology, endocrine changes associated with human POF very consistent, and dose and time to clinical doses of chemotherapy drugs to maintain minimum safety requirements entirely consistent;2. Cetrorelix preventive applied before chemotherapy, on the rat ovarian function, reproductive function, embryonic development has a range of protective effects, enhanced ovarian reserve capacity, to improve ovarian function and fibrosis, necrosis. Rick West Music also can improve the preventive application of pregnancy after chemotherapy in rats, reduced the incidence of stillbirths and birth rate of absorption, suggesting that gonadotropin-releasing hormone antagonist in patients with chemotherapy can improve long-term quality of life;3. Cetrorelix POF chemotherapy injury prevention role and inhibition of ovarian granulosa cell mitochondrial pathway of apoptosis and apoptosis related genes BAX, caspase-3, Bcl-2 expression is closely related. Envisaged to inhibit the ovarian granulosa cell apoptosis in ovarian gene into cells, the POF may be a very promising method of gene therapy, the purpose of ultimately cure POF, it will be a challenging and attractive subject.4. Cetrorelix on cultured human luteinized granulosa cells play a direct role to improve the endocrine function of granulosa cells induced by chemotherapy reduced.