| Premature ovarian failure (POF) is a syndrome clinically defined by failure of the ovary before the age of 40 yr. The patients with POF have a typical menstrual history of normal menarche or delayed adolescent age and normal secondary sexual character. The disease thereafter presents either with oligomenorrhea or abrupt amenorrhea. The serum levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) are elevated greatly, and the level of FSH is greater than LH, but the level of estradiol (E2) is decreased. Patients with POF are mainly troubled by infertility. Most patients experience climacteric symptoms, such as hot flushes and sweating boosts due to the hypoestrogenic status. Other troubled symptoms include atrophy of vagina and osteoporosis. At present, patients with POF are almost treated with hormonal replacement therapy and assisted reproductive technology. Some scholars also consider that immunosuppressive therapy for autoimmune POF is effective. However, these methods are not the foundational treatment to POF, and it will bring some serious side effects if people treated by immunosuppressive therapy for a long time. So it is becoming an urgent task for doctors to find a new way to treat patients with POF. There are three objectives in this study, the first is to probe the pathogenicity of POF, the second is to discover a convenient method to establish the animal model of POF, and the last is to find a new way to treat autoimmune POF with oral tolerance. Immunologic abnormality and experimental autoimmune oophoritis can be induced in mouse by immunization with ovarian zona pellucida (ZP). According to this principle, in this experiment we establish the mouse model of POF by hypodermic injection of ovarian ZP antigens. The peptide amino acid sequence of ZP3330-342 (NSSSSQFQIHGPR) was synthesized. The mice were divided into model group, adjuvant control group and blank control group randomly. Each mouse of model group was injected subcutaneously 50nM of pZP3 emulsified in complete Freund's adjuvant (CFA), the mice of adjuvant control and blank control groups were injected subcutaneously CFA and Milli-Q water respectively. The changes of estrous cycles, antibody of anti-ZP and ovarian histopathology of POF were detected. Among the mice of model group, 70% of estrous cycle of mice became irregular, 80% of mice presented positive antibody of anti-ZP in the serum and 70% of mice positive in the ovary, 90% of mice developed autoimmune oophoritis with different degrees. The morbidities were significantly different among model group, adjuvant control group and blank control group. It demonstrated that the mouse model of autoimmune POF could be induced by injection of pZP3 subcutaneously. Oral tolerance refers to systemic Ag hyporesponsiveness that occurs after oral Ag administration. Oral tolerance is an effective method in suppressing autoimmune diseases. According to it, in this study we suppress autoimmune POF of mouse with oral pZP3 administration. The mice were divided into control group, model group, low dose therapeutic group, middle dose therapeutic group and high dose therapeutic group randomly. Each mouse of model group was injected subcutaneously 50 nM of pZP3 emulsified in CFA except the mice of control group. The mice of control group were injected Milli-Q water. In addition, the mice of each therapeutic group receivedintragastric administration of different doses of pZP3 for seven times before and after hypodermic injection of pZP3. The dose of each mouse received was 1μg, 10μg and 100μg respectively. In this study, the therapeutic effect was evaluated by estrous cycle, autoantibody of anti-ZP, the CD25+CD4+ T cells rate in the peripheral blood, and ovarian tissue. The estrous cycle was determined by vaginal cytology. Autoantibody of anti-ZP was detected by Immunofluorescence method. CD25+CD4+ T cells were analyzed by flow cytometry. After being treated, 80% of mice of middle dose group and 75% of high dose group presented regular mestrual cycle, 15% of middle dose group and 20% of high dose group presented positive ZP antibody in the serum, and 10%, 5% in the ovary respectively, only 40% of middle dose group and 55% of high dose group developed autoimmune oophoritis. The percentage of CD25+CD4+ T cells of the middle and the high dose group were 2.69±0.64 and 2.41±0.57 respectively. There were significant different morbidities between model group and the therapeutic groups of middle dose and high dose. In this study, we demonstrated that autoimmune POF could be treated with oral pZP3 administration. 21 patients with POF and 20 females with unexplained infertility were selected, for detection of their serum levels of ZP antibody, IFN-γ, IL-4 and IL-10. The results were compared with those of 20 healthy women. The ZP antibody was seen in 33.3% of POF group and 15.0% of infertility group respectively, whereas in the healthy controls there was only 5.0% positive cases. Compared with healthy controls, the serum levels of IFN-γof POF group and of infertility group increased significantly, and the level of IL-10 of those two groups decreased significantly. The serum levels of IL-4 were no difference among the three groups. The rate of IFN-γ/ IL-10 of POF group and infertility group were significantly higher than that of healthy controls. We suggested that the ZP antibody might interfere the development of ovarian folliculles, also ovulation and fertilization, and in the end resulted in POF and... |
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