The Research On Clinical Diagnosis And Treatment Of Auto-immunity Of POF

Objective:To establish the immunologic diagnostic methods of autoimmune premature ovary failure(POF)and to induce the animal model of autoimmune POF and provide theoretical evidence for treatment of autoimmune POF by glucocorticoids and androgen.Methods:Part 1 Study of diagnosis of autoimmune POF:52 patients with POF complicating infertility were chosen for test group,and 20 patients with infertility caused by others factors were chosen for control We detect and analyze the serum levels of ANA,AoAb,AcA,A-TG,ds-DNA, immunoglobulins IgG,IgA,IgM,IgE,complement C3,C4 and AzpAb.Part 2 Induction of animal model of autoimmune POF:We bought 130 seven-to nine-week old female inbred line mice(C57BL/6J)from the SLACCAS animal centre,and divided them into two groups:10 mice in the control group and 120 in the model group.Each mouse accepted subcutaneous injections in both posterior sole of feet.The model group was injected with CFA—pZP3 mixed solution,which contained CFA: 0.05ml,pZP3:72.225ug.And the control group was injected with 0.1ml of distilled water.Observation indices included:①Smear of the vaginal cast-off cell of the mice for sexual cycle observation;②Detection of AzpAb serum level by ELISA;③Observation of the histological changes of the mice by HE slicing under optical microscope; ④Evaluation of the severity of autoimmune oophoritis via examining infiltration of cells expressing CD45 in the ovary by immunohistochemisty to.Part 3 Study of therapy methods of glucocorticoids or androgen for autoimmune POF:We choose 80 mice of autoimmune POF,and randomly distributed them into 4 groups,20 mice per group.Three groups were treated by glucocorticoids,androgen,or therapeutic alliance of glucocorticoids and androgen repsectively.We evaluated and analyzed the treatment effectiveness of these three therapeutic methods according to the index above.Results:Part 1 The serum level of AzpAb and positive rates of ANA, AoAb,ds-DNA and A-TG in the test group were 110.42±10.38pg/ml, 38.5%,40.4%,35.5%,44.2%respectively,which were all higher than the control group(P＜0.05).Abnormality rates of ACA,immunogloblin IgM,complement C3 in the test group were 28.8%,23.1%and 25% respectively,which were found to be higher than the control group. However,the difference was not significant(P＞0.05).Abnormality rates of immunogloblin IgG,IgA,IgE and complement C4 were 5.8%,1.9%, 1.9%,3.8%respectively,which was found to be lower than the control group,but not significantly so(P＞0.05).Part 2 Eighty percent of mice in the model group were found to have disordered sexual cycles.The mean serum level of AzpAb was at 17.55±3.52ng/ml,whereas the proportion of growing follicles was only 22.44±5.88%.In addition,ninety percent of mice suffered from ovary infiltration of cells positively expressing CD45,which supported autoimmune oophoritis.Thus,all theindices in the model group were significantly different from the control group(P＜0.01).suggesting that the animal model of autoimmune POF was successful.Part 3①Comparison of sexual cycles of each group after treatment: Sexual cycle of 60%of mice in the glucocorticoids group was normal, while 70%in the androgen group,65%in the therapeutic alliance group, but 10%model control group were normal.Each therapeutic group was significantly different from the control group(P＜0.01)However,the differences among the three therapeutics groups were not significant(P＞0.05)②Comparison of serum levels of AzpAb of each group after treatment:The serum levels of AzpAb in glucocorticoids group,androgen group and therapeutic alliance group was 21.18±6.72ng/ml,16.12±4.64 ng/ml and18.32±5.28ng/ml respectively.All of the above values were significantly different from the model control group(32.61±8.77 ng/ml)(P＜0.01).The difference between the glucocorticoids group and androgen group was significant(P＜0.01).But the difference between the glucocorticoids group and the therapeutic alliance group,or androgen group and the therapeutic alliance group were not significant(P＞ 0.05).③Comparison of the proportion of growing follicles in each group after treatment:The proportion of growing follicles in glucocorticoids group,androgen group and therapeutic alliance group was36.8±8.00%, 37.5±6.06%and30.71±4.36%respectively,all of which was significantly different from the model control group(25.72±5.31%,P＜0.01).The difference between the glucocorticoids group and androgen group was not significant,(P＞0.05).But the difference between the glucocorticoids group and the therapeutic alliance group,or androgen group and the therapeutic alliance group were significant,(P＜0.01).④Comparison of infiltration of lymphocytes in the ovaries:Twenty five percent of mice in the glucocorticoids group suffered from oophoritis,that was,the infiltration of cells expressing CD45 in the ovaries,while 30%in the androgen group,50%in the therapeutic alliance group,90%in the model group suffered from oophoritis.Each therapeutic group was significantly different from the control group(P＜0.01).But the differences among the three therapeutics groups were not significant(P＞0.05).Conclusion:1.We found many kinds of autoimmune antibodies in patients with POF.Detection of ANA,AOAb,ds-DNA,A-TG and AzpAb was of clinical significance to diagnosing autoimmune premature ovary failure.2.We successfully developed animal model of autoimmune premature ovary failure by active immunization of adult mouse with a murine ZP3 peptide(pZP3)in complete Freund's adjuvant(CFA),and provided conditions for further research of POF in diagnostics and therapeutics.3.We found that both glucocortcoids and androgen could significantly redress the clinical symptons and immunical indicators of autoimmune POF.But the differences between them were not significant (P＞0.05).4.We found that the therapeutic alliance of glucocorticoids and androgen was not better than either glucocorticoids or androgen alone.