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Objective To investigate the presence of apolipoprotein A 1 (APOA1) and its function in human spermatozoa. Methods The expression assays for APOA 1 were carried out by immunofluorescence and Western blotting in human sperm cells . Set up a blank control group , rabbit polyclonal IgG group , which contain anti-APOA1 antibody 10 μg/ml, 20 μg/ml and 40 μg/ml, to treat normal semen samples and incubated at 37 ℃for 1 h, 2 h and 3 h.The progressive motility of spermatozoa was determined bya computer-assisted motion analyzer ( CASA ) , apoptosis rate by flow cytometry and ultrastructural changes by electron microscopy .Comparisons of sperm progressive motility and apoptosis rate were performed by independent sample t tests.Results The study showed APOA1 protein mainly located in the sperm head , the molecular size was 31000.A significant decline in sperm progressive motility was observed after 1,2 and 3 hours of incubation with APOA1 antibody.There was a statistically significant difference between the blank control group and the APOA 1 antibody concentration of 20 μg/ml and 40 μg/ml groups [ 1 hour after incubation , blank control group ( 68.65％ ±15.70％) with 20 μg/ml group (48.45％±5.20％), 40 μg/ml group(39.25％±7.89％), t=2.442, 3.345 , both P<0.05;2 hours after incubation, blank control group(55.33％±10.12％) with 20 μg/ml group(28.68％±11.70％), 40μg/ml group(18.13％ ±10.52％), t=3.445, 5.097,both P<0.05; 3 hours after incubation, blank control group(35.73％±14.08％) with 20μg/ml group(15.53％±8.42％), 40μg/ml group(9.98％± 7.08％), t=2.462, 3.268,both P<0.05].After incubation 2 hours, the percentage of apoptotic cells increased from 16.02％ ±4.28％ in the blank control group to 21.72％ ±2.67％ ( 20 μg/ml APOA1 antibody)and 28.01％±3.93％(40 μg/ml APOA1 antibody), respectively (t=3.177, 5.834, both P<0.01).Treatment with 40 μg/ml APOA1 antibody for 4 hours resulted in major morphological changes to sperm cells observed by electron microscope , including membrane distension ,vacuole formation and different periods of apoptosis cells .Conclusion APOA1 plays an important role in maintaining sperm motility and survival rate,however the mechanism needs further study .
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Objective To reveal the mechanisms of immunological infertility,the method of coimmunoprecipitation(CO-IP) and liquid chromatogram mass/mass (LC-MS/MS) was used to screen sperm membrane proteins which interacting with antisperm antibodies (ASA).Methods This study was designed as a case-control.The disease group including 56 serum samples from 521 cryptogenic infertile patients were screened ASA positive by ELISA and conformed with mixed antiglobulin reaction(MAR).The controls were 31 serum samples which ASA is negative and already possessed healthy offspring.All subjects were enrolled from September 2015 to December 2015 in China PLA General Hospital.Spermatozoa samples from 48donors with normal sperm parameters were from January 2016 to April 2016 in China PLA General Hospital.The purified human sperm membrane proteins were then mixed with serum from disease group (positive for ASA) and control group (not containing ASA).The binding proteins of antisperm antibodies were enriched using CO-IP assay.The immunoprecipitates were separated on sodium dodecyl sulfate-polyactylamide gel (SDS-PAGE),then the binding proteins were cut from the gel and analyzed by LC-MS/MS after the enzymolysis.These proteins could bc idcntified as definition,biological function (s) and subccllular localization with Uniprot database.Results The serum samples from infertile persons (39 females and 17males) were screened ASA positive by ELISA and conformed with MAR.The healthy controls (17 females and 14 males) were ASA-negative in ELISA.Forty proteins that interact with ASA were obtained from the study and these could be divided into three groups:11 antigens detected by control serum samples only,14antigens recognised by both infertile patients and control sera,and 15 antigens specific for patients with ASA.These 15 proteins are Sperm Cation channcl protein 1,Sperm Cation channel protein 3,Sperm Cation channel protein 4,Sperm associated antigen 9,Apolipoprotein A-I,Dynein heavy chain 14,Cylicin-2,Izumo sperm-egg fusion protein 4,Thioredoxin domain-containing protein 2,IQ domain-containing protein H,IQ domain-containing protein F1,Spermatogenesis-associated protein 5,Spermatogenesis-associated protein 5-like protein 1,Sperm acrosome membrane-associated protein 1,E3 ubiquitin-protein ligase RNF 114.Conclusion Fifteen proteins discovered with CO-IP technology and LC-MS/MS analysis could be referred as male immunoinfertility-related antigens and they may hold the great importance in revealing the secret of immunological infertility.
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Objective Through the analysis of the special thromboelastography (TEG) graphics to improve the ability of analysis and identification the TEG and provide more valuable results for clinical practice.Methods This study is a retrospective study.Retrospectively analysis of 15 430 cases of TEG results from People's Liberation Army General Hospital,using the parametrs as reaction time (R),the clotting time (K),the solidification angle (Angle),the maximum amplitude (MA),the percentage of blood clot dissolution (EPL),the rate of 30 minutes clot amplitudereduce (LY30),the coagulation index (CI) and other parameters to analyze the hyperfibrinolysis group,the heparin graphics group,the secondary activation of fibrin group,the platelet function group and the anti-platelet drug monitoring group.Results In the hyperfibrinolysis group,the EPL value,LY30 value and CI value of primary hyperfibrinolysis group was 19.3,19.3 and 0.4 respectively and the secondary hyperthyroidism group was 11.6,11.6 and 3.2 respectively.In the heprin application graphic group,R value of CKH group was shorter than that of CK group (10.9 vs.50.8),R value of CKH group was longer than that of CK group (12.2 vs.15.7).In fiber protein secondary activation graphics groups,the MA value of the first time detection was 35.2,then replacing the channel repetition MA value was 34.7 in true functional fiber protein secondary activation group.For the pseudo fiber protein secondary activation group the first time detection MA value was 73.4,then replacing the channel repetition MA value was 20.0.The MA value of platelet function reduce group and hyperfunction group was 21.6 and 79.2 respectively in platelet function group.In anti-platelet drug monitoring group,the inhibition rate of adenosine diphosphate (ADP) of ADP inhibitor (clopidogrel) ineffective group was 10.9％ ; And the inhibition rate of arachidonic acid (AA)of AA metabolic pathway inhibitor (aspirin) ineffective group was 7.5％.Conclusions The correct judgment on the abnormal thromboelastography graphics is helpful to ensure the accuracy of the results andto reduce the occurrence of adverse events.
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Objective To establish the enzyme-linked immunosorbent assay (ELISA) for the detection of serum alcohol dehydrogenase (ADH) antibody and evaluate its role in its diagnosis of autoimmune hepatitis( AIH ). Methods The reactivity between yeast ADH and human anti-ADH serum antibody was tested by Western blot analysis. ELISA was established using yeast ADH. The method was applied in serums of 67 AIH patients,94 primary biliary cirrhosis(PBC) patients, 199 chronic hepatitis B (CHB) patients, 132 chronic hepatitis(CHC) patients, 24 alcohol hepatitis disease(ALD) patients, 99 connective tissue disease(CTD) patients and 31 healthy individuals. The positive rate of ADH antibody in the patients and healthy individuals was measured. The χ2 test was used to compare the positive rates. Results The ELISA method for detecting human anti-ADH serum antibody was established successfully and the optimum reaction conditions were defined. Western blot showed that yeast ADH has cross reactivity with human anti-ADH antibody. The positive rate of anti-ADH antibody in the AIH group [59. 7% ,40/67 ] was higher than that in the normal control group(0,χ2 = 31. 271 ,P <0. 05), PBC group (6. 4% ,χ2 =54. 492,P <0. 05), CHB group( 14. 1% ,χ2 =54. 848,P <0. 05) ,CHC group(21.2% ,χ2 = 29.269,P<0.05), ALl) group ( 25. 0% ,χ2 =8.512,P <0.05)and CTD group ( 43. 4% ,χ2 =4.229, P <0. 05). Conclusions Compared with the PBC, CHB, CHC, ALD and CTD group, the anti-ADH antibody positive rate in the serums of AIH was significantly increased. The antibody may be helpful to the diagnosis of autoimmune hepatitis.
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Objective To investigate the significance of antibody to Ro-52 in patients with autoim-mune liver disease(AILD). Methods One hundred and fifteen patients with abnormal liver functions, who had anti-Ro-52 detection by immunological blotting, were reviewed retrospectively. According to types of AILD, the clinical features were compared between patients with and without anti-Ro-52, respectively, κ test of concordance was used to provide a chance-corrected valve for immune-serological results. Results The rates of anti-Ro-52 in autoimmune hepatitis( AIH), primary sclerosing cholangitis(PBC) and AIH/PBC o-verlap syndrome groups were 32.43%, 24.56% and 33.33%, respectively, there were no significant differ-enees among three groups ( x2 = 0. 949, P >0. 05). The rate of anti-soluble hver antigen/liver-pancreas ( an-ti-SLA/LP) in AIH patients with anti-Ro-52 (58.33%) was higher than AIH patients without anti-Ro-52 ( 16.00% ,P < 0.05 ). The rate of anti-SLA/LP in AIH/PBC overlap syndrome patients with anti-Ro-52 (85.71%) was also higher than that of control group (28.57% ,P <0.05). Anti-Ro-52 and anti-SLA/LP had concordance according to κ test( κ >0.40, P <0.05). The average level of IgG in AIH/PBC overlap syndrome patients with anti-Ro-52 was higher than patients without anti-Ro-52 ( t = 2. 508, P < 0.05 ). Conclusion The rates of anti-Ro-52 in AIH, PBC and AIH/PBC overlap syndrome were of no significant differences. Anti-Ro-52 may have correlation with anti-SLA/LP. AIH/PBC overlap syndrome patients with anti-Ro-52 shewed higher IgG level than patients without anti-Ro-52.