[Isolation and immunology identification of spermagglutinating antibodies from human serum]. / Izolace a imunologická identifikace spermaglutinacní protilátky z lidského séra.

OBJECTIVE: Isolation of spermagglutinating antibodies and their assesment. DESIGN: Retrospective study. SETTING: Special consulting for reptoduction immunology, Department of Obstetrics and Gynecology, Charles University and Faculty Hospital, Plzen. METHODS: Fractionation of serum samples by liquid exclusion chromatography, examination of full sera and their chromatographic fractions by Friberg teste (Tray Agglutination Test--TAT), indirect antiimmunoglobulin reaction test (i-MAR test) and by supplementar radial immunodiffusiona (RID). RESULTS: Isolation of spermagglutinating fractions of antisperm antibodies positive sera preserved spermagglutinating aktivity and confirmed great spermagglutinating potential of IgM. CONCLUSION: According to assesment of the presence of IgG and IgG we reported possible states of immunisation: actual immunisation with IgM activity, perpetual stimulation (IgG and IgM) and, finaly, anamnestic titres in IgG. These findings can help us to choose an optimal way of treatment. Excluding gel chromatography is suitable method for serum proteins fractionation, but not their identification--presence of antisperm antibodies does not affect the chromatographic spectrum, nor the RID patterns.

[Serum antibodies against annexin V and other phospholipids in women with fertility failure]. / Sérové protilátky proti annexinu V a jiným fosfolipidum m u zen s poruchou plodnosti.

THE AIM: To study serum antibodies against annexin V and against other phospholipids in women with fertility failure. DESIGN: Prospective study. SETTING: Department of Gynecology and Obstetrics, Institute of Imunology and Alergology, Institute of Forensic Medicine, Charles University and Faculty Hospital, Pilsen, Czech Republic. PATIENTS AND METHODS: We investigated sera from a total of 1550 women aged 26-41 years who attended the Division for Infertility and Reproductive imunology, in the Department of Obstetrics and Gynecology, Charles University, and Faculty Hospital, Pilsen, Czech Republic. ELISA was used for detection of antibodies against annexin V, phosphatidic (ph)-acid, ph-ethanolamine, D-L glycerol, ph-inositol, L-serine, cardiolipine, beta-glycoprotein, and prothrombine), antibodies against zona pellucida by passive hemmagglutination. Antibodies against annexin V in ovulatory cervical mucus were studied only in 47, and in follicular fluid in 168 women. Eighty-four fertile healthy women were included in the control group. RESULTS: Antibodies against annexin V were found in 13% (in 170 infertile women). Anamnestic data showed two or more unexplained pregnancy losses in 48 (28.2%), 108 (63.5%) patients had two or more unsuccessfull in vitro fertilization, polycystic ovaries (PCO) was diagnosed in 3 (1.7%), and endometriosis in 11 (6.6%) of them. The increased levels of antibodies against ph-inositole (28.8%), and L-serine (19.4%) were detected. 353% of patients had positive levels against zona pellucida. In ovulatory cervical mucus only in six (6/47) antibodies against annexin V were detected, in 5.55% in follicular fluid (6/108). CONCLUSION: Our study also demonstrated serum positivity against annexin V as a significant risk factor mainly in patiens with repeated reproductive failure.

[Electrophoretic analysis (SDS PAGE) of ovulatory cervical mucus in patients with fertility failure and after unsuccessful IVF]. / Elektroforetická analýza (SDS PAGE) ovulacního cervikálního hlenu pacientek s poruchou fertility a po neúspesné IVF.

OBJECTIVE: To evaluate the presence and activity of local sperm antibodies and electrophoretic analysis of ovulatory cervical mucus (OCM) to prove the correlation of results of Kremer mucus sperm capillary penetration test and i-MAR (mixed antiimmunoglobulin reaction) test with SDS-PAGE. DESIGN: Retrospective study. SETTING: Department of Obstetrics and Gynecology, Medical Faculty of Charles University, Pilsen. METHODS: 94 patients aged 22-40 (average age 32.3 years) were chosen for our study. Ovulatory cervical mucus was taken from uterine cervical canal in Consultation for Reproductive Immunology. Kremer test and indirect mixed antiglobulin reaction test for IgG, IgA, IgM and IgE were used for detection of sperm antibodies. For the SDS-PAGE analysis, OCM was incubated with sodium dodecyl sulfate. We studied separated protein fractions from OCM. RESULTS: Sperm-capillary ovulatory mucus penetration test (Kremer) was 0-10 cm/hour in our group (average value in patiens without sperm antibodies was 2.43 cm/hour, with sperm agglutinating antibodies 1.4 cm/hour), significant levels (> 45%) of spermagglutinating antibodies were detected in IgA in 6 patients (6.38%), IgG in 5 (5.32%) patients, sperm-cytotoxic levels (IgA and/or IgG) in 5 patients (5.32%); levels of IgE less than 30% in 3 patients (3.19%). Individual immunological factors gained by SDS-PAGE showed the spectrum of various molecular weights with range of 14.4- 350 kDa. The presence of IgG and/or IgA was in 53 cases (56.38%), with 13 ASA positivities (24.53% correlation with i-MAR test) and no ASA activity in 40 cases (75.47%); 6 ASA positive patiens (31.58%) were not detected by SDS-PAGE. CONCLUSIONS: Analysis of 94 OCM by SDS-PAGE showed several significant correlates, but their specifications will be based on further immunoblot research.

[Fructose and glucose in follicular fluid and serum of women undergoing stimulation in an in vitro fertilization program]. / Fruktóza a glukóza ve folikulární tekutine a séru stimulovaných zen v programu IVF.

OBJECTIVE: To find out if there is some amount of fructose in the follicular fluid of IVF stimulated patients. If so then to compare fructose and glucose levels with IVF outcome, type of stimulation and infertility factors. DESIGN: Retrospective study. SETTING: Department of Gynaecology and Obstetrics, Charles University, the Czech Republic. METHODS: 202 women undergoing IVF treatment since 11/1999 till 02/2001 were evaluated. We measured a level of fructose and glucose in a sample of serum and follicular fluid (FF) obtained at ovum pick-up. Then the results were compared with the IVF outcome, the length of stimulation and infertility factors. The control group consists of 23 women with a pure andrologic factor of infertility. RESULTS: At the day of pick-up the serum concentration of glucose and fructose taken on an empty stomach is on average 5.43 +/- 1.35 mmol/l and 25.05 +/- 9.26 micrograms/ml compared to 5.26 +/- 1.00 mmol/l and 23.69 +/- micrograms/ml in control group. The concentration of both glucose and fructose in FF correlates with their serum level. The FF concentration of glucose is on average 3.75 +/- 1.32 mmol/l (70% of serum concentration) and fructose 11.34 +/- 5.29 micrograms/ml (48% of serum concentration) by comparison with 3.78 +/- 0.91 mmol/l (73%) and 12.37 +/- 7.06 micrograms/ml (53%) in control group. CONCLUSION: There is a possible influence of follicular fluid saccharides like glucose and fructose in process of fertilization. We derived this hypothesis from studies evaluating the role of saccharides in endometrial secretion, cervical mucous and seminal plasma. We assumed that patients with a successful IVF cycle have a lower fructose concentration in FF. The clinical pregnancy is really mildly lower but this difference is not statistically significant. We confirmed FF fructose is in close correlation with its serum concentration. But we did not confirm the FF glucose and FF fructose concentration is related to the IVF outcome.

Beta 2-glycoprotein I is a good indicator of certain adverse pregnancy conditions.

OBJECTIVE: To compare levels of beta2-glycoprotein I antibodies with six different antiphospholipid antibodies (aPL) in sera from patients with certain adverse pregnancy conditions. PATIENTS AND METHODS: aPL levels were examined in pregnant women with anti-phospholipid syndrome (26), pre-eclampsia (32), autoimmune disease (12), or diabetes mellitus (23) and in a group with physiological pregnancy (38). A commercial ELISA was used to determine the serum levels of anti-beta2-GPI (Immunotech) in isotypes IgG and IgA, and anti-cardiolipin levels (Milenia) in IgG and IgM. aPL screening also included L-alpha-phosphatidic acid, L-alpha-phosphatidylethanolamine, L-alpha-phosphatidyl-DL-glycerol, L-alpha-phosphatidylinositol, and L-alpha-phosphatidyl-serine (Sigma, U.S.A.) in IgG and IgM. Statistical analysis of all aPL levels was made by cut-off levels for Ig isotypes by using 3 SD or 95th percentile calculated using STATGRAPHICS. RESULTS: Positive levels of antibodies against beta2-GPI in IgA are more frequently associated with a diagnosis of anti-phospholipid syndrome, pre-eclampsia, and autoimmune disease in pregnant women than with diabetes mellitus in pregnancy. Very high interindividual differences in aPLs (against inositol, L-serine, cardiolipin, and beta2-glycoprotein in IgG and IgA) were found in serum from women with pregnancy complicated by anti-phospholipid syndrome, pre-eclampsia, and autoimmune disease. Pregnant patients with diabetes mellitus had higher serum levels in aPLs to DL-glycerol, inositol, L-serine, and beta2-glycoprotein. Positive aPL levels predominate in isotype IgG. Very low levels of aPLs to phosphatidic acid and phosphatidyl-ethanolamine were detected in all groups studied. CONCLUSION: Serum levels of anti-beta2-GPI could serve as a better prognostic marker in complicated pregnancy than the panel of seven different anti-phospholipid antibodies. Detection of anti-beta2-GPI is proposed as a first step of the screening for aPLs.