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Objective: An increased number of NK cells is associated with autoimmune disorder and is known to play a role in infertility. The aim of our research was to monitor the density of NK cells CD56+ and CD16+ in ovulatory cervical mucus (OCM) and in endometrium in infertile women as well as in connection with the actual status of antibodies against phospholipids, sperm and HHV-6 antibodies., Type of Study: Original aticle., Setting: Genetika - Plzeň., Methods: Seventy-two randomly selected women aged 20-39 (mean age: 32.3) years old resulted in fifty-seven patients with repeated unexplained miscarriages, and fifteen fertile healthy women. The hormonal status was studied including ovulation, the humoral autoimmune responses to eight phospholipids, trombophilia, karyotyping, hysteroscopy, and endometrium immunohistology. Patients were without any clinical and laboratory symptoms of vaginitis at the time of OCM sampling and endometrium study. In one patient antiphospholipid syndrome was present, and in one woman diabetes mellitus was identified. Uterine NK cells CD56+ , CD16+ and NK cells in OCM were identified by immunocytochemistry, antiphospholipid antiboides by ELISA. We used indirect MAR-test for study of local spermagglutinating antibodies in OCM. Indirect immunofluorescent method was used for detection of serum and OCM IgM, IgG antibodies against HHV-6 levels at the time of ovulation., Results: We found both high density of NK cells CD56+ and CD16+ in OCM and in endometrium in only two infertile women with repeated abortions. NK cells in OCM were missing in other samples of patients. The prevalence of high density of NK cells CD56+ in the endometrium was seen in twenty three (40%), NK cells CD16+ in eleven (19%), NK cells 56+ and NK cells 16+ together in eight (14%). Levels of serum and OCM IgG against HHV-6 in all examined patients were not elevated, no cervical sperm antibodies were found., Conclusion: We compared density of NK cells CD56+ and CD16+ in OCM and secretory endometrium in all infertile patients. Our results show that cell mucosal activity in the cervical area at the time of ovulation in two infertile patients was evident. We excluded the abnormal number of NK cells owing to local and general viral infection (HHV-6). But our question still remains - are cervical NK cells fixed or still migrating from endometrium into OCM? New research is planned.

Introduction: This statements follows the first part of our publication entitled Screening of endometrial NK cells in selected infertile patients (First part - Methods and current results), where we dealt with methodology and endometrial findings of the natural killer cells CD16+ and CD56+. These cells are among the most important in preimplantaion, and in implantation period, in early pregnancy too, but can also negatively influence above mentioned processes., Objective: We focused on the immunomodulatory treatment with intramuscular immunoglobulins in 21 infertile patients with a high density of endometrial NK cells CD16+ and CD56+., Design: Original work-prospective study., Setting: Genetics-Pilsen., Methods: From the originally investigated 57 women aged 25-41 (average 34) years with their history of 3-9 abortions, we target on a high selective subgroup of 21 infertile patients with pathological density of endometrial NK cells CD56+ and CD16+., Results: Twelve patients underwent immunomodulatory treatment with intramuscular immunoglobulin in time of positive HCG and went on the therapeutic scheme, nine infertile women continued with IVF-ET but did not achieve HCG positivity despite of initial treatment with immunoglobulins. Of the 12 treated patients, seven of them gave a birth to healthy babies, as to the next five pregnancies - one was extrauterine pregnancy, one genetically defective fetus, three pregnancies go on successfully.Nine women from the IVF program did not get pregnat., Conclusion: Immunomodulatory treatment with immunoglobulins also influences endometrial NK cells CD56+ and CD16+ associating with an overproduction of embryocytoxic cytokines. Treatment of our patients with the pathological endometrial density of NK cells must be solved strictly individually.

Introduction: Generaly, natural killer cells (NK cells) are among the most important cells of our immune defense system. They are present in the blood, decidua but also in secretory endometrium., Objective: We investigate an association between high density of NK cells CD56+ and NK cells CD16+ into blood and secretory endometrium in patients with up to now unexplained recurrent miscarriage. At the same time, we focused on diagnosis of antiphospholipid syndrome, congenital trombophilic factors, stress factor, and lenght of hormonal contraception before conception., Design: Original work-prospective study., Setting: Genetics-Pilsen., Methods: We investigated 59 patients aged 25-41 (average 34) years with their history of 3-9 abortions. We monitored their blood cells by flow cytometry and endometrium obtained by hysteroscopy performed on the 22nd-24th day of the menstrual cycle. We concentrated on the cellular immunity focused on the identification of lymphocytes CD56+ and CD16+. ELISA method was used for identification of antiphospholipid antibodies., Results: Our study goes on, we are publishing our preliminary results. We found a high density of endometrial NK cells CD56+ in 41/57, NK cells CD16+ in 40/57, both NK cells CD56+ and CD16+ in 36/57 patients. In 13 women, we identified only sporadically these cells in the secretory endometrium. NK cells CD56+ and NK cells CD16+ in the blood were all normal number in all our patients. Three of them have a homozygot form of Leiden mutation, and 18/57 primary antiphospholipid syndrome, 12 women have university education, four of them, medical doctors, have regular night services in the hospital., Conclusion: Overproduction of endometrial NK cells is associated with increased local activity of embryocytotoxic cytokines that may negatively affect pregnancy. Treatment of patients with pathological immunological findings must be solved individually, as we will show soon in part two of our study.

Objective: Ovarian inhibins (INH) are hormones participating in the regulation of gametogenesis. We monitored inhibin A and B levels in serum (S) and follicular fluids (FF), depending on the type of fertility failure and treatment outcome., Material and Methods: We examined INHA and B levels in S and FF of 72 women undergoing ovarian stimulation for in vitro fertilization, including embryo transfer We took serum samples at the time of egg collection (S1), embryo transfer (S2), and diagnostics of early pregnancy (S3). FF samples were obtained during egg collection. INH A and B levels were measured by ELISA set kit in all media., Results: Healthy women had median of INHA S1 592.02pg/ml INHA S2 593.58pg/ml, INHA S3 15.17pg/ml and INH B S1 242.46pg/ml, INH B S2 and INH B S3 zero levels. Women with ovarian disorders had significantly lower levels of INH A S1 and INH A S2 (p<0.05). Women with polycystic ovaries had significant higher INH B S2 levels (p<0. 05). No statistically significant differences were found in women with endometriosis. Presence of oocyte in the dominant follicle positively correlated with INH B FF levels (p<0.05)., Conclusions: WE confirmed differences in the levels of inhibins in sera depending on type of fertility failure. Inhibin B better reflected the presence of an oocyte. The potential paracrine role of inhibins needs to be examined to improve preparation for the in vitro fertilization treatment (IVF).

Background: In this study, we investigated pH, levels of IgA, IgG, IgM, lysozyme and spermagglutinating antibodies in human saliva of healthy women, with or without hormonal contraception (HC) and their effect on sperm motility., Methods and Results: Saliva was collected immediately after waking up from 59 healthy randomly selected women. We measured pH in sera and in saliva as well as immunoglobulin G, A and M levels in saliva by radial immunodiffusion. Sperm - saliva capillary penetration test in native saliva served for examination of sperm motility, indirect-MAR test for detection of sperm antibodies in sera, and in saliva, both observed by inverted microscope. The average pH values in native saliva according to the menstrual cycles were: from 2-5 days 6.896, from 6-17 days 7.027, and from 18 and more days 7.17. Rapid decrease of sperm motility was registered in acidic saliva in comparison with alkaline. The average concentration of IgA, IgG, IgM and lysozyme slightly differed in women with or without hormonal contraception. Moderate decreasing of sperm motility was found in saliva with higher levels of IgA. No sperm agglutinating antibodies in saliva and in serum were detected in all subjects., Conclusions: The pH of saliva is influenced by several causes (hormonal, immunological, microbiological factors, by meal and drinks, smoking, etc.). We demonstrated that sperm motility is lower in acidic saliva. pH is markedly changed during various days of menstrual cycles. We proved individual high levels of saliva IgA. Human saliva used as coital lubricant decreases sperm motility, but does not replace a local form of contraception.

Objective: To determine whether patients with recurrent spontaneous abortions and IVF suffer from latent celiac disease., Design: Perspective study., Settings: Department of Gynecology and Obstetrics and Faculty Hospital, Charles University, Pilsen., Material and Methods: Sera from 442 infertile patients aged 23-47 years and 86 healthy fertile controls aged 22-38 years were examined by commercial ELISA sets for the presence (absence) of antibodies against gliadin IgG, IgA and tissue transglutaminase in IgG and IgA., Results: We found a statistically significant difference between the control group and patients with decreased fertility. They have a positive values in anti-tissue transglutaminase IgA (p = 0.0096). Frequent, very low positive reaction of antigliadin IgG was also found in the control group of women., Conclusion: Patients with unexplained repeated miscarriages and repeated unsuccessful IVF with IgA positive tissue transglutaminase have new complement of other tests for autoimmunity and examination of a total IgA, and gastroenterological examination. Eleven patients with significant positive anti-tissue transglutaminase IgA were successfully conceived only under strict gluten-free diet.

Objective: The aim of our preliminary study was to compare the levels of total local sIgA and IgG with activity of detected sperm antibodies in ovulatory cervical mucus (OCM)., Setting: Department of Gyneacology and Obstetrics, Medical School and Faculty Hospital, Charles University, Plzen., Methods: We screened samples of OCM from 12 pacients aged 26-31 (29,6 years on average) by radial immunodifusion (RID) to determine s IgA and IgG. Indirect MAR test was used for detection of spermagglutinationg antibodies., Results: We found out by RID the average concentration of sIgA in OCM 567,84 mg/l (0 -1250,47) and the average concentration of IgG in OCM 23,57 mg/l (8,74-47,99). Antibody activity against sperm cells dominates in IgA with 6 pacients, in IgA with 1 patient, in IgA and IgG together with 1 infertile woman and in IgA and IgM isotypes together with 1 patient. No local sperm antibodies were determined with 3 patients., Conclusion: We proved the hypothesis, that the levels of spermagglutinating antibodies do not correlate with findings of total sIgA and IgG in OCM with our patients.

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.

Objective: To study of IgG antibodies against laminin-1 (IgG-a-Ln-1) in patients with decreased fertility, especially with endometriosis and with diagnosis of polycystic ovaries., Design: Prospective study., Setting: Special Consultation for Immunology of Reproduction and Research Laboratories for Reproductive Immunology, Department of Gynaecology and Obstetrics, Medical School of Charles University and Faculty Hospital, Pilsen., Methods: Commercial ELISA kits for detection of IgG antibodies against laminin-1 in serum (S) and peritoneal fluid (PF) in 120 patients after diagnostic laparoscopy, and in serum in 277 patients after 2-3 in vitro fertilizations and with diagnosed endometriosis., Results: Only 16 positive results in IgG-a-Ln-1 antibodies (more than 15 IU/ml) were detected in sera and peritoneal fluids in the group of 120 women after laparoscopy. We have not detected any serum IgG-a-Ln-1 positivity in the group of 277 patients after 2-3 unsuccesfull IVF and with anamnestical date about endometriosis together., Conclusion: Increased levels of IgG-a-Ln are in particular found in endometriosis II-III and in diagnosis of polycystic ovaries, in female diseases also related to autoimmune process.

Background: Up to now, it does not exit whole look at the life quality of the Pilsner female University students, especially in connection with gynecological history. It is reality that in last years the number of gynecological problems among young women increases such as the number of diseases with immunological background., Method: We prepared questionnaire with 36 questions directed to life style, gynecological history, partner's life, food and hygienic habits, and nicotinism., Results: 28 female students from the total group of 141 suffered from vaginal mycosis--22 (78.6%) use pills, 6 (21.4%) no hormonal contraception. 17 (60.7%) prefer flyaway cloth and 9 (32.1%) tight cloth. 141 students mentioned various number (0-25) of their sexual partners: 1-3 (79 students) in 15 cases (19%) mycoses, more than 4 partners (49 students) in 13 (26.5%) mycoses appeared. The first menstruation appeared in o 13 years (10-17 years). High incidence of vaginal mycosis was proven in students using only tampons than hygienic towels., Conclusion: Vaginal mycosis is affected by many endogenous and exogenous factors. The main factors in pilsner university female students are hormonal contraception, promiscuity and hygienic habits.

Objective: The aim of this study was to compare levels of PGE2 in cervical ovulatory mucus in patients with various frequency of spontaneous miscarriages, and in healthy fertile women., Setting: Special Consultation for Immunology of Reproduction and Research Laboratories for Reproductive Immunology, Department of Gynaecology and Obstetrics, Medical Faculty of Charles University and Faculty Hospital, Pilsen., Methods: We used commercial, high sensitive method of ELISA (PGE2 HS, R&D Systems) for detection of PGE2 in cervical ovulatory mucus. Statistic analysis (T-test, ANOVA-test and Kruskal-Wallis test) also provided our study., Results: It was taken in total 120 measurements. The R & D Systems company does not mention physiological levels of PGE2 in cervical ovulatory mucus. Therefore we compared all results of our studied groups with average concentration (197356 pg/ml PGE2) gained from control group. We found that in patients with one miscarriage PGE2 levels are approximately 6 times higher, with two miscarriages 13 times higher, and in patients with three and more miscarriages even 21 times higher than controls (p < 0.0327, alpha 5%)., Conclusion: In present literature does not exist any study concerning of PGE2 in cervical ovulatory mucus. We prove that some patients mainly with repeated "unexplained" miscarriages have ovulatory levels of PGE2 significantly higher than controls.

Background: Decreasing quality of the semen represents a big problem for human population. Parameters of spermiogramms are used for obtaining of information about the human semen quality. The aim of our study was to evaluate parameters of semen and its immunological characteristics in relation to data from personal history of our students., Methods and Results: Basic analysis of semen samples of 110 students was performed according to World Health Organization criteria. Direct mixed-antiimmunoglobulin-reaction-tests for detection of sperm-agglutinating antibodies, intraacrosomal proteins by specific monoclonal antibodies and immunoflurescence examination were used. Data from anamnesis were obtained by a questionnaire. Normozoospermia was found in 72% cases, oligozoospermia in 14%, asthenozoospermia in 5%, teratozoospermia in 4%, oligoasthenozoospermia in 5% of men. We have not confirmed negative influence of smoking, drug abuse, wearing tight clothes, hot baths, and using sauna on sperm quality. There was significantly higher percentage of students consuming presevered and spicy food more than twice a week in the group with pathological spermiogramm in comparison with the normozoospermic group. Acrosomal proteins were found in 75% men with normozoospermia and in 43% of pathological spermiogramms. Findings of sperm-agglutinating antibodies were not significant., Conclusions: Semen quantity as well as quality is affected by many intrinsic and extrinsic factors. Our study shows that fertility of our students is better than we expected.

Objective: Study of antibodies against laminin-1 in IgG in patients with habitual abortion., Design: Prospective study., Methods: Commercial ELISA method for detection of IgG antibodies against laminin-1 in ovulatory cervical mucus, in sera in women with repeated spontaneous abortions, in sera in patients during spontaneous miscarriages., Results: Positive levels of antibodies against laminine-1 in IgG in sera were found in patients during spontaneous abortions, as to ovulatory mucus of infertile women in time out of pregnancy, we did not find any positivity, only in one serum sample in patient with three pregnancy losses in her history., Conclusion: Antibodies against adhesive molecule as laminin-1 presents, can participate during diagnosis of repated spontaneous miscarriages in context of neuro-endocrinne-immune dysregulation, which is not seen during physiological pregnancy.

The Aim: To study placenta annexin V receptors, and serum antibodies against ph-acid, ph-ethanolamine, ph-glycerol, ph-inositol, ph-serine, cardiolipin, beta2-glycoprotein I, prothrombin a annexin V in patients with recurrent pregnancy loss., Setting: Department of Gynecology and Obstetrics and Faculty Hospital of Charles University, Plzen., Methods: We examined sera and placenta tissue from 156 patients in reproductive age with unexplained repeated pregnancy loss. Control group of eighty-four fertile healthy women was included. ELISA methods were used for detecting a panel of sera of anti-phospholipid antibodies (aPLs). Immunolocalization of annexin V receptors in 143 trophoblast-placenta specimen of 156 patients was investigated by the immunofluorescence technique using Annexin V-FITC, Apoptosis and Annexin V-CY3 commercial kits., Results: Positivity for anti-phospholipid antibodies mainly against ph-serine, ph-inositol, and ph-ethanolamine was found together in 80.8%, anti-prothrombin antibodies in 12%, and anti-annexin V antibodies in 13.5% women. No significant levels of aPLs were found in 6 controls. Immunohistopathology of placenta also exhibited some changes manifested by the presence of apoptotic and necrotic cells in trophoblast, and very few microtrombotization in some intervillous spaces and in placenta vessels., Conclusion: Our detailed study demonstrated the prevalence of majority of antiphospholipid antibodies as one of the high risk factor of repeated reproductive failure. Very low microthrombosis in placenta could be explained by the changes of haemocoagulation properties out of uterus.

The Aim: To monitor the basic andrologic and immunologic sperm factors and the levels of inhibin B in serum and in seminal plasma in men from the couples with infertility disorders., Setting: Department of Gynecology and Obstetrics, Medical School, Charles University and University Hospital, Plzen, Institute of Molecular Genetics, AV CR, Prague, Institute of Clinical Immunology and Allergology, LF UK a FN, Plzen., Methods: We used conventional methods for estimation of sperm quality according to WHO and we detected the intra-acrosomal proteins by monoclonal antibodies (Hs8 and Hs14, immunofluorescent method), spermantibodies by direct mixed antiimunoglobulin reaction (MAR) test, and we examined inhibin B in serum (< or =400 pg/ml= A) and in seminal plasma (< or = 600 pg/ml= N) by ELISA in 355 men aged 21-52 years (ø 34 years) with normal levels of FSH, LH and testosterone. The control group was created by 56 health sperm donors., Results: We found 65% normospermatics in the group of 355 patients, 34.9% men with various kind of pathologies. Predominance of spermagglutinating antibodies was found in 15.77% in IgG, in 19.44% in IgA, in 8.44% in IgA and IgG together. Normal intraacrosomal proteins were reached in 74.65% for Hs8, in 20.85% pathologic, in 86.2% normal findings for Hs14, in 4.23% pathologic. The immunological results in control group were completaly negative. Pathological levels of inhibin B in seminal plasma was found in 37.2% (152 men), in 25% in serum, and in 5.6% in serum and in seminal plasma together. In 54.7% of patients we found physiological levels of inhibin B in both biological fluids. We also compared physiological 109/152 (71.71%), and pathological spermiogrammes 43/152 (28.29%) with abnormal levels of inhibin B in seminal plasma, with intraacrosomal proteins to levels of inhibin B in serum. Our detailed study shows high interidividual results, which must be studied in complex with diagnosis of decreased fertility in man., Conclusion: Andrologic and immunologic analysis in the group of 355 men showed normal parameters of spermiogrammes in 231 patients (65%), in the rest of men the immunologic profil was in various parts pathologic. Only 105 men have got excellent spermiogrammes. Inhibin B as hormon regulates in back the secretion of FSH, and serves as good indicator in male reproductive failures.

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.