Biomedical subjects

R G Roussev

Publications and source records attributed to R G Roussev.

14 recordsLinked to original sources

PubMed1999

Murine embryos as a direct target for some human autoantibodies in vitro.

The involvement of one or another autoantibody in reproductive failure have long been thought to be through post-implantation thrombosis and/or peri-implantation trophoblast dysfunction and/or maternal hormonal imbalance. It can be postulated that the embryo may be a direct target for some autoantibodies prior to implantation. Mouse embryos have been labelled and cultured with affinity purified immunoglobulin (IgG) and IgA from positive for antiphospholipid antibody sera, as well as IgG from positive for antinuclear antibody sera and positive for antithyroid antibody sera. Intact IgG and IgA from healthy individuals were used as controls. All embryos cultured with purified antiphospholipid IgG or IgA, and anti-nuclear IgG exhibited strong immunofluorescence. No difference in fluorescent intensity was observed whether antiphospholipid or anti-nuclear antibodies were used, but the pattern of antibody distribution seemed to be different. Antiphospholipid IgG was more dominant on the zona pellucida, while antiphospholipid IgA and antinuclear IgG had predominant distribution on the embryonic cells. None of the embryos cultured with antithyroid IgG or with control immunoglobulins showed strong immunofluorescence. Embryos cultured with purified antiphospholipid and antinuclear immunoglobulins experienced significant growth impairment or death compared to those cultured with antithyroid or control immunoglobulins.

Animals↗

PubMed1999

Immunodiagnostic evaluation in women with reproductive failure.

PROBLEM: Several immunological factors have been associated with diagnostic subpopulations of reproductive failure. It is important to determine a trend of immunological abnormalities among these subpopulations. The purpose of this study is to assist in the selection of treatment for patients suspected of having specific diagnoses of reproductive failure. METHOD OF STUDY: Blood samples from 591 patients were evaluated for the presence of antiphospholipid (APA), antinuclear (ANA), and antithyroid (ATA) antibodies, as well as for lupus anticoagulant (LA), embryotoxic factors (ETF), and elevated levels of natural killer (NK) (CD56+) cells, and all tests were performed as a panel. The patients were grouped into the following diagnostic categories: recurrent pregnancy loss (n = 302), IVF/ET failure (IVFf, n = 122), unexplained infertility (n = 97), ovarian dysfunction (n = 47), and endometriosis (n = 23). The thresholds for positivity and the prevalence of the tested factors among normal healthy populations have been established by testing 100 or more healthy male and female individuals with each one of the tests used (general population control). All tests as panel were performed on 20 normal fertile female individuals as controls (fertile female controls). RESULTS: Of all patients with reproductive failure, 75.6% had at least one abnormal test. The most frequent abnormal result was found to be the elevation of NK (CD56+) cells (37%), followed by ANA (34%), APA (24%), ATA (19%), and ETF (11%). Of the recurrent pregnancy loss patients, 74.2% had at least one positive abnormal result from all of the tests performed: overall, 70% of women with IVF failure had at least one abnormal test; of patients diagnosed with unexplained infertility, approximately 81% had at least one abnormal result; 74.4% of the patients with ovarian dysfunction and 52% of the patients with endometriosis had at least one abnormal result. From normal fertile controls, 10% showed at least one abnormal test result. CONCLUSION: APA, ANA, ATA, ETF, and elevated NK (CD56 ) cells are significantly more prevalent among women experiencing reproductive failure than among the control group and normal healthy individuals.

Abortion, Habitual↗

PubMed1998

Anti-endothelial cell antibodies: another cause for pregnancy loss?

PROBLEM: Previous studies demonstrated that unique tissue-specific antigens expressed on vascular endothelial cells can serve as immunogens, and the apparent association between transplant rejection and antiendothelial cell (EC) antibodies is well established. A common feature of some placentas from women with recurrent pregnancy loss is the diffuse formation of microthrombi associated with changes in thromboresistant properties of endothelial cells, similar to the findings in rejected organs. Therefore, the prevalence of anti-EC antibodies in patients with recurrent pregnancy loss and the role of these antibodies in cultured human endothelial cells from umbilical cord vein were studied. METHOD OF STUDY: To evaluate the frequency of anti-EC antibodies, sera from 160 nonpregnant patients with recurrent pregnancy loss after absorption with pooled platelets and pooled leukocytes were tested using cultured human umbilical cord vein endothelial cells (HUVEC) by flow cytometry. To study the role of anti-EC antibodies, purified anti-EC IgGs were added to HUVEC cultures and hemostatic, fibrinolytic, and anticoagulation pathway markers (tissue factor, tissue plasminogen activator, palsminogen-activator inhibitor, thrombomodulin, heparan-sulfate proteoglican, antithrombin III, von Willebrand factor, CD54, human leukocyte antigens-DR, and transferin receptor) were detected by specific antibodies and flow cytometry. Immunoblotting analyses were done by using purified anti-EC IgGs against cell membrane proteins from endothelial cells and leukocytes extracted by detergent solubilization. RESULTS: Thirty-nine (24%) of the patients were positive for EC antibodies. Antipaternal lymphocyte antibodies were tested as well and were found in 37 (23%) of the patients as 25 sera (15.6%) showed reactivity with both EC and lymphocytes. Certain patient sera were reactive with HUVEC lines from some but not other umbilical cords, which suggests allotypy. Sera from 70 normal healthy male and nonpregnant female controls did not react with any of the individual HUVEC lines used. The purified anti-EC immunoglobulin G (IgGs) recognized bands, from HUVEC surface membrane protein preparations, with molecular weights of 120 kDa. Both hemostatic and fibrinolytic pathway markers were found activated in the presence of anti-EC IgGs, suggesting an altered endothelial cell surface activation state. CONCLUSIONS: The results indicate that anti-EC antibody is another marker for a subset of recurrent spontaneous aborters who may have activation of hemostasis and fibrinolysis as a mechanism involved in their losses.

Abortion, Habitual↗

PubMed1997

Antiphospholipid antibodies associated with implantation failure after IVF/ET.

PURPOSE: Our purpose was to determine the specific antiphospholipid antibodies (APAs) that should be evaluated to identify individuals at risk for implantation failure associated with reproductive autoimmune failure syndrome (RAFS). METHODS: The prevalence of APAs among 312 women with implantation failure was compared with that of 100 fertile control women. To be included in the implantation failure group, each woman had to have had at least 12 embryos transferred without subsequent positive pregnancy test. Enzyme-linked immunoabsorbant assay was used to measure IgG, IgM, and IgA anticardiolipin, antiphosphatidyl ethanolamine, antiphosphatidyl inositol, antiphospatidic acid, anti-phosphatidyl glycerol, antiphosphatidyl choline, and antiphosphatidyl serine. RESULTS: When the values for each of the seven APAs in three isotypes were compared between women with implantation failure and the control population, all of the APAs tested had a significantly higher frequency among women with implantation failure. Positive APAs were detected in 69 (22%) of the 312 women with implantation failure compared with 5 (5%) of the 100 control women (P < 0.0001). Anticardiolipin antibodies were found in 13 (4%) of the 312 women with implantation failure and none of the controls. Fifty-six (18%) of the 312 with implantation failure were negative for anticardiolipin antibodies but had positive values of other APAs. CONCLUSIONS: A complete APA panel using seven isotypes is necessary for diagnosing implantation failure associated with RAFS. If only anticardiolipin antibody is measured, 4% (13/312) of the positive APAs are detected, and 81% (56/69) of women with implantation failure associated with RAFS will have the diagnosis missed.

Antibodies, Antiphospholipid↗

PubMed1996

Embryonic origin of preimplantation factor (PIF): biological activity and partial characterization.

Preimplantation factor (PIF) is detected in the serum of women shortly after fertilization; its origin, however, has not been established. In this study, the embryonal origin of PIF was investigated and partial characterization of the factor was carried out. Culture media from viable human 2-8-cell stage embryos and mouse 2-cell-blastocyst stage embryos were analysed using the lymphocyte/platelet binding assay (LPBA). The assay was performed by combining culture media with donor O+ type blood-derived lymphocytes/platelets, complement and an antibody against CD2. Increased autorosette formation between lymphocytes and platelets (> 9%) was an indication for the presence of PIF. In addition, the effect of platelet-activating factor (PAF) and chaperonin 10 on PIF activity was determined. Partial purification of PIF was carried out using gel filtration and reverse-phase high purification liquid chromatography (HPLC), followed by mass spectrometry. Culture media of single human viable fertilized oocytes were negative for PIF; however, the 10-fold concentrated medium was positive for PIF. In medium in which five or more mouse embryos were cultured, PIF activity was observed starting at the morula stage and was higher by the blastocyst stage. Addition of PAF or chaperonin 10 to the PIF assay did not elicit a specific effect on PIF activity. Chromatographic data suggest that PIF activity is due to low molecular weight proteins. PIF appears to be a low molecular weight protein which is derived from viable preimplantation embryos. It is different from PAF or chaperonin 10. Its final characterization will be valuable for better understanding of maternal recognition of pregnancy and implantation.

Animals↗

PubMed1996

Development and validation of an assay for measuring preimplantation factor (PIF) of embryonal origin.

PROBLEM: Tests to determine presence of embryos prior to implantation are needed. METHODS: Sera from women after embryo transfer were tested for preimplantation factor (PIF) using the lymphocyte/platelet binding assay. Autorosettes were counted using blood type O+ donor lymphocytes and platelets incubated with blinded serum in the presence of antiCD2 antibody and rabbit complement. Human chorion gonadotropin (hCG) concentrations were determined 7 days later and compared with results of the lymphocyte/platelet assay. Implantation was confirmed by ultrasonographic evidence of presence of an intrauterine gestational sac. The roles of platelet activating factor (PAF) and chaperonin 10 in the observed phenomena were studied experimentally. RESULTS: Significantly more lymphocyte/platelet rosette formations were observed when sera from women who successfully implanted were compared to sera from women who failed to implant. Neither PAF nor chaparonin added to the tested sera controls influenced the percentage of lymphocyte/platelets rosettes. CONCLUSIONS: PIF is a likely candidate to be the next frontier of diagnosing the presence of viable preimplantation embryos in vivo.

Biological Factors↗

PubMed1996

Antiphospholipid antibody prevalence in patients with IVF failure.

Antiphospholipid antibodies (APAs) have been associated with reproductive wastage. The purpose of this study was to establish the prevalence of APAs in women who have had at least 12 embryos transferred during several in vitro fertilization (IVF) cycles without ensuing pregnancy. Sera from 42 women with IVF failure and 42 women who successfully conceived after IVF were tested for the presence of APAs by ELISA. Successful post-IVF pregnancy was determined by obtaining two consecutive rising beta-hCG levels followed by an ultrasound to confirm a viable conceptus. The sera were tested for three isotypes of antibody: IgA, IgG, and IgM against seven phospholipids: cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic Acid (PA), phosphatidyl-glycerol (PG), phosphatidylcholine (PC), and phosphatidyl-serine (PS). From the IVF failure group, 11/42 (26.2%) were positive for APAs. From the control group, 2/42 (4.8%) were found positive only for IgA against PE. The difference between IVF failure and successful IVF groups was significant (P = 0.01). These results suggest that antiphospholipid antibodies should be considered an important marker for increased risk of IVF failure. Patients who are involved with an IVF program should be tested for the presence of APAs prior to initiation of an IVF cycle.

Antibodies, Antiphospholipid↗

PubMed1996

Laboratory evaluation of women experiencing reproductive failure.

Reproductive life table analysis indicates that the majority of reproductive failures result from post fertilization failures, whether before or after implantation. It is important to have a set of tests to clarify the diagnosis of the reproductive failure so that appropriate therapy can be instituted. To determine the frequency of abnormal immunologic tests among women experiencing reproductive failure, 108 patients were evaluated for the presence of antiphospholipid antibodies (APA); lupus anticoagulant (LA); thyroid-thyroglobulin and microsomal antibodies (TGT); embryotoxic factor (ETA); and systemic CD56+/CD16- cells. The frequency of abnormal results obtained from testing for APA, LA, TGT, ETA, and CD56+/CD16- cells among 108 patients with diagnoses of recurrent pregnancy loss (RPL)(n = 45), unexplained infertility (n = 45) including IVF failure (n = 10), endometriosis (n = 10), premature ovarian failure (n = 5), and polycystic ovaries (n = 3) were compared with 15 normal controls. Seventy of one hundred eight (65%) women experiencing reproductive failure had at least one positive test, compared to 1 of 15 (7%) controls (P = 0.0001). Presence of phospholipid antibodies was the most frequently abnormal result followed by elevated CD56+/CD 16 cells. The prevalence of a particular abnormal test varied among the diagnoses. The most frequent abnormal test among women with RPL was an increased percentage of CD56+/CD16- cells (40%), followed by APAs (29%), TGT (9%), and ETA (7%). The most frequent abnormal result among women with unexplained infertility was the presence of APAs (42%), followed by CD56+/CD16- cells (16%), ETA (16%), and TGT (9%). APA, CD56+/CD16- cells, ETA, and TGT are useful tools to assist in the diagnosis of reproductive failure.

Abortion, Habitual↗

PubMed1995

Validation of an embryotoxicity assay.

PROBLEM: Culture of mouse blastocysts has served as a tool for identifying various embryotoxic factors in human serum. While inactivated, sera from recurrently aborting women inhibit mouse blastocyst development in vitro. Variation in results from individual serum samples has limited the usefulness of this assay in establishing a new classification of idiopathic recurrent spontaneous abortion (RSA). METHOD: Two-cell embryos were collected from superovulated mated CB6F1/J mice and cultured in Ham's F-10 media supplemented with 10% fetal bovine serum (FBS) or tested human serum at 37 degrees C with 5% CO2 and high humidity. Each sample was assayed in triplicate using three mice with at least five embryos from the same mouse per dish. Development was evaluated at 72 h and the frequency of atretic embryos was recorded. RESULTS: Intrasample (interassay) variation yielded a coefficient of variation of 9%. When repeated, samples from a given individual were evaluated and the coefficient of variation was 8.7%. Interoperator variability was 4% interassay and 2% intrassay. Atresia of embryos was 23% when incubated with FBS (N = 122), 21% in FC (N = 122), and in the sera of patients with RSA 34.6% (N = 95). Results of percentage of atresia from the fertile control group had a nonparametric distribution. Using 2.2 multiples of the median to determine the 95% confidence interval, a threshold at 44.0% of atresia was established. CONCLUSIONS: The critical step in maintaining low variability in this bioassay is to control mouse variability by averaging the percentage atresia from different mice as embryo donors for each tested serum. A subgroup of 24% (23/95) RSA patients who displayed embryotoxic activity was identified with a specificity of 95% and positive predictive value of 83%, P = 0.001.

Abortion, Habitual↗

PubMed1995

Preimplantation factor (PIF) predicts subsequent pregnancy loss.

PROBLEM: To evaluate the ability of preimplantation factor (PIF) measured in the lymphocyte/platelet binding assay (LPBA) to predict subsequent spontaneous abortion. METHOD: Serum from 57 women experiencing first trimester pregnancy losses were studied using the LPBA (46 women conceived after in vitro fertilization and embryo transfer for treatment of infertility and 11 with a history of unexplained recurrent spontaneous abortion conceived spontaneously). The assay employs a combination of heat inactivated sera with donor O+ lymphocytes and platelets, complement and an antibody against CD2. Chromosome analysis was performed on 32 of the abortuses. Results of PIF assay were compared between karyotypically normal and abnormal abortuses. RESULTS: PIF assay was negative in all 57 women at the time of abortion. Among 12 karyotypically normal abortuses only 1 woman (8%) had an initial positive PIF, 11 (92%) had negative PIF. Serial PIF assays were performed on 15 women. PIF assay became negative a minimum of two weeks prior to demonstration of intrauterine demise at a time when hCG concentrations remained elevated. A trend to subnormal was seen in women with normal when compared to those with abnormal abortus karyotype, but the numbers were too small to reach statistical significance (P = 0.09). CONCLUSION: Measurement of PIF throughout the first trimester of pregnancy predicts subsequent pregnancy loss.

Abortion, Habitual↗

PubMed1995

Prevalence of embryotoxic factor in sera from women with unexplained recurrent abortion.

PROBLEM: The presence of embryotoxic factors in sera from women with recurrent spontaneous abortion (RSA) has been proposed as a basis for classification of unexplained RSA. To determine the prevalence of circulating embryotoxins among women with idiopathic RSA, sera from 160 women were studied using the mouse blastocyst assay. METHODS: Two-cell embryos were collected from superovulated mated (CB6F1/J mice and cultured in media supplemented with fetal bovine serum (FBS) or 10% serum at 37 degrees C with 5% CO2 and high humidity. Each assay was run in triplicate using three mice with at least five embryos from each mouse. Results were determined by calculating the average percentage atresia for each mouse. FBS, known to support embryo proliferation, was used to control in each assay. RESULTS: The prevalence of embryotoxic factors among women experiencing RSA was 24.4% (39/160). There is no correlation found between the presence of embryotoxicity and phospholipid antibodies, lupus anticoagulant, and thyroglobulin/microsomal antibodies. CONCLUSION: The embryotoxicity assay can serve as a basis for a new approach for classification of unexplained recurrent spontaneous abortion.

Abortion, Habitual↗

PubMed1995

Systemic CD56+ cells can predict pregnancy outcome.

PROBLEM: To evaluate differences in circulating CD56+ cells between successful and unsuccessful pregnancies, 114 pregnant women were studied prospectively. METHOD: Seventy women had a history of infertility (INF) and 44 had two or more previous spontaneous abortions (RSA). Among the infertile women, 12 were donor egg recipients (DER) and 15 underwent intracytoplasmic sperm injection (ICSI) for treatment of male factor infertility. Nineteen women were carrying multiple gestations (MG) and 55 had singleton gestations (SG). Thirteen additional women were receiving intravenous immunoglobulin (IVIg). RESULTS: The percentage of CD56+ cells was determined in 310 blood samples from 114 pregnant women by flow cytometry. The prevalence of women with persistent elevation of percent of 56+ cells (> 12%) was 58% among DER, 73% among ICSI, 37% among MG, 22% among SG, 18% among RSA, and 39% among INF. Thirteen women with SG received IVIG, 10 had CD56+ cells greater than 12% and all 13 experienced live births. Women with percentage CD56+ cells persistently greater than 12% who were not DER, not ICSI, not receiving IVIg, and not carrying MG had a live birth rate of 11%. Women with greater than 12% CD56+ cells had normal karyotype in 78% of concepti studied in contrast to women less than 12% CD56+ cells who had 68% abnormal karyotypes (P = 0.04). CONCLUSION: Elevated CD56+ cells in pregnant women who are not DER, not ICSI, not receiving IVIg, and not carrying MG predicts loss of a karyotypically normal conceptus with a specificity of 87% and positive predictive value of 78%. While the specificity value of this test is high in both infertile and RSA populations, the sensitivity is 86% in RSA and only 54% in INF suggesting this test does not identify all losses among INF. It may identify a subset of pregnancies at risk for loss of a karyotypically normal embryo that may respond to treatment with IVIg.

Abortion, Habitual↗

PubMed1995

A novel bioassay for detection of preimplantation factor (PIF).

PROBLEM: To identify the presence of vital preimplantation embryos in vivo in humans, a newly observed phenomenon based on autorosette formation between lymphocytes and platelets, when treated with pregnant sera, was used as a marker. METHOD: Serum samples were obtained from 65 patients on the fourth day after embryo transfer (ET). Sera from 10 healthy males and 47 nonpregnant women were used as controls. The preimplantation factor (PIF) was detected by using blood group O+ donor lymphocytes and platelets incubated with blinded serum in the presence of anti-CD2 antibody and rabbit complement. Human chorion gonadotropin (hCG) concentrations were determined 7 days later and compared with results of the lymphocyte-platelets assay. Implantation was confirmed by ultrasonographic evidence of presence of an intrauterine gestational sac. The role of platelet activating factor (PAF) in the observed phenomena was studied experimentally. RESULTS: Significantly more lymphocyte-platelet rosette formations were observed when sera from women who successfully implanted were compared to sera from women who failed to implant. This assay yielded a specificity of 95%, sensitivity of 88%, positive predictive value of 94%, and negative predictability of 90%. PAF added directly to the cell suspension and tested sera controls did not influence the percentage of lymphocyte/platelets rosettes. CONCLUSION: The application of PIF assay will enable the identification and study of early pregnancy events before the implantation occur. PAF by itself is not responsible for the rosette formation.

Biological Assay↗

PubMed1993

Phenotypic characterization of normal human placental mononuclear cells.

The placenta is a rich source of immunocompetent cells. We have studied the phenotype, number and origin of placental mononuclear blood cells isolated from 32 normal term placentae using 4 color flow cytometry. Respective maternal and cord blood leucocyte preparations were also compared. Placental tissue without extraembryonic membranes was cut into small pieces and divided. One portion was washed extensively with ice-cold PBS. Both tissue portions were disrupted in a blender and cells were dissociated by using a 180 mu sieve. Leucocytes were isolated by Ficoll-Hypaque density gradient centrifugation. Maternal and cord bloods were HLA typed and in cases of HLA-A2 or B7/40 disparity, monoclonal anti-HLA antibodies to these antigens showed that unwashed placental tissue contained 35% maternal and 65% fetal cells. This ratio, however, was not reflected for a given cell phenotype. In comparison, washed placental tissue contained cells of fetal origin only. Both unwashed and washed placental tissue contained fewer CD3 and CD4, but more CD8 cells than maternal and cord blood. Markers of NK cells such as, CD16, CD56, and CD57 showed this cellular phenotype to be 15 times more abundant in the placental preparations than in cord and maternal blood. The quantitative differences between peripheral blood and placental CD8 and NK cells were further explored with an antiprogesterone receptor antibody in combination with anti-CD8, anti-CD57 and anti-HLA-DR. The number of progesterone receptor (PGR) positive cells was three times higher in placental tissues than in cord or maternal blood. These data indicate that the phenotypic frequencies of certain placental leucocytes are significantly different from maternal and fetal peripheral blood. Progesterone and the presence of PGR may be important in the differential retention of placental leucocytes.

Antigens, CD↗