Roumen G. Roussev

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.

Multiple Thrombophilic Gene Mutations Rather than Specific Gene Mutations are Risk Factors for Recurrent Miscarriage

Recurrent miscarriage is a heterogeneous condition. While the role of acquired thrombophilia has been accepted as an etiology of recurrent miscarriage, the contribution of specific inherited thrombophilic genes to this disorder has remained controversial. We compared the prevalence of 10 thrombophilic gene mutations among women with a history of recurrent miscarriages and fertile control women.

Immunodiagnostic Evaluation in Women with Reproductive Failure

Kaider AS, Kaider BD, Janowicz PB, Roussev RG. Immunodiagnostic evaluation in women with reproductive failure. AJRI 1999; 42:335–346

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+/CD16 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.

Multiple thrombophilic gene mutations are risk factors for implantation failure

While the role of inherited thrombophilia has been accepted as a cause of recurrent late pregnancy complications, the contribution of mutated thrombophilic genes to implantation failure has not been studied. Proteins involved in fibrinolysis are necessary for trophoblast invasion into the endometrium. This study compared the prevalence of 10 thrombophilic gene mutations among 42 women with a history of recurrent implantation failure after IVF–embryo transfer with 20 fertile control women. Buccal swabs were taken from all of the women for DNA analyses. Women with a history of implantation failure after IVF–embryo transfer displayed a higher prevalence of PAI-1 4G/5G mutations than controls (P = 0.007). No differences in the frequency of the other specific gene mutations were detected. However, the prevalence of total gene mutations among patients with implantation failure was significantly higher than among controls. More than three gene mutations among the 10 genes studied were observed in 74% of women with implantation failure and 20% of controls (P = 0.0004). It is concluded that inherited thrombophilias are associated with implantation failure. This association is manifest by total number of mutations as well as with PAI-1 mutations.

Which thrombophilic gene mutations are risk factors for recurrent pregnancy loss

Problem  Thrombophilia has been associated with poor obstetrical outcomes. To determine the association of specific inherited thrombophilias and recurrent pregnancy loss, 10 thrombophilic genes were investigated.

Correlation of NK Cell Activation and Inhibition Markers with NK Cytoxicity Among Women Experiencing Immunologic Implantation Failure After In Vitro Fertilization and Embryo Transfer

AbstractPurpose: The pivotal event in determining successful from unsuccessful cycles after in vitro fertilization is implantation. The purpose of this study was to compare the percentage of circulating NK cells expressing activation and inhibition markers between infertile and fertile control women and to determine the correlation between these markers and those of the NK cytotoxicity activation assay. Lastly, we wish to determine the ability of each of these markers to predict pregnancy outcome after IVF/ET (in vitro fertilization/embryo transfer). Methods: Blood samples from 22 infertile women undergoing IVF/ET during the November 2001 cycle were drawn on cycle Day 9 and analyzed for expression of CD69+, HLA-DR, CD161+, CD94+, and CD158a+ as well as NK cytotoxicity using immunoflluorescent labeling and flow cytometry. Results were compared with those from 26 fertile control women and correlated to pregnancy outcome that of cycle. Results: Infertile women had significantly higher expression of NK cell activation markers of CD69+ and CD161+ than fertile women. NK cytotoxicity correlated inversely with expression of NK cells bearing the inhibition marker of CD94+. None of the successfully pregnant women of that cycle had elevated levels of NK cytotoxicity whereas 50% of those experiencing a chemical pregnancy loss and those not becoming pregnant had elevated levels of NK cytotoxicity. Conclusions: Immunologic markers can identify mechanisms involved in implantation failure. Activation markers of CD69+ and CD161+ expressed on NK cells as well as NK cytotoxicity can be added to the previously reported risk factors for immunologic implantation failure.

Antiphospholipid antibodies associated with implantation failure after IVF/ET.

Purpose: Our purpose was to determine the specific anti-phospholipid 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 ethanol-amine, antiphosphatidyl inositol, antiphospatidic acid, antiphosphatidyl 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.

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), phosphatidyle‐thanolamine (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.

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.