Showing papers in "American Journal of Reproductive Immunology in 2003"

The Role of Cytokines in Endometriosis

Abstract: PROBLEM: To review the literature on the role of peritoneal cytokines in the pathogenesis and endometriosis-related infertility. METHODS OF STUDY: A MEDLINE search was conducted by the key words of cytokine and endometriosis in the English publications, and references identified within the identified papers were also reviewed. RESULTS: Several cytokines including interleukin (IL)-1, 6, 8, 10, tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF) were reported to be increased in the peritoneal fluid (PF) of women with endometriosis. Those cytokines may be involved in macrophage activation, inflammatory change and enhanced angiogenesis. However, some cytokines were less expressed such as IL-2, and interferon (IFN)-γ. They reflect the impaired T- and natural killer (NK)-cell function. Endometriotic implants produce some factors, e.g. matrix metalloproteinases (MMPs), Bcl-2, and affect their capacity to implant into the peritoneum. CONCLUSION: Peritoneal cytokines, which are produced by mesothelial cells, leukocytes and ectopic endometrial cells, interwork locally and systemically in women with endometriosis. More studies about the specific role and interactions of these cytokines are needed to improve the understanding of endometriosis and to develop novel therapies.

Immunology and endometriosis.

Abstract: Problem: Accumulating data suggests that aberrant immune responses during retrograde menstruation may be involved in the development of endometriosis. Method of Study: The role of immunology in the etiology of endometriosis is reviewed and summarized from the available literature. Results: Immunologic factors may affect a woman's susceptibility to implantation of exfoliated endometrial cells. Immune alterations include increased number and activation of peritoneal macrophages, decreased T cell reactivity and natural killer cell cytotoxicity, increased circulating antibodies, and changes in the cytokine network. Conclusion: There is substantial evidence that immunologic factors play a role in the pathogenesis of endometriosis and endometriosis-associated infertility. Decreased natural killer cell cytotoxicity leads to an increased likelihood of implantation of endometriotic tissue. In addition, macrophages and a complex network of locally produced cytokines modulate the growth and inflammatory behavior of ectopic endometrial implants.

Pre-conceptional natural killer cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype

Abstract: Problem: The aim of the present study was to determine whether pre-conceptional natural killer (NK) cell activity and percentage are predictive of subsequent spontaneous abortion in women with recurrent spontaneous abortion (RSA). Method of study: Pre-conceptional NK cell activity and percentage in peripheral blood of women who had a history of two or more RSA was prospectively assessed. The 51Cr release assay and flow cytometric analysis were performed. A total of 113 RSA women were recruited, and 85 conceived later. Results: Pre-conceptional NK cell activity/percentage values in women whose next pregnancies ended in biochemical pregnancy and spontaneous abortion with normal fetal karyotype (n = 17, median 47%/17.1%), but spontaneous abortion with abnormal karyotype (n = 9, 27%/15.7%), were higher than those in live births (n = 59, 33%/13.1%). High values of pre-conceptional NK cell activity (>46%; relative risk 3.6, 95%CI 1.6–8.0) and percentage (>16.4%; 4.9, 1.7–13.8) were found to predict biochemical pregnancy and spontaneous abortion with normal karyotype in the next pregnancy. Conclusion: Pre-conceptional NK cell abnormalities were predictive of spontaneous abortion with normal fetal karyotype.

Identification of Possible Mediators of Embryonic Mortality Caused by Mastitis: Actions of Lipopolysaccharide, Prostaglandin F2α, and the Nitric Oxide Generator, Sodium Nitroprusside Dihydrate, on Oocyte Maturation and Embryonic Development in Cattle

Abstract: PROBLEM Mastitis and immunization against constituents of organisms causing mastitis can reduce fertility of cattle and sheep, respectively. For the current experiments, it was hypothesized that these effects are mediated via actions of lipopolysaccharide (LPS), prostaglandin F2alpha (PGF2), and nitric oxide on oocyte maturation and embryonic development. METHOD OF STUDY To evaluate effects on oocyte maturation, oocytes were matured with various concentrations of LPS, PGF2alpha, or the nitric oxide (NO) generator, sodium nitroprusside (SNP). Following maturation, oocytes were fertilized and cultured until day 8 after fertilization. To test effects on embryo growth, oocytes were matured and fertilized and cultured after fertilization with LPS, PGF2alpha, or SNP. RESULTS Addition of 100 and 1000 ng/mL LPS and 50 and 100 ng/mL PGF2alpha to oocyte maturation medium reduced the proportion of oocytes that became blastocysts at day 8 after fertilization. When added after fertilization, in contrast, neither LPS nor PGF2alpha reduced development to the blastocyst stage. Unlike for LPS and PGF2alpha, addition of SNP during oocyte maturation was without effect on the proportion of oocytes that became blastocysts at day 8 after fertilization. However, addition of 10 microM SNP to culture medium after fertilization completely prevented development to the blastocyst stage while 0.1 and 1 microM SNP did not affect development. CONCLUSIONS Results indicate that increased local concentrations of LPS, PGF2alpha, and NO can have deleterious consequences on oocyte function (LPS, PGF2alpha) and embryonic development (NO). Thus, these molecules are putative mediators of effects of infectious disease or inflammation, including mastitis, on fertility of cattle.

Cytokine secretion patterns of NK cells and macrophages in early human pregnancy decidua and blood: implications for suppressor macrophages in decidua.

Abstract: During pregnancy, the woman carries a fetus partly foreign to her immune system, because of the expression of paternal antigens. Despite this, the fetus is normally tolerated and not rejected, as is often the case with organs in allogeneic transplantations. Systemic changes in maternal blood occur during pregnancy but, perhaps of greater importance, are changes in tissues locally in the uterus. The pregnant uterine endometrium, the decidua, is infiltrated by large numbers of leukocytes, mainly natural killer (NK) cells but also macrophages and T lymphocytes. Further, various cytokines are known to be secreted at the fetomaternal interface. However, the functions of these cells and the cytokine networks are not fully understood. The aim of this thesis was to investigate the local immune balance in normal human pregnancy decidua, both in the early phase of pregnancy and at parturition. First trimester decidual mononuclear cells, NK cells and macrophages were all shown to secrete IFN-γ, IL-4 and IL-10, as detected by ELISPOT. The secretion was not mirrored in blood from the same subjects. A significantly larger number of decidual macrophages secreted IL-10 than did their blood counterparts, indicating potential regulatory functions of this cell type. Further examination of early pregnancy decidual macrophages by microarray revealed 120 genes being differentially regulated at the transcriptional level in decidual compared to blood monocytes/macrophages. Several genes were associated with alternative activation/M2 polarization of macrophages, including CCL-18, CD209, IGF-1, MRC-1 and FN-1. Genes connected to immune regulation and tissue remodelling were common, in line with the potential functions for this cell type in utero. In addition, some molecules not previously connected to decidual macrophages, such as TREM-2, A2M and PGDS, were found to be upregulated, gaining new insights into the regulatory functions of decidual macrophages. Term decidual mononuclear cells spontaneously secrete IFN-γ, TNF, IL-4, IL-10, and TGF-β. No differences were seen between tissues obtained before and after the onset of labour, indicating that decidual mononuclear cells are not the main cell population responsible for plausible cytokine regulation in the process of labour induction. Placental and fetal membranes as well as cells in the maternal systemic circulation may instead contribute to a possible shift in immune balance prior to pregnancy termination. In conclusion, decidual leukocytes, including NK cells and macrophages, are potential producers of both Th1-like/pro-inflammatory and Th2-like/anti-inflammatory cytokines in early pregnancy as well as at parturition. Decidual macrophages are of a specialized phenotype with effector functions contributing to a proper invasion of the placenta and to immunological protection of the semi-allogeneic fetus. This thesis adds new knowledge on local immune balance during normal human pregnancy, however, the clinical significance of the presented data needs to be clarified.

Cloning Adult Farm Animals: A Review of the Possibilities and Problems Associated with Somatic Cell Nuclear Transfer

Abstract: In 1997, Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal. To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer. The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo. The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte. Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals. Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes. Moreover, genetically modified farm animals have created a new form of 'pharming' whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors. A major limitation of cloning procedures is the extreme inefficiency for producing live offspring. Dolly was the only live offspring produced after 277 attempts. Similar inefficiencies for cloning adult animals of other species have been described by others. Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life. Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans.

Pro-inflammatory maternal cytokine profile in preterm delivery.

Abstract: PROBLEM: The objective of this study was to determine the levels of cytokines produced by maternal peripheral blood mononuclear cells (PBMC) upon stimulation with a mitogen, with autologous placental cells and with a trophoblast antigen extract. METHOD OF STUDY: Peripheral blood mononuclear cells from 54 women with a history of successful pregnancy and 30 women undergoing preterm delivery (PTD) were stimulated with the mitogen and antigens, and the cytokine levels in mitogen-stimulated culture supernatants assessed. RESULTS: Significantly higher levels of the type 1 cytokines, interferon (IFN)-γ and interleukin (IL)-2, were produced by the PTD group than by the normal pregnancy group, which on the contrary showed significantly greater production of the type 2 cytokines, IL-4, IL-5 and IL-10. A comparison of the ratios of type 2 to type 1 cytokines is indicative of a type 1 cytokine bias in PTD. CONCLUSIONS: These data are suggestive of a maternal type 1 cytokine bias in PTD.

Cytokines, implantation and early abortion: re-examining the Th1/Th2 paradigm leads to question the single pathway, single therapy concept.

Abstract: Problem: Human in vitro fertilization (IVF) embryo transfer is accompanied by a low implantation rate even after a very successful IVF, and there are a certain number of ‘idiopathic sterilities’ which are due to repeated implantation failures. In the very same vein, the question of improving implantation rates is of prime importance in agricultural research to improve the management of livestock. Pre-implantation prenatal diagnosis cannot be accomplished in individuals who have a high rate of implantation failure, whether women undergoing IVF, or animals, during genetic cloning. Implantation cytokine networks need to be known in such a perspective. Methods: We review the evolution and theories in reproductive immunology, briefly deal with the complexity of implantation as a step by step developmental event, and then present some of our recent data in mice and human. Conclusions: We conclude that the T helper cell type 1/2 (Th1/Th2) paradigm, as useful as it has been to explain pregnancy, is no longer sufficient in view of the emerging complexity of the cytokine network at the materno-fetal interface. This is peculiarly true for implantation, which, as a step by step developmentally regulated process, involving inflammatory molecules, cannot fit into such a scheme.

Actions of tumor necrosis factor-alpha on oocyte maturation and embryonic development in cattle.

Abstract: Problem Infertility can accompany mastitis in cattle. Involvement of tumor necrosis factor-alpha (TNF-alpha) in this phenomenon is suggested by observations that circulating concentrations of TNF-alpha are elevated after intramammary infection or infusion of endotoxin. It was hypothesized that (1) TNF-alpha acts on the oocyte during maturation to decrease the percent of oocytes that cleave and develop following fertilization; (2) exposure of embryos to TNF-alpha after fertilization reduces development to the blastocyst stage; and (3) TNF-alpha increases the proportion of blastomeres that undergo apoptosis in a stage-of-development dependent manner. Method of study In one experiment, oocytes were matured with various concentrations of TNF-alpha and then fertilized and cultured without TNF-alpha. In another study, embryos were cultured with TNF-alpha for 8 days beginning after fertilization. Finally, embryos were collected at the two or four-cell stage (at 28-30 hr after insemination) or when > or = 9-cells (at day 4 after insemination) and cultured +/- TNF-alpha for 24 hr. The proportion of blastomeres undergoing apoptosis was then determined by the TUNEL procedure. Results Addition of TNF-alpha to maturation medium did not affect the proportion of oocytes that cleaved. However, the percent of oocytes that developed to the blastocyst stage at day 8 after insemination was reduced (P = 0.05) at all TNF-alpha concentrations tested (0.1-100 ng/mL). When added during embryo culture, there was no significant effect of TNF-alpha on the proportion of oocytes that became blastocysts. In addition, TNF-alpha did not induce apoptosis in two and four-cell embryos. For embryos > or = 9-cells, however, 10 and 100 ng/mL TNF-alpha increased (P Conclusion TNF-alpha can have deleterious actions on oocyte maturation that compromise development of the resultant embryo. While exposure of fertilized embryos to TNF-alpha did not inhibit development to the blastocyst stage, TNF-alpha increased the percentage of blastomeres undergoing apoptosis when exposure occurred for embryos > or = 9-cells. Increased blastomere apoptosis could conceivably compromise subsequent embryo survival.

Heme oxygenases in pregnancy II: HO-2 is downregulated in human pathologic pregnancies.

Abstract: Problem: We previously reported a diminished expression of the heme-degrading enzymes heme oxygenases (HO)-1 and HO-2 in decidua and placenta from mice undergoing Th1-mediated abortion, strongly indicating the protective effect of HO in murine pregnancy maintenance. Here we investigated whether the expression of HO-1 and HO-2 is also reduced at the feto-maternal interface of pathologic human pregnancies. Method of Study: Immunohistochemistry was used to detect HOs expression in placental and decidual first-trimester tissue from patients with: spontaneous abortion (n = 14), choriocarcinoma (n = 14), hydatidiform mole (H-mole) (n = 12), compared with normally progressing pregnancies (n = 15). Further, we investigated early third-trimester decidual and placental tissue from patients with pre-eclampsia (n = 13) compared with fetal growth retardation (n = 14) as age-matched controls. Results: In first trimester tissue, we observed a significant reduction of HO-2 expression in invasive trophoblast cells, endothelial cells, and syncytiotrophoblasts in samples from patients with spontaneous abortion compared with normal pregnancy. H-mole samples showed a diminished expression of HO-2 in invasive trophoblast cells and endothelial cells in comparison with NP, whereas choriocarcinoma samples showed no significant differences compared with the control. In third trimester tissue, HO-2 was also reduced in syncytiotrophoblasts and invasive trophoblast cells from pre-eclampsia compared with samples from fetal growth retardation. HO-1 expression was diminished in all pathologies investigated; however, the differences did not reach levels of significance. Conclusions: Our data indicate that HOs play a crucial role in pregnancy and low expression of HO-2, as observed in pathologic pregnancies, may lead to enhanced levels of free heme at the feto-maternal interface, with subsequent upregulation of adhesion molecules, allowing enhanced inflammatory cells migration to the feto-maternal interface.

Natural Killer (NK) Cell Receptors' Repertoire in Couples with Recurrent Spontaneous Abortions

Abstract: PROBLEM: Natural killer (NK) cell receptors (NKRs) have been suggested to protect trophoblast, but their function at the fetomaternal interface remains unknown. To investigate if the outcome of pregnancy depends on women's NKRs, we studied the NKR repertoire in couples with recurrent spontaneous abortions (RSA). METHODS: Twenty-six childless couples with ≥2 abortions, characterized by alloimmune abnormalities, and 26 control couples were genotyped for five killer immunoglobulin-like receptors (KIR) and two CD94/NKG receptors, known to have as ligands human leukocyte antigen (HLA) class I molecules with trophoblastic expression: inhibitory 2DL1,2,3 and activating 2DS1,4 KIRs, inhibitory NKG2A and activating NKG2C. Detected repertoires of women and partners were compared between the two groups. RESULTS: Less aborters than controls were found to have all three inhibitory KIRs (30.77% versus 69.23%, P = 0.01), some of them had only one inhibitory KIR (19.23% versus 3.85%, P = 0.08) and most of them were lacking inhibitory KIRs possessed by their husbands (57.69% versus 15.38%, P = 0.001). CONCLUSIONS: Women with alloimmune abortions have a limited inhibiting KIR repertoire and such miscarriages may occur because trophoblastic HLA class I molecules are recognized by decidual NK cells lacking the appropriate inhibitory KIRs.

Placental trophoblast from successful human pregnancies expresses the tolerance signaling molecule, CD200 (OX-2).

Abstract: PROBLEM: Th1 cytokine-dependent abortions in the CBA x DBA/2 mouse model have been linked to down-regulation of expression of the CD200 (OX-2) 'tolerance' signal on trophoblast and in decidua prior to onset of the abortion process. Abortions could be prevented by administration of a soluble CD200. Is CD200 expressed on trophoblast in successful human pregnancy? METHOD OF STUDY: As one cannot easily obtain trophoblasts in large quantities from successful human pregnancies in the first trimester prior to the onset of the abortion process at 6 weeks gestation, we examined as a first step, trophoblast isolated from term placentae (i.e. successful pregnancies). CD9 - trophoblasts were isolated by affinity column and stained for intracellular cytokeratin, and surface CD200 using PE-anti-human CD200 monoclonal antibody. mRNA was extracted from CD9 + and CD9 - cells and tested by reverse transcription--polymerase chain reaction for CD200 mRNA. CD9 - placental cells were separated by velocity sedimentation and test for CD200-dependent suppression of an allogeneic human mixed lymphocyte culture where cytotoxic T cell (CTL) generation, and Th1 → Th2 cytokine production shift were measured. RESULTS: CD9 - but not CD9 + placental cell populations contained cells with mRNA for CD200, both a normal length transcript and a truncated transcript. Flow cytometry showed a CD200 + cytokeratin + moderate-to-large-sized cell population compatible with trophoblasts and a smaller subset of cytokeratin - cells that expressed CD200 at normal and at high levels. The moderate-sized population proved most potent at inhibiting CTL generation and caused a Th1 → Th2 cytokine shift. These effects were blocked by monoclonal anti-CD200. CONCLUSIONS: A subpopulation of cytokeratin + placental trophoblasts express bioactive CD200 able to alter maternal immune responses in a favorable (Th2 > Thl) direction. Two populations of CD200 + small- and medium-small-sized cytokeratin - placental cells remain to be identified. Studies of karyotyped first trimester elective termination and spontaneous miscarriage tissues are needed.

GM-CSF and pregnancy: evidence of significantly reduced blood concentrations in unexplained recurrent abortion efficiently reverted by intravenous immunoglobulin treatment.

Abstract: PROBLEM: Certain Th-2 cytokines and granulocyte-macrophage colony-stimulating factor (GM-CSF) are propitious for the success of pregnancy and recurrent spontaneous abortion (RSA) is often characterized by a failure of Th-2 type responses. These considerations as well as the use of intravenous immunoglobulin (IVIg) in RSA induced us to evaluate the levels of GM-CSF in normal pregnancies, in pregnant women affected with unexplained RSA and the effects of IVIg treatment. METHOD OF STUDY: Peripheral blood free GM-CSF was measured by means of a sandwich enzyme immunoassay in 39 healthy women (13 non-pregnant, 26 pregnant) and in 53 RSA patients (11 non-pregnant, 42 pregnant). In 14 pregnant RSA patients GM-CSF was studied also after the very first IVIg infusion (0.5 g/kg body weight). RESULTS: In healthy women we found a significant increase of GM-CSF during pregnancy, in pregnant RSA patients such an increase was not detected. After IVIg, GM-CSF concentrations were almost doubled. CONCLUSIONS: GM-CSF is found increased in normal pregnancy and is very low during pregnancy in RSA. IVIg infusions are capable of increasing GM-CSF in pregnant recurrent aborters.

Demonstration of the presence of IL-16, IL-17 and IL-18 at the murine fetomaternal interface during murine pregnancy.

Abstract: PROBLEM: To determine if interleukin-16 (IL-16), IL-17, and IL-18 are present at the murine fetomaternal interface during pregnancy as a first step towards investigating their roles in fetomaternal relationship. METHODS: Expression of IL-16, IL-17, and IL-18, was assessed by immunohistochemistry (IHC) in the BALB/c x BALB/k (H2d x H2k), and the CBA/J x BALB/c non-abortion prone, and CBA/J x DBA/2 abortion prone matings. Enzyme-linked immunosorbent assay (ELISA) were performed for the two latter cytokines to compare local production in the abortion prone CBA/J x DBA/2 versus the non-abortion prone CBA/J x BALB/c matings. RESULTS: Expression of IL-17 was borderline. The anti-IL-16 staining specifically localized in the uterine stroma and glandular epithelium and was rather low in the placenta. IL-18 staining started in the peri-implantation uterus in the basal proliferative stroma, and was also traced, although weaker, in the glandular epithelium. In the immediate post-implantation period, a weak stromal staining persisted but there was a strong labeling of the ectoplacental cone. Interestingly, when the ectoplacental cone differentiates into placenta having a major histocompatibility complex (MHC) class I + spongiotrophoblast and a (MHC class I-) labyrinth, a very strong transient labeling of uterine natural killer (u-NK) cells was found. Later in gestation, IL-18 was also produced by giant cell and spongiotrophoblast. Finally, we compared by ELISA the production of IL-17/-18 in CBA/J x DBA/2 and CBA/J x BALB/c matings. We detected significantly more IL-18 in the non-abortion prone combination decidua or placenta. CONCLUSION: The three cytokines IL-16, IL-17, and IL-18 were detected at the fetomaternal interface with a tissue specific, stage-dependent distribution. The predominance of IL-18 secretion in the non-resorption prone matings lead us to question the general validity of the classical T-helper (Th)1/2 paradigm.

Increased T-cell activation in decidua parietalis compared to decidua basalis in uncomplicated human term pregnancy.

Abstract: PROBLEM: The aim of this study was to quantify and compare activated T cells in term decidua basalis and parietalis using flow cytometry. METHOD OF STUDY: Term decidua basalis and parietalis samples were obtained from 20 placentas collected after elective caesarean section. Percentages of leukocyte subclasses within the CD45+ cell fraction and activated T cells were determined by flow cytometry. RESULTS: There was no significant difference in CD45+, CD14+, CD19+, and CD3+ cell percentages. However, within the CD3+ population, there were significantly more T-cell receptor-γδ+ (TCR-γδ+) and CD8+ cells in decidua parietalis compared with decidua basalis. More importantly, percentages of T cells expressing CD25, human leukocyte antigen (HLA)-DR, CD45RO, and CD69 markers were significantly increased in decidua parietalis. CONCLUSION: These findings suggest that there are more activated T cells in decidua parietalis than in decidua basalis. Further investigation into differences between the two decidual sites may expand our understanding of the immunology of the maternal–fetal interface.

Antiprothrombin Antibodies are Associated with Pregnancy Loss in Patients with the Antiphospholipid Syndrome

Abstract: OBJECTIVE: To document the clinical association between the history of pregnancy loss in patients with the diagnosis of primary or secondary antiphospholipid syndrome (APS) and the presence of different antiprothrombin antibody subtypes [immunoglobulin G (IgG), IgM and IgA] in a cohort of patients with APS. METHODS: Records of 170 female patients with primary APS, or APS secondary to systemic lupus erythematosus (SLE) or secondary to other autoimmune diseases were studied. RESULTS: In female APS patients with IgG antiprothrombin antibodies (n = 105) significant associations to pregnancy loss (p < 0.0001), early pregnancy loss (p < 0.0001) and a negative association to thrombocytopenia (p < 0.01) could be identified. In the group of patients with IgG antiprothrombin antibodies and at least one pregnancy (n = 84) a significant association with pregnancy loss (p < 0.005) and especially with early pregnancy loss (p < 0.0001) was demonstrated. No association with other immunoglobulin subtypes of antiprothrombin antibodies could be documented. In the subgroup of patients with primary APS and at least one pregnancy in the history, pregnancy loss (p < 0.005) and early pregnancy loss (p < 0.0001) were found to be highly associated with the presence of IgG antiprothrombin antibodies. IgG antiprothrombin antibodies represent the highest independent risk factor for pregnancy loss with an odds ratio of 4.5. There was no statistically significant association with venous or arterial thrombosis in all IgG antiprothrombin antibody positive patients CONCLUSION: The results of this study document the association of IgG antiprothrombin antibodies with pregnancy loss and in particular early pregnancy loss in a large and well-characterized cohort of patients. We would recommend routine testing for antiprothrombin antibodies in young female patients with APS.

Evidence for a Correlation between Trophoblast Invasiveness and STAT3 Activity

Abstract: Problem: Extravillous trophoblast cells are capable of invading decidual tissue during early pregnancy. This property is reminiscent of cancer cells. The invasiveness of trophoblasts, however, extends only to a well-regulated limit. Signal transduction processes underlying this phenomenon are as yet poorly characterized. Many factors involved in trophoblast invasiveness are known to trigger intracellular signaling cascades in other cell types that ultimately lead to the activation of signal transducers and activators of transcription (STATs). STAT3 activity was recently found related to the malignant phenotype of different tumor cells and potentially contributes to their invasive properties. Method of Study: We investigated the status of STAT3 activity in ex vivo trophoblast cells from first trimester and term placentae employing an electrophoretic mobility shift assay (EMSA) and compared it with that of a highly malignant choriocarcinoma cell line. Results: Specific DNA binding activity of two STAT3 variants (STAT3α and β) was observed in immature trophoblasts and appeared to be lost in term placentae. The malignant phenotype of choriocarcinoma cells coincides with a high degree of STAT3 activity. Conclusion: These results suggest a connection between STAT3 activity and trophoblast invasiveness.

Inherited thrombophilia: impact on human reproduction.

Abstract: The development of thrombotic disorders is a major threat for young women during pregnancy. It is one of the main causes of pregnancy-related disorders, which may also result in harm for the conceptus. Successful pregnancies require an even balance of coagulation and fibrinolysis, in order to secure stabilization of the basal plate as well as adequate placental perfusion. Thrombophilia is a laboratory definition for pre-disposing factors of thrombosis, which can be inherited or acquired. Many individuals, who carry a thrombotic defect remain asymptomatic, at least until additional boosting factors arise. The documentation of thrombophilic causes of recurrent miscarriage or pregnancy-related disorders is important, because of availability of effective early treatment. There is a rapidly growing awareness on the relationship of genetic factors influencing hemostasis and pregnancy-related disorders. The aim of our review is to summarize this knowledge, focusing on common genetic variations.

Characterization of spermatozoa surface antigens by antisperm antibodies and its influence on acrosomal exocytosis.

Abstract: PROBLEM: Antisperm antibodies (ASA) are the main cause of immunological infertility, they impair sperm functions by binding to the sperm membrane. The aim of this study was to characterize highly enriched sperm membrane proteins by 2-D-electrophoresis and to identify membrane antigens binding ASA and to evaluate the influence of ASA on the acrosome reaction (AR). METHOD OF STUDY: Sperm membrane proteins were separated by 2-D-electrophoresis and antigens were identified by immunoblotting with ASA from seminal plasma samples of infertile men. The influence of ASA on the AR were observed and determined by means of flowcytometry. RESULTS: A total of 18 antigens were identified by using ASA from seminal plasma. Six of the recognized proteins were analyzed by means of mass spectrometry and peptide matching: HSP70 and HSP70-2, disulfide-isomerase-ER60, caspase-3 and two subunits of the proteasome (component-C2 and zeta-chain). ASA from seminal plasma are able to enhance the AR in donor-spermatozoa. CONCLUSION: The biochemical identification of these proteins will be helpful to understand the mechanism by which ASA impair sperm function and the fertilization process. Spermatozoa, in which the AR was prematurely induced by ASA, will not be able to fertilize anymore.

TNF‐α Protects Embryos Exposed to Developmental Toxicants

Abstract: BACKGROUND Tumor necrosis factor alpha (TNF-alpha) has been implicated in mediating post-implantation embryo loss or the embryonic maldevelopment induced by development toxicants or maternal metabolic imbalances. In order to clarify the role of TNF-alpha further, a comparative study was performed in TNF-alpha, knockout and TNF-alpha, positive mice, exposed to a reference teratogen, cyclophosphamide (CP). METHODS Cyclophosphamide was injected on day 12 of pregnancy and 18-day fetuses were examined for external structural anomalies. Apoptosis and cell proliferation were measured by TdT-mediated biotin-dUTP nick-end labeling and 5'-bromo-2'-deoxyuridine incorporation, respectively, in the brain (an organ, sensitive to the teratogen) of embryos 24 hr after CP injection. NF-kappaB DNA-binding activity by electrophoretic mobility shift assay (EMSA) and the expression of Re1lA (an NF-kappaB subunit) and I(kappa)B(alpha) proteins by Western blot analysis were assessed in the brain of embryos tested 24 and 48 hr after CP treatment. RESULTS Surprisingly, the proportion of fetuses with craniofacial, trunk and severe limb reduction anomalies were significantly higher in TNF-alpha -/- females, than in TNF-alpha,+/+ mice. Excessive apoptosis and suppression of cell proliferation was found in the brain, and they were more prominent in TNF-alpha -/- than TNF-alpha +/+ embryos, when examined 24 hr after CP injection. Finally, CP-induced suppression of NF-kappaB DNA-binding activity was found to be enhanced in the brain of TNF-alpha -/- embryos, and the restoration of NF-kappaB DNA-binding activity was compromised. CONCLUSION This work demonstrates for the first time that TNF-alpha may act as a protector of embryos exposed to teratogenic stress. One possible mechanism may be restoration of NF-kappaB activity in embryonic cells surviving the teratogenic insult.

Antiphospholipid antibodies in implantation failures

Abstract: PROBLEM: The amino phospholipids (PL), phosphatidylserine (PS) and phosphatidylethanolamine (PE) are distributed asymmetrically in the plasma membranes of eucaryotic cells. This arrangement involves active transport of PS and PE from the outer to inner membrane leaflet by an aminophospholipid translocase (flipase). Cell activation, injury and programmed cell death (apoptosis) cause collapse of the PS/PE asymmetry by activation of another enzyme system, scramblase. Unlike other cells, the developing trophoblast exteriorizes PS during its differentiation. METHODS OF STUDY: An analysis of published and unpublished data. RESULTS: The trophoblast is targeted by antiphospholipid antibodies (aPL), especially to PS (aPS). Cardiolipin is not present in the trophoblast plasma membrane, nonetheless, anticardiolipin (aCL) has been implicated in trophoblast pathology. The aPS and aCL are often crossreactive. Both animal and in vitro experimental models have shown monoclonal and polyclonal aPS and aCL to specifically destroy trophoblast, inhibit syncytium formation, halt human chorionic gonadatropin (hCG) production, and limit trophoblast invasion. Antibodies to PE (aPE) have not been well characterized, however, recent reports from several independent laboratories document that aPE are associated significantly with very early (embryonic) recurrent pregnancy loss (RPL). Umeda and coworkers have shown that during cytokinesis (late telophase) of Chinese hamster ovary (CHO) cells, formation of PE rafts in cleavage furrows is required for completion of cell division and formation of daughter cells. This raises the question whether aPE might interfere with implantation and cell division during embryogenesis. CONCLUSIONS: A role for aPL in implantation failure and occult pregnancy loss constitutes the basis of this overview.

Th1 cytokines and the prothrombinase fgl2 in stress-triggered and inflammatory abortion.

Abstract: Problem The immune system contributes to the outcome of pregnancy by complex immunological interactions. Cytokines especially influence the immune milieu pro or contra pregnancy. T helper 1 (Th1) cytokines [tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] cause inflammation and together are thought to threaten the maintenance of pregnancy. It has been proposed that increased levels of these Th1 cytokines activate coagulation via up-regulating the novel prothrombinase, fgl2. This study further investigates the Th1 cytokine up-regulation of fgl2 expression in a pathophysiological, stress induced abortion model, and an inflammatory, interleukin-12 (IL-12) triggered abortion model. Method The DBA/2J-mated CBA/J female mice were exposed to sonic sound stress or were injected with IL-12 during early gestation. On day 13.5 of pregnancy the uteri were removed and the resorption rate was calculated. We evaluated TNF-alpha, IFN-gamma, fgl2 as well as IL-12 messenger RNA (mRNA) expression in decidual samples of all mice by quantitative, real-time polymerase chain reaction (PCR). Results A similar resorption rate of 24% was detected in stressed mice, as well as in IL-12 injected mice compared with approximately 11% in non-stressed, non-injected control mice. In stressed mice compared with controls, we observed on day 13.5 up-regulated TNF-alpha, unchanged IFN-gamma down-regulated fgl2, and a slightly increased levels of IL-12. In the IL-12 triggered abortion model, we observed up-regulated levels of TNF-alpha, IFN-gamma and fgl2. Conclusion These novel data suggest two distinct cytokine patterns leading to similar abortion rates. A physiological cascade associated with up-regulation of TNF-alpha, and an IL-12-triggered cascade characterized by persistent up-regulation of TNF-alpha and IFN-gamma as well as a persistent increase in fgl2.

Intravenous immunoglobulin treatment in women with recurrent abortions: increased cytokine levels and reduced Th1/Th2 lymphocyte ratio in peripheral blood.

Abstract: PROBLEM The aim of this study was to investigate changes in peripheral blood Th1/Th2 cytokine levels and lymphocyte ratios after massive intravenous immunoglobulin (MIVIg) treatment for women with recurrent spontaneous abortion (RSA) of unexplained etiology. METHOD OF STUDY Serum Th1 (IFN-gamma, TNF-alpha) and Th2 cytokine (IL-4, IL-10) levels were assessed by ELISA methods (n = 9) and peripheral blood Th1/Th2 lymphocyte ratios (n = 4) by flow cytometry before and after MIVIg treatments in women with four or more consecutive RSA. RESULTS Pre-treatment serum IFN-gamma (0.06 +/- 0.09 pg/mL, mean +/- SD), TNF-alpha (0.21 +/- 0.45 pg/mL), IL-4 (0.70 +/- 1.16 pg/mL), and IL-10 (1.12 +/- 1.67 pg/mL) increased to 0.17 +/- 0.16 pg/mL, 0.77 +/- 0.28 pg/mL, 1.82 +/- 0.89 pg/mL, and 3.44 +/- 0.48 pg/mL, respectively, after MIVIg treatments (P < 0.05). CD4-positive IFN-gamma/IL-4 lymphocyte ratios (17.3 +/- 9.1) were reduced to 11.5 +/- 7.1 after treatment (P < 0.05). CONCLUSIONS Massive intravenous immunoglobulin treatments increased peripheral blood cytokine levels and decreased Th1/Th2 lymphocyte ratios; thus, MIVIg treatments modify the peripheral Th1/Th2 balance.

Estrogen and progesterone modulate monocyte cell cycle progression and apoptosis.

Abstract: PROBLEM: Pregnancy is characterized by dramatic immunologic changes most commonly characterized as suppression of cell-mediated immunity. Mechanisms of this immunosuppression are obscure but may be caused by increases in pregnancy-associated sex steroids such as 17-β-estradiol or progesterone. METHOD OF STUDY: Using five myelomonocytic cell lines in various stages of differentiation, the effects of 17-β-estradiol and progesterone on cell cycling, apoptosis, and bcl-2 expression in randomly cycling cells before and after lipopolysaccharide (LPS) activation were examined. RESULTS: Lipopolysaccharide alone inhibited cell cycle progression in THP-1 monocyte-like cells and U-937 histiocyte-like cells. Estrogen alone produced cell cycle arrest in all myelomonocytic cells except HL-60 pro-myelocyte-like cells. Progesterone had effects predominantly on pro-myelocytic-like HL-60 cells, inducing apoptosis. Estrogen and progesterone both decreased levels of bcl-2 in KG-1α, HL-60, and THP-1 cells. LPS partially antagonized both estrogen-induced THP-1 apoptosis and its suppression of bcl-2 protein. CONCLUSIONS: Sex steroid-induced effects on cell cycle transition and apoptosis are potential mechanisms by which pregnancy-induced cell-mediated immune suppression may occur. Further investigation should provide a better understanding of pregnancy-induced immune changes and, perhaps, sex-based differences in monocyte function and immunologic responses.

MD-1 is a critical part of the mechanism causing Th1-cytokine-triggered murine fetal loss syndrome.

Abstract: PROBLEM Fetal loss syndrome (abortion/resorption) occurring on or after gestation day (gd) 9.5 in CBA/JxDBA/2 matings is dependent upon presence of TNF-alpha + IFN-gamma, which act by increasing expression of fg12 prothrombinase at the feto-maternal interface. The magnitude by which the abortion rate can be boosted by an injection of these cytokines on gd 7.5 depends on endogenous rate of loss, and appears to depend on microbial flora. Is cytokine-triggered abortion dependent upon a third signaling pathway that senses 'danger'? METHODS Female CBA/J were mated to DBA/2 males and, C57B1/6 and C57B1/6 TNFalphaR1-/-Mak were mated to C57B1/6 control or TNFalphaR1-/-Mak males. LPS from Escherichia coli and Salmonella enteritidis, or the combination of TNF-alpha + IFN-gamma, was injected to stimulate abortions. The effect of anti-MD-1, which interferes with expression of CD14 and, hence, with signaling by LPS via the CD14-tlr4 complex, on TNF-alpha + IFN-gamma was tested. The presence of MD-1 in the uterus was evaluated by in situ hybridization, and effect of lipopolysaccharide (LPS) on mice lacking TNF-alphaR1 was tested. RESULTS Anti-MD-1 completely abrogated TNF-alpha + IFN-gamma-induced abortions. MD-1 was expressed on trophoblast and in deciduas on gd 8.5 but LPS could not abort mice that lacked the type 1 receptor for TNF-alpha. Pregnant CBA/J females had classical resorptions (abortions) countable on gd 13.5-14.5 in response to LPS from E. coli or S. enteritidis, but C57B1/6 strain mice resorbed only in response to the latter, and E. coli LPS appeared to induce 'occult' losses. 'Occult' loss did not require TNF-alphaR1. CONCLUSIONS TNF-alpha + IFN-gamma could not induce murine abortions without co-presence of a 'danger' signal such as LPS acting via CD14 on toll receptors, and LPS could not act without co-signaling by TNF-alpha. Classical resorptions/abortions and 'occult' losses have a different mechanism in these models as reflected in type of endotoxin and requirement for TNF-alphaR1 signaling.

Progesterone regulates IL12 expression in pregnancy lymphocytes by inhibiting phospholipase A2.

Abstract: Par G, Geli J, Kozma N, Varga P, Szekeres-Bartho J. Progesterone regulates IL12 expression in pregnancy lymphocytes by inhibiting phospholipase A2. AJRI 2003; 49:1–5 © Blackwell Munksgaard, 2003 PROBLEM: Progesterone-induced blocking factor (PIBF) is one of the pathways that mediate the immunological effects of progesterone. PIBF inhibits natural killer (NK) cytotoxic activity. Recently we showed that neutralization of PIBF results in an increased interleukin (IL)-12 expression, which is corrected by cyclooxygenase inhibitors. As exogenous arachidonic acid (AA) voids the NK blocking effect of PIBF, it is likely that PIBF acts before the level of the cyclooxygenase enzyme. Therefore in this study we investigated the effect of PIBF neutralizing antibody and simultaneous phospholipase A2 inhibitor quinacrine (Q) treatment on IL-12 production. METHODS: Pregnancy lymphocytes were treated with anti-PIBF antibody or lipopolysaccharide (LPS) as a positive control, in the presence or absence of Q. IL-12 expression by PBMC was detected by immunocytochemistry. RESULTS: Neutralization of PIBF as well as LPS treatment resulted in an increased IL-12 expression, which was corrected by simultaneous Q treatment. Pre-treatment of lymphocytes with progesterone prevented the stimulating effect of LPS on IL-12 production. CONCLUSION: Progesterone binding of the lymphocytes is followed by the release of PIBF that inhibits AA release. The subsequent block of prostaglandin synthesis reduces IL-12 production and results in a lowered cytotoxic NK activity, which may contribute to a normal pregnancy outcome.

Germ Cell Apoptosis in Autoimmune Orchitis: Involvement of the Fas–FasL System

Abstract: PROBLEM: The aim of this study was to determine the mechanism of germ cell death in experimental autoimmune orchitis (EAO) and the involvement of the Fas–FasL system in this process. METHOD OF STUDY: The EAO was induced in rats by immunization with testis homogenate and adjuvants. Apoptosis was studied by light microscopy, in situ end labeling of apoptotic DNA and DNA fragmentation techniques. Fas, FasL and caspase 3 expression was detected by immunohistochemistry. RESULTS: In rats with orchitis the number of Fas+ and FasL+ apoptotic germ cells increased from day 50, when the lesion develops, to 150 days, and correlates with the degree of testicular damage. Most spermatocytes expressing Fas were apoptotic. Many Fas+ germ cells were also immunoreactive for FasL. Moreover, these cells also expressed caspase 3. CONCLUSIONS: In rats with EAO germ cell death occurs through an apoptotic mechanism preceding germ cell sloughing. Immunohistochemical data suggest that the Fas–FasL system mediates germ cell apoptosis in an autocrine and/or paracrine way.

Macrophages in human reproductive tissues contain luteinizing hormone/chorionic gonadotropin receptors.

Abstract: PROBLEM: To determine whether macrophages in human reproductive tissues contain luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor mRNA and receptor protein that can bind 125I-hCG. METHOD OF STUDY: Macrophages isolated from term pregnancy human decidua were used for LH/hCG receptor detection by in situ hybridization for receptor mRNA and immunocytochemistry for a macrophage marker, CD68, performed alone and in combination, reverse transcription-nested polymerase chain reaction, Western and ligand blotting. The LH/hCG receptor presence in macrophages in late luteal phase human endometria and corpora lutea was determined by sequential performance of in situ hybridization and immunocytochemistry. RESULTS: The macrophages present in term pregnancy human decidua and late luteal phase human endometria and corpora lutea contain LH/hCG receptors. CONCLUSIONS: This is the first demonstration of macrophages present in human reproductive tissues containing LH/hCG receptors. The receptor presence suggests that LH and hCG may regulate macrophage functions in gonadal as well as in non-gonadal target tissues.

Concentrations of interferon-gamma-induced protein-10 (IP-10), an antiangiogenic substance, are decreased in peritoneal fluid of women with advanced endometriosis.

Abstract: Problem: To assess whether interferon-γ-induced protein-10 (IP-10), a chemokine that has antiangiogenic activities, may be involved in the pathogenesis of endometriosis. Method of Study: A total of 120 patients undergoing laparoscopy for pain and/or infertility were recruited, and peritoneal fluid (PF) and bone marrow derived cells in PF were collected. Concentrations of IP-10 in PF were measured with a specific enzyme-linked immunosorbent assay. Expression of IP-10 and IP-10 receptor, CXCR3, in bone marrow derived cells in PF, peritoneum and endometriotic cells was analyzed by reverse transcription-polymerase chain reaction. Results: All of the PF samples examined contained detectable concentrations of IP-10. In women with advanced stages of endometriosis, IP-10 concentrations in PF were significantly lower than those of women in early stages (P = 0.02). The IP-10 concentrations in women with advanced endometriosis also appeared to be lower than those without endometriosis although the difference was statistically marginal (P = 0.06). The expression of both IP-10 and CXCR3 was clearly detected in the bone marrow derived cells in PF, peritoneum and endometriotic stromal cells. Conclusions: Decreased concentrations of IP-10 in PF from women with advanced stages of endometriosis may imply that the peritoneal environment of these women is permissive to the development of the disease by enhancing angiogenesis and/or modulating inflammatory/immunological responses.

Effect of intravenous immunoglobulin treatment on the Th1/Th2 balance in women with recurrent spontaneous abortions.

Abstract: PROBLEM: The way by which intravenous immunoglobulin (IvIg) acts to prevent immunlogically mediated recurrent spontaneous abortions (RSA) has not been clarified. In the present study, a possible effect of IvIg on the T helper cell (Th1/Th2) balance was investigated in abortions of either alloimmune or autoimmune abnormalities. METHOD OF STUDY: The study included 21 women treated with IvIg before conception because of a history of RSA characterized by alloimmune abnormalities (n = 15) or associated with anti-phospholipid antibodies (APA) (n = 6). Peripheral blood samples, collected before and 5 days after the first IvIg infusion, were stimulated, and Th1 and Th2 cells were detected by flow-cytometric analysis using a combination of monoclonal antibodies against T-cell surface markers and intracellular interferon (IFN)-γ and interleukin (IL)-4. The percentage of IFN-γ-producing (Th1) and IL-4-producing (Th2) cells and the Th1/Th2 ratio were compared between pre- and post-infusion samples. RESULTS: A decrease of Th1 percentage in 66.6% of the cases and a concurrent Th2 percentage increase (47.61%) resulted in a decrease in the Th1/Th2 ratio in most of the cases (76.1%) (p < 0.01). Similar results were found in Group A (Th1/Th2 decreased in 60% of the cases, p < 0.05), while in Group B the effect of IvIg was not clear (Th1/Th2 increased in three and decreased in another three cases). CONCLUSION: Our finding suggests that IvIg administration in women with alloimmune RSA enhances Th2 polarization. This is not always the case with APA-associated abortions.