Trophoblast Antigens Research Articles

Second-trimester maternal serum invasive trophoblast antigen: a marker for Down syndrome screening.

More than 2 million pregnancies in the United States are screened for Down syndrome each year, most often in the second trimester, by a test using three biochemical markers in combination with maternal age (1). The “triple” test includes measurements of serum α-fetoprotein (AFP), unconjugated estriol (uE3), and human chorionic gonadotropin (hCG). In the general pregnancy population, the triple test can identify ∼65% of Down syndrome pregnancies at a 5% false-positive rate (2)(3). Recently, a fourth test, dimeric inhibin-A (DIA), was added to form the “quadruple” test, which has a higher sensitivity of up to 75% (4)(5). Combining first-trimester biochemistry (free β-subunit of hCG and pregnancy-associated plasma protein A) and ultrasound markers can yield higher performance (6)(7)(8), but the highest performance reported to date is from a combination of both first- and second-trimester markers into the “integrated” test (9). The search continues for even better markers that can be used to both increase the detection rate and reduce false positives. One such marker was originally identified as hyperglycosylated hCG. Cole et al. (10) used a specific antibody to measure hyperglycosylated hCG [recently called invasive trophoblast antigen (ITA)] in random urine specimens from women with Down syndrome pregnancies. When these authors used only the creatinine-corrected ITA multiple of the median (MoM), 78% of cases were detected at a false-positive rate of 5% (10). The current practice of Down syndrome screening is based on serum samples. Although collecting random urine is not difficult, the additional sample type may be inconvenient and adds expense beyond the measurements of ITA and creatinine. In addition, obtaining a serum sample for AFP analysis would still be required to screen for open neural tube defects. We therefore investigated the possibility of using maternal serum ITA as a Down syndrome …

Sialic acid-deficient invasive trophoblast antigen (sd-ITA): a new urinary variant for gestational Down syndrome screening.

Invasive trophoblast antigen (ITA), also known as hyperglycosylated hCG, is a glycosylation variant of hCG produced by invasive cytotrophoblast cells. Publications show that ITA is potentially a sensitive test for Down syndrome screening, reportedly detecting 38 and 80% of cases at a 5% false-positive rate in the first and second trimesters of pregnancy respectively. ITA has six oligosaccharides varying greatly in terminal sialic acid content (0-19 residues), leading to wide-charge heterogeneity. With the objective of developing an improved Down syndrome screening test, we examined the charge, or sialic acid variants, of ITA. Twenty urine samples were collected from the 16th to 18th week of gestation from 10 patients with trisomy 21 fetuses, demonstrated by karyotype analysis, and from 10 patients with normal karyotype pregnancies. Proteins in each sample were separated by preparative isoelectric focusing. Fractions were collected, pooled into the pl ranges of 3.0 to 4.0, 4.1 to 5.0, 5.1 to 6.0 and 6.1 to 7.0 and assayed for ITA. A difference in the distribution of ITA charge variants was demonstrated between cases and controls, most apparent in the less acidic (pl 5.1-7.0) range. The controls and Down syndrome samples contained 26.7 +/- 14% versus 45.6 +/- 20%, respectively, of the less acidic form of ITA (P = 0.02). The ITA in Down syndrome cases is composed of a much higher proportion of less acidic ITA. This could be the major source of elevated levels of ITA in Down syndrome pregnancies. It is postulated that the development of a new assay specifically measuring ITA that is deficient in sialic acid might enhance the utility of the already highly sensitive screening tests for Down syndrome.

Invasive trophoblast antigen (hyperglycosylated human chorionic gonadotropin) in second-trimester maternal urine as a marker for down syndrome: preliminary results of an observational study on fresh samples.

Down syndrome screening is commonly performed in the US using maternal age and three or four second-trimester maternal serum markers that can identify up to 75% of affected pregnancies by offering diagnostic studies to 5% of women. Invasive trophoblast antigen [ITA; hyperglycosylated human chorionic gonadotropin (hCG)] is a promising marker that can be measured in urine or serum in the first or second trimester. We report preliminary results for urinary ITA in an ongoing observational study. Women undergoing second-trimester amniocentesis for reasons not associated with biochemical testing provided consent and a urine (and possibly serum) sample that was tested within a few days. Demographic and pregnancy-related information was collected, along with karyotype. Screening performance was modeled for ITA alone and in combination with serum markers Twelve recruitment centers collected urine from 2055 women with singleton pregnancies between 15 and 20 weeks of gestation (2023 unaffected, 28 Down syndrome, and 4 pregnancies with other chromosome abnormalities). After correction for gestational age, urine concentration, and maternal race and weight, the ITA measurements were higher in women with a Down syndrome pregnancy (median ITA, 4.33 multiples of the median). At a 75% detection rate, the false-positive rate could be reduced by substituting ITA for hCG measurements (from 5.6% to 2.6% for the triple test) or by adding ITA measurements to existing combinations (from 3.3% to 2.0% for the quadruple test). Our data provide preliminary confirmation of the potential usefulness of urinary ITA measurements in detecting Down syndrome in a setting that simulates routine usage.

Pro-inflammatory Maternal Cytokine Profile in Preterm Delivery

Obstetrical & Gynecological Survey: November 2003 - Volume 58 - Issue 11 - p 700-701 doi: 10.1097/01.OGX.0000093668.78362.90

Executive summary

Objectives To identify the most effective, safe and cost-effective method of antenatal screening for Down's syndrome using nuchal translucency (NT), maternal serum and urine markers in the first and second trimesters of pregnancy, and maternal age in various combinations.Design A prospective study of women who booked for their antenatal care at about 8-14 weeks of gestation, with follow-up to identify pregnancies with Down's syndrome ascertained through second trimester screening or at birth.Setting Twenty-five maternity units (24 in the UK and one in Austria) offering second trimester Down's syndrome serum screening that agreed to collect observational data in the first trimester.Participants The results were based on 47,053 singleton pregnancies, including 101 pregnancies with Down's syndrome.Measurements and tests NT measurements were included if obtained between 9 and 13 weeks of pregnancy; serum and urine samples were also taken and stored. Another pair of serum and urine samples was collected in the second trimester and included if obtained between 14 and 20 weeks. Urine and serum samples from each affected pregnancy and five matched controls were tested for:Serum:alphafetoprotein (AFP)total human chorionic gonadotrophin (hCG)unconjugated oestriol (uE(3))pregnancy associated plasma protein A (PAPP-A)free beta-hCG.dimeric inhibin-A.Urine:invasive trophoblast antigen (ITA)beta-core fragmenttotal hCGfree beta-hCG.The matching criteria were gestation (using an ultrasound crown-rump length or biparietal diameter measurement), duration of storage, and centre. Screening performance of the individual markers and combinations of markers together with maternal age was assessed using standard methods. In addition pairs of first and second trimester serum samples from 600 controls were tested to secure a larger set in which screening performance could be determined using distribution parameters based on dates (time since first day of the last menstrual period).Main outcome measures The following were determined for different combinations of markers:efficacy (by assessing screening performance, focusing on the false-positive rate (FPR) for an 85% detection rate (DR))safety (focusing on the number of fetal losses due to amniocentesis (or chorionic villus sampling) in 100,000 women screened)cost-effectiveness (focusing on the cost of screening 100,000 women and the cost per Down's syndrome pregnancy diagnosed).Results Efficacy (screening performance) The false-positive rates for an 85% detection rate for the main screening tests are shown in the above table, in decreasing order of screening performance:With the serum integrated test, 10 weeks is the preferred time in pregnancy for the PAPP-A measurement. For the integrated test and the combined test, the timing of the measurement of the first trimester markers is less critical.Safety The lower false-positive rate with the integrated test compared with other tests means that at an 85% detection rate there would be nine diagnostic procedure-related unaffected fetal losses per 100,000 women screened compared with 44 using the combined test or 45 with the quadruple test.Cost-effectiveness Screening using the integrated test is less costly than might be expected because the extra screening costs tend to be offset by savings in the cost of diagnosis arising from the low false-positive rate. It was estimated that to achieve an 85% detection rate the cost to the UK NHS would be pound15,300 per Down's syndrome pregnancy detected. The corresponding cost using the second trimester quadruple test would be pound16,800 and using the first trimester combined test it would be pound19,000.Conclusions Implications for healthcare The results showed that screening performance in the first trimester of pregnancy was virtually the same as that in the second trimester, and in either it was much less effective than integrating screening measurements from both trimesters into a single test. In applying these results to screening practice several conclusions can be drawn. The following tests offer the most effective and safe method of screening:overall: the integrated testif an NT measurement is not available: the serum integrated testfor women who do not attend for antenatal care until the second trimester of pregnancy: the quadruple testfor women who choose to have a screening test in the first trimester: the combined test.At a constant detection rate, the cost-effectiveness of these four tests is broadly similar, any extra screening costs tending to be offset by fewer diagnostic costs. The evidence presented in this report does not support retaining the double test, the triple test, or NT measurements on their own (with or without maternal age) because each would lead to many more women having invasive diagnostic tests, without increasing the proportion of Down's syndrome pregnancies detected.

Persistent low levels of human chorionic gonadotropin: A premalignant gestational trophoblastic disease

Objective: This study was undertaken to evaluate the significance of persistent low-level human chorionic gonadotropin (hCG) titers (usually <50 IU/L) in the absence of clinical evidence of pregnancy or gestational trophoblastic disease. Study Design: The USA hCG Reference Service consulted on 114 cases with persistent low levels of hCG; 51 had false-positive hCG results. The remaining 63 cases had real hCG results and are presented here. Results: Antecedent gestational events included hydatidiform mole (27), pregnancy (35), and gestational trophoblastic neoplasm (1). Forty of the 63 (64%) cases received therapy, including chemotherapy (38), hysterectomy (2), or both (10). Despite treatment, in all cases, low hCG titers persisted. After 1 to 4.5 years of low titers, four women had a sudden rapid increase in hCG levels, and malignant disease was confirmed or clearly suggested (gestational trophoblastic neoplasm [3] and placental site trophoblastic tumor [1]). Invasive trophoblast antigen (ITA) is a marker of invasive cytotrophoblast cells. ITA was measured in 38 of the cases with persistent low hCG, in all cases ITA accounted for less than 25% of the hCG concentration. It was also determined in the 4 cases indicated with malignant disease, accounting for more than 80% of the hCG. Conclusion: The presence of persistent low-level hCG titers defines a subset of women with preinvasive or quiescent gestational trophoblastic disease. ITA effectively detected the presence or absence of invasive cells in these cases. The recommended management of the quiescent disease is close surveillance without therapy until malignant disease detected. (Am J Obstet Gynecol 2003;188:1254-9.)

Fully automated chemiluminometric assay for hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen).

Hyperglycosylated human chorionic gonadotropin (HhCG) is a glycoprotein hormone secreted during embryonic implantation and trophoblast invasion of the uterine wall and is an early marker of pregnancy (1). Relative to hCG, HhCG has a higher molecular mass (38.5–40 kDa, depending on the amount of carbohydrate) and a higher number of asparagine (N)-linked triantennary carbohydrates and serine (O)-linked tetrasaccharide core structures in the β-subunit (2). Although both are secreted from the placenta and choriocarcinoma, HhCG is produced by mononucleated cytotrophoblasts, and hCG is produced by syncytiotrophoblast cells (3)(4)(5)(6). Because the cytotrophoblasts are primitive and invasive in nature, HhCG is also called invasive trophoblast antigen (ITA) (5). Birken et al. (7) described a monoclonal antibody (B152) specific for the β-subunit C-terminal peptide and the O-linked oligosaccharide of HhCG. Although the epitope for this antibody does not require sialic acid, the presence of the O-linked tetrasaccharide core structure is essential (1). Using IRMAs and ELISAs, investigators showed that (a) HhCG rapidly increases in early pregnancy, attaining substantially higher concentrations and decreasing earlier than hCG (1)(8); (b) HhCG is increased in Down syndrome-affected pregnancies in both the first and second trimesters (9)(10)(11); and (c) the HhCG:hCG ratio appears to be higher in those with invasive vs noninvasive trophoblastic disease (12). The above HhCG assays were performed manually using large sample volumes (200 μL) and long incubation …

Pro-inflammatory maternal cytokine profile in preterm delivery.

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. 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. Significantly higher levels of the type 1 cytokines, interferon (IFN)-gamma 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. These data are suggestive of a maternal type 1 cytokine bias in PTD.

Placental Trophoblast and Endothelial Cells as Target of Maternal Immune Response

Pregnancy is a unique physiologic condition that guarantees the survival of the semiallogenic embryo during the long period of gestation. The placenta plays a key role in the maintenance of local tolerance and allows the mother to accept the embryo until completion of pregnancy. The complex process of tolerance accompanying the survival of the foetus is controlled at the embryo-maternal interface by factors deriving from decidualized endometrium and from the trophoblast itself. Trophoblasts develop various strategies to evade the damaging attack by the maternal immune response including expression of non-classical MHC class I antigens and of complement regulatory proteins. Also, cytokines released at the feto-meternal interface play an important role in regulating embryo survival controlling not only the maternal immune response but also angiogenesis and vascular remodelling. The delicate equilibrium established between the mother and the foetus can be compromised in pathological condition of pregnancy as a result of humoral and/or cellular response of the mother against trophoblast antigens leading to sponstaneous miscarriage. Cytotoxic cells and antibodies to trophoblast and endothelial cells are frequently found in patients with recurrent spontaneous abortion. This review article focuses on the delicate equilibrium established at the feto-maternal interface during pregnancy examining the various strategies devised by the embryo to evade the maternal immune attack, and the pathological conditions in which this equilibrium is compromised leading to serious complications of pregnancy.

Identification of the White Blood Cell Populations Responsible for Th1 Immunity to Trophoblast and the Timing of the Response in Women with Recurrent Pregnancy Loss

Aim: The purpose of this study was to identify the white blood cell populations responsible for Th1 immunity to trophoblast as evidenced in our in vitro assays following trophoblast activation and the timing of this response. Study Design: Peripheral blood mononuclear cells (PBMC) were isolated from 32 nonpregnant women with a history of at least three prior first trimester spontaneous abortions of unknown etiology except their PBMC secreted Th1 embryotoxic cytokines in response to trophoblast stimulation. White blood cell populations were separated from PBMC by magnetic immunobeads and cultured with and without a trophoblast antigen extract. Supernatants from these cultures were added to two cell mouse embryos and after four days of culture assessment of blastocyst development was made to determine the white blood cell population responsible for embryotoxicity. In separate experiments trophoblast-activated PBMC culture supernatants were prepared over seven time points and individual Th1 cytokines (IL-2, INF-γ, TNF-α) were measured by ELISA to determine the timing of the response to trophoblast stimulation. Results: The white blood cell (CD45) populations responsible for embryotoxicity in response to trophoblast were T cells (CD3) and NK (CD56) cells. Levels of IL-2 peaked in the first 24 h of culture followed by TNF-α and IFN-γ levels which peaked at 96 h of culture. Conclusions: Our data demonstrated that the white blood cell populations responsible for embryotoxicity in our in vitro assays, were both T and NK cells. The kinetics of the cytokine response to trophoblast found in our study parallels the time course of a typical Th1 cytokine response. The profile of secreted cytokines support our hypothesis that trophoblast can produce Th1 immunity in some women with recurrent pregnancy loss that have embryotoxic effects in vitro.

Procoagulants in fetus rejection: the role of the OX-2 (CD200) tolerance signal

The spontaneous loss of normal karyotype embryos may be initiated or prevented by the maternal immune system. In mice, loss between the time of implantation (day 4.5) and formation of a vascularized placenta (day 9.5) when the embryo is too large to survive by diffusion alone, is analogous to occult pregnancy failure in humans. They are called occult because usually the woman does not know she is pregnant. From studies in mice, these early losses have a different mechanism than abortion of a vascularized placenta (analogous to clinically evident human spontaneous miscarriage). The latter depend on the activation of the novel prothrombinase fgl2 on the fetal trophoblast and in maternal decidua by the T helper-1 (Th1) type cytokines TNF- α+γ -interferon that arise from NK cells and NKγδ T cells; conversion of prothrombin to thrombin which in turn generates IL8 that activates polymorphonuclear leukocytes leads to embryonic death. These inflammatory processes are counteracted by Th2/3-type cytokines that arise in part from V γ 1 δ 6 T cells reacting to, as yet, unidentified trophoblast antigens in the presence of the ‘tolerance signaling molecule’ OX-2. By contrast, peri-implantation losses (between implantation and formation of a vascularized placenta, analogous to occult losses in humans) appear to be dependent upon perforin+cells, complement activation, and products of αβ T and NK αβ T cells, but not on TNF-α or procoagulant activation. Similarities and differences between findings in the mouse and human, and the potential evolutionary significance of mechanisms affecting reproductive success are reviewed.

Cytokine Patterns in Maternal Blood After Premature Rupture of Membranes

In Brief OBJECTIVE To compare two types of cytokines, type 1, which activate cell-mediated reactions and are important in cytotoxic and delayed-type hypersensitivity reactions, and type 2, which encourage vigorous antibody production and are commonly found in association with humoral immune responses, in blood of women with premature rupture of membranes (PROM). METHODS Forty-four women with histories of at least three successful pregnancies and who currently delivered normally served as controls. The PROM group consisted of 30 women with spontaneous rupture of fetal membranes at term. Peripheral blood mononuclear cells were stimulated separately with a mitogen, placental cells, and a trophoblast antigen extract, and the supernatants examined for type 1 and type 2 cytokines. RESULTS Mitogen-stimulated blood cells produced significantly higher levels of type 1 cytokines in PROM women than in normal controls. Higher levels of the type 1 cytokine interferon-γ were produced by PROM samples stimulated with autologous placental cells and with trophoblast antigens. Ratios of type 1 to type 2 cytokines were higher in PROM compared with normal pregnancy, and in some cases as much as 25-fold higher. CONCLUSION Women in the PROM group had a stronger type 1 reactivity whereas normal women were more predisposed to type 2 immunity; thus, PROM appears to be associated with a maternal type 1 bias. Pregnancies ending in premature rupture of membranes were more likely to have had elevated maternal production of type 1 cytokines than normal pregnancies.

Cytokine patterns in maternal blood after premature rupture of membranes

OBJECTIVE: To compare two types of cytokines, type 1, which activate cell-mediated reactions and are important in cytotoxic and delayed-type hypersensitivity reactions, and type 2, which encourage vigorous antibody production and are commonly found in association with humoral immune responses, in blood of women with premature rupture of membranes (PROM). METHODS: Forty-four women with histories of at least three successful pregnancies and who currently delivered normally served as controls. The PROM group consisted of 30 women with spontaneous rupture of fetal membranes at term. Peripheral blood mononuclear cells were stimulated separately with a mitogen, placental cells, and a trophoblast antigen extract, and the supernatants examined for type 1 and type 2 cytokines. RESULTS: Mitogen-stimulated blood cells produced significantly higher levels of type 1 cytokines in PROM women than in normal controls. Higher levels of the type 1 cytokine interferon-γ were produced by PROM samples stimulated with autologous placental cells and with trophoblast antigens. Ratios of type 1 to type 2 cytokines were higher in PROM compared with normal pregnancy, and in some cases as much as 25-fold higher. CONCLUSION: Women in the PROM group had a stronger type 1 reactivity whereas normal women were more predisposed to type 2 immunity; thus, PROM appears to be associated with a maternal type 1 bias.

Urinary hyperglycosylated hCG in first trimester screening for chromosomal abnormalities.

Hyperglycosylated human chorionic gonadotrophin (H-hCG), also known as Invasive Trophoblast Antigen or ITA, is a unique metabolic variant of hCG with more complex oligosaccharide side chains. Concentrations are independent of regular hCG. Urine H-hCG has recently proved to be a highly sensitive marker for Down syndrome screening in the second trimester of pregnancy. We evaluated H-hCG as a potential marker in the first trimester of pregnancy. Maternal urine samples were collected from 10(+0) to 11(+6) weeks of gestation prior to genetic analysis and stored in frozen form. Samples from eight cases of Down syndrome, two cases of trisomy 13, one case of trisomy 18, and 55 control pregnancies were hand-carried frozen to the USA and tested blindly. Samples were tested in a specific H-hCG immunoassay and values were normalized to creatinine concentration. Values were plotted against gestational age, and multiples of control pregnancy median (MoM) calculated. The median level of the MoMs of the eight Down syndrome cases was 3.6 MoM. Five of the eight Down syndrome cases exceeded the 90th centile of the 55 unaffected cases. The MoMs of the trisomy 13 and 18 pregnancies were 0.2, 0.2 and 0.3. All three cases were under the 10th centile of unaffected pregnancies. The results of this study indicate that H-hCG testing may be useful in screening for Down syndrome in the first trimester of pregnancy. Further studies are needed to assess the potential screening values of urine H-hCG and the combination of this test with free beta-subunit, PAPP-A and other markers for Down syndrome in the first trimester of pregnancy.

Hyperglycosylated Human Chorionic Gonadotropin (Invasive Trophoblast Antigen) Immunoassay: A New Basis for Gestational Down Syndrome Screening

Serum human chorionic gonadotropin (hCG) and hCG free beta-subunit tests are used in combination with unconjugated estriol and alpha-fetoprotein in the triple screen test, and with the addition of inhibin-A in the quadruple marker test for detecting Down syndrome in the second trimester of pregnancy. These tests have a limited detection rate for Down syndrome: approximately 40% for hCG or free beta-subunit alone, approximately 60% for the triple screen test, and approximately 70% for the quadruple marker test, all at 5%, or a relatively high, false-positive rate. New tests are needed with higher detection and lower false rates. Hyperglycosylated hCG (also known as invasive trophoblast antigen or ITA) is a new test. It specifically detects a unique oligosaccharide variant of hCG associated with Down syndrome pregnancies. We evaluated this new Down syndrome-directed test in prenatal diagnosis. Hyperglycosylated hCG was measured in urine samples from women undergoing amniocentesis for advanced maternal age concerns at 14-22 weeks of gestation, 1448 with normal karyotype and 39 with Down syndrome fetuses. The median hyperglycosylated hCG value was 9.5-fold higher in Down syndrome cases (9.5 multiples of the normal karyotype median). The single test detected 80% of Down syndrome cases at a 5% false-positive rate. Urine hyperglycosylated hCG was combined with urine beta-core fragment (urine breakdown product of serum hCG free beta-subunit), serum alpha-fetoprotein, and maternal age-related risk. This urine-serum combination detected 96% of Down syndrome cases at a 5% false-positive rate, 94% of cases at a 3% false-positive rate, and 71% of cases at a 1% false-positive rate. These detection rates exceed those of any previously reported combination of biochemical markers. Hyperglycosylated hCG is a new base marker for Down syndrome screening in the second trimester of pregnancy. The measurement of hyperglycosylated hCG can fundamentally improve the performance of Down syndrome screening protocols.

Transcervical recovery of fetal cells from the lower uterine pole: reliability of recovery and histological/immunocytochemical analysis of recovered cell populations.

The aim of this work was to isolate, enumerate and attempt the identification of fetal cells recovered from the lower uterine pole. Immediately before elective termination of pregnancy at 7-17 weeks gestation, samples were recovered by transcervical flushing of the lower uterine pole (n = 108) or transcervical aspiration of mucus from just above the internal os (n = 187), and their contents examined using histological, immunohistochemical and molecular techniques. Syncytiotrophoblasts were identified morphologically in 28 out of 89 (31%) and 50 out of 180 (28%) flushings and aspirates respectively (mean 29%). Immunocytochemistry with monoclonal antibodies (mAbs) recognizing trophoblast or epithelial cell antigens on a smaller number of samples (n = 69) identified putative placental cells in 13 out of 19 (68%) and 25 out of 50 (50%) flushings and aspirates respectively (mean 55%). These included groups of distinctive cells with a small, round, hyperchromatic nucleus, strongly reactive with mAbs PLAP, NDOG1 and FT1.41.1. Smaller groups of larger, amorphous cells, usually containing multiple large, pale staining nuclei, reactive with mAb 340 and to a lesser degree with mAb NDOG5 were also observed. Taking cellular morphology and immunophenotype into consideration, the smaller uninucleate cells were likely to be villous mesenchymal cells, while the larger cells were possibly degrading villous syncytiotrophoblast. There was no significant difference in the frequency of fetal cells obtained by the two recovery methods. Squamous or columnar epithelial cells, labelled strongly with antibodies to cytokeratins or human milk fat globule protein, were observed in 97% (29 out of 30) of aspirates. The use of cervagem in a small number of patients prior to termination of pregnancy did not appear to influence the subsequent recovery of placental cells. Y-specific DNA was detected by polymerase chain reaction (PCR) in 13 out of 26 (50%) flushings and (99 out of 154) 64% aspirates analysed (mean 62%). In-situ hybridization (ISH) revealed Y-specific targets in 40 out of 69 (60%) of aspirates analysed. A comparison of PCR data obtained from transcervical recovered samples and placental tissues showed a concordance of 80% (76 out of 95), with 10 false positives. Comparing the PCR data from tissues with data derived by ISH from 41 aspirates gave a concordance of 90% with two false positives. Although syncytiotrophoblasts were much more likely to be present in samples containing immunoreactive placental cells, the detection rates of fetal-derived DNA were similar regardless of the morphological and/or immunological presence of placental cells. We conclude that the transcervical recovery of fetal cells, while promising, requires considerable additional effort being expended in further research and development, particular in the sampling procedure.

Reactivity of human trophoblast monoclonal antibodies with marmoset monkey trophoblast cultures.

The aim of this study was to determine whether cultured trophoblast tissues, derived from the trophectoderm of marmoset monkey blastocysts, contain homologues of human trophoblast antigens. This is an essential prerequisite to determine whether the marmoset may be a suitable model for preclinical testing of a human antitrophoblast antigen for fertility regulation. Previously evaluated monoclonal antibodies from the Flinders University laboratory, which reacted with human trophoblast with a high degree of specificity, were tested for immunohistochemical reactivity using an immunoperoxidase detection method on both frozen and paraformaldehyde-fixed sections of the cultured marmoset monkey trophoblast. All monoclonal antibodies raised against human placenta reacted positively, when compared to controls, suggesting that human and marmoset trophoblast cells share common epitopes. The specificity of the monoclonal antibodies was investigated by determining whether there was cross-reactivity with other marmoset monkey tissues, including adrenal, spleen, kidney, liver, muscle, ovary and testis. The specificities of the monoclonal antibodies on these marmoset tissues were similar to those previously found on the corresponding human tissues. We have concluded that marmoset monkey trophoblast exhibits homologues of human trophoblast antigens. The findings also suggest that marmoset monkeys should be evaluated further as a primate model to test suitable target antigens for antitrophoblast vaccines that may be useful contragestation agents in humans.

Enhanced expression of cytokines may play a fundamental role in the mechanisms of immunologically mediated recurrent spontaneous abortion

Objectives. To determine circulatory levels of 1L‐1 β, TNFα, IFN‐γ and Neopterin in immunologically mediated recurrent abortion. Design. Blood samples were withdrawn from both groups of first trimester recurrent abortion and controls for the determination of serum IL‐lβ, TNFα, IFN‐γ by the corresponding IRMA and serum neopterin by a double antibody RIA. Setting. Department of Obstetrics &amp; Gynecology, Cairo University Hospitals. Patients. Thirty pregnant women in their first trimester who presented with inevitable abortion with history of at least 3 prior spontaneous consecutive abortions, in addition to twenty‐three pregnant women in their first trimester presented with their first miscarriage due to chromosomal anomalies (controls). Every attempt was made to eliminate the possible known contributing factors of recurrent abortion except for the presence of cervical mucus sperm antibodies which were present in 24 out of 30 cases. Interventions. None. Results. Serum ILiβ, TNFα and IFN‐γ levels of the abortion group were significantly elevated when compared to the corresponding levels of controls. The incidence of abnormal high values of these cytokines varied between 40 to 70%. There was no significant difference of serum neopterin between abortion and control groups. The increase of IFNγ, the most active principle inducing neopterin release, was below the concentration needed for neopterin stimulation. Conclusions. In view of the proven findings that cytokines do have detrimental effects on implantation, trophoblast proliferation, embryo development and fetal survival, our results suggest a potential mechanism of immunologic recurrent abortion which involves the secretion of IL‐iβ. TNFα, IFNγ and other cytokines by activated endometrial lymphocytes and macrophages in response to either trophoblast or sperm antigens and these cytokines could partially defuse or penetrate into the systemic circulation.

Enhanced expression of cytokines may play a fundamental role in the mechanisms of immunologically mediated recurrent spontaneous abortion.

To determine circulatory levels of IL-1 beta, TNF alpha, IFN-gamma and Neopterin in immunologically mediated recurrent abortion. Blood samples were withdrawn from both groups of first trimester recurrent abortion and controls for the determination of serum IL-1 beta, TNF alpha, IFN-gamma by the corresponding IRMA and serum neopterin by a double antibody RIA. Department of Obstetrics & Gynecology, Cairo University Hospitals. Thirty pregnant women in their first trimester who presented with inevitable abortion with history of at least 3 prior spontaneous consecutive abortions, in addition to twenty-three pregnant women in their first trimester presented with their first miscarriage due to chromosomal anomalies (controls). Every attempt was made to eliminate the possible known contributing factors of recurrent abortion except for the presence of cervical mucus sperm antibodies which were present in 24 out of 30 cases. None. Serum IL1 beta, TNF alpha and IFN-gamma levels of the abortion group were significantly elevated when compared to the corresponding levels of controls. The incidence of abnormal high values of these cytokines varied between 40 to 70%. There was no significant difference of serum neopterin between abortion and control groups. The increase of IFN gamma, the most active principle inducing neopterin release, was below the concentration needed for neopterin stimulation. In view of the proven findings that cytokines do have detrimental effects on implantation, trophoblast proliferation, embryo development and fetal survival, our results suggest a potential mechanism of immunologic recurrent abortion which involves the secretion of IL-1 beta, TNF alpha, IFN gamma and other cytokines by activated endometrial lymphocytes and macrophages in response to either trophoblast or sperm antigens and these cytokines could partially defuse or penetrate into the systemic circulation.

A fresh look at the causes and treatments of recurrent miscarriage, especially its immunological aspects

The scientific basis for many traditionally accepted causes of recurrent miscarriage (RM) is weak. A significant proportion of RM cases with relatively few miscarriages can presumably be attributed to the random occurrence of consecutive chromosomally abnormal conceptions. New insights in the immunological interactions taking place at the feto-maternal interface provide us with the opportunity to propose detailed pathophysiological models for immunologically mediated RM. Scientific support for the theory that RM is a consequence of graft rejection-like alloimmune reactions against paternal human leukocyte antigens on the fetus is sparse. Conversely, there is considerable evidence that decidual natural killer cells play a role in the implantation and early invasion of the trophoblast and in the pathogenesis of RM. T helper (Th) cells from women with RM react against trophoblast antigens in vitro with the secretion of mainly interleukin-2 and interferon-gamma (a so-called Th1 response), which are known to inhibit trophoblast growth. The predisposition to a Th1 response against a given antigen may be determined by an individual's class II histocompatibility genes. In accordance with this, case-control, prospective and family studies indicate that maternal histocompatibility haplotypes comprising DR1 and DR3 alleles confer susceptibility to RM. The frequent occurrence of autoantibodies in women with RM is compatible with the theory of a Th1 response against trophoblast as a cause of the syndrome, but the autoantibodies themselves probably do not cause RM.