Sperm antibodies in men from infertile couples. Analysis of sperm agglutinins and immunofluorescent antibodies in 657 men.

Sera from 657 men from infertile couples were tested for sperm agglutinins and spermatozoal antibodies detectable by the indirect immunofluorescense technique (IFT), and the results were correlated to the clinical examinations of the couples. Sperm agglutinins were found in 6.7%. Spontaneous agglutination of the ejaculated spermatozoa was observed only among these men, most commonly among those with high serum titres. IF-antibodies against the four spermatozoal antigens located beneath the cell membrane occurred in 15.2% of the patients. Antibodies against the front part of the acrosome and the postnuclear cap were mainly IgM. Antibodies against the equatorial segment of the acrosome were predominantly IgG and in a few cases IgA, whereas straining of the main tail piece was caused by IgG antibodies. Considering the clinical fertility status of the couples, sperm agglutinins in high titres (greater than or equal to 10) against the equatorial segment and the main tail piece of the spermatozoa were found significantly more often among men from couples with unexplained infertility than among clinically normal men from couples where the findings in the women could be assumed to cause infertility. These results support the view that sperm agglutinins can cause infertility, whereas the significance of the IF-antibodies is still unclarified as, in some cases, these can be found even in high titres in men with proven fertility. The possible mechanism of autosensitization were evaluated by means of an anamnestic study.     

Identification of selectively solubilised syncytiotrophoblast plasma membrane proteins as potential antigenic targets during normal human pregnancy

Syncytiotrophoblast plasma membranes prepared from term placentae were selectively solubilised in non-ionic detergents. The solubilised proteins and the insoluble residue were tested in an ELISA assay for their ability to function as antigenic targets for anti-trophoblast antibodies present in normal first trimester pregnancy sera. The soluble proteins were fractionated by gel filtration and four major antigen forms were identified. The antigens were reactive with affinity purified anti-trophoblast antibody isolated from maternal sera and hence were termed maternally-recognised trophoblast antigens (MRTA); these were designated MRTA-I (Mr = 400,000 D), MRTA-II (Mr = 142,000), MRTA-III (Mr = 50,000) and MRTA-IV (Mr = 13,000). The relationship between MRTA-I, II, III and IV and antigens identified in maternal sera in the form of immune complexes is discussed.     

Trophoblast debris extruded from preeclamptic placentae activates endothelial cells: A mechanism by which the placenta communicates with the maternal endothelium

Introduction

Preeclampsia is characterized by maternal endothelial dysfunction. While the mechanisms leading to preeclampsia are unclear, a factor(s) from the placenta is responsible for triggering the disease. One placental factor implicated in triggering preeclampsia is trophoblast debris which may transmit pathogenic signals from the placenta to endothelial cells. In this study, we investigated whether trophoblast debris from preeclamptic placentae triggered endothelial cell activation.

Methods

Trophoblast debris from preeclamptic or normotensive placentae, or trophoblast debris from normal placental explants that had been cultured with preeclamptic (n = 14) or normotensive sera (n = 14) was exposed to endothelial cells. Activation of the endothelial cells was quantified by cell surface ICAM-1 and U937 adhesion to endothelial cells. The levels of IL-1β, pro-caspase-1 and active caspase-1 in the trophoblast debris were measured.

Results

Compared to controls, the levels of ICAM-1 and U937 adhesion to endothelial cells were significantly increased following exposure of the endothelial cells to trophoblast debris from preeclamptic placentae or placentae treated with preeclamptic sera. The levels IL-1β, pro-caspase-1 and active caspase-1 were significantly increased in both trophoblast debris from preeclamptic placentae and placentae treated with preeclamptic sera.

Discussion

These results provide the first direct evidence that trophoblast debris produced from preeclamptic placentae or placentae treated with preeclamptic sera can activate the endothelium.

Conclusions

Trophoblast debris from preeclamptic but not normotensive placentae can induce endothelial cell activation. This may be one mechanism by which the preeclamptic placenta communicates with the maternal endothelium to induce activation of the endothelium.     

Sera from women with histories of repeated pregnancy losses cause abnormalities in mouse peri-implantation blastocysts

Incubation of peri-implantation mouse blastocysts in the presence of untreated human sera resulted in destruction of the blastocysts. Heating the serum resulted in deactivation of the non-specific toxic factor. Whereas heat-treated serum from women with normal obstetrical histories, and men, supported normal trophoblast attachment and outgrowth, sera from women with reproductive dysfunction resulted in inhibition of attachment or disruption of the trophoblast cells. The inner cell masses were not adversely affected by the sera which were toxic to trophoblast. Fractionation of a serum sample by affinity chromatography resulted in removal of the toxic factor with the IgG fraction. Absorption of the toxic serum with human trophoblast membranes resulted in serum that supported trophoblast outgrowth indicating that the toxic factor was an antibody directed against trophoblast antigens.     

Antigen expression by trophoblast populations in the human placenta and their possible immunobiological relevance

Antigen expression by villous and extravillous human trophoblast populations at discrete anatomical sites has been reviewed. The various different antigenic phenotypes have been highlighted using a panel of monoclonal antibodies reactive with characteristic trophoblast membrane antigens, a trophoblast-leucocyte common antigen, class I MHC antigens, epithelial cell cytokeratin and epithelial membrane markers. This approach has allowed three separate fetal trophoblast populations to be identified within term amniochorionic membranes, and also has facilitated further definition of trophoblast populations in maternal uterine tissues. Furthermore, antigenic alterations have been noted in the maternal uterine gland epithelium in pregnancy leading to the expression of a trophoblastic phenotype, thereby suggesting a mechanism of extrinsic regulation of gene expression in these tissues. The possible involvement in the immunoregulatory control of maternal responses in pregnancy of MHC-linked gene products expressed by trophoblast has been discussed.     

Microsatellite analysis provides efficient confirmation of fetal trophoblast isolation from maternal circulation

Fetal trophoblasts can be found in maternal circulation from an early stage of pregnancy and thus provide a potential source of DNA for non-invasive prenatal diagnosis. We have developed a two-step method for trophoblast isolation between the 8th and 12th week of pregnancy. Blood was sampled from 14 women undergoing termination of pregnancy or spontaneous abortion. Immunomagnetic beads precoated with HLA class I and II, and with anti-cytokeratin-18 monoclonal antibodies, were used to remove CD8+ and other maternal cells, and to select for fetal trophoblasts, respectively. Microsatellite analysis was performed on DNA extracted from the isolated, maternal, paternal and placental cells after PCR amplification. Recovery of the trophoblasts was confirmed in 13/14 cases (93%) by the identification of an identical microsatellite pattern for fetal and placental cells. Further evidence was the presence of heterozygous alleles of both maternal and paternal origin. The correct prediction of gender in all five male fetuses was an additional confirmation of trophoblast recovery. We conclude that trophoblasts can be effectively isolated from maternal blood in the first trimester, and by using polymorphic microsatellite markers to confirm sample purity, this method has potential future application in prenatal diagnosis.     

Leptin Expression in Primary Trophoblast Cells in Response to Incubation with the Serum of Preeclamptic Women

Objective. We investigated whether the increase of leptin expression in preeclamptic placentas is additionally influenced by soluble maternal factors under hypoxic and nonhypoxic conditions. Methods. Term trophoblast cells were isolated and stimulated with sera from preeclamptic women under normoxic or hypoxic conditions. Levels of leptin mRNA and protein were evaluated by real-time RT-PCR or ELISA and Western blot analysis. Results. Leptin concentrations were increased in the serum of patients with preeclampsia and gestational diabetes. Hypoxia, insulin, and dexamethasone induced leptin expression in trophoblast cells. The incubation with sera from preeclamptic women led to a small, though, significant, increase of leptin gene expression. The effect of preeclamptic serum on leptin gene expression in trophoblast cells was lost under hypoxia. The serum of women with gestational diabetes did not increase leptin expression neither in normoxic nor hypoxic primary trophoblast cells. Conclusion. Our results can not exclude a soluble maternal factor in the serum of women with preeclampsia accounting for increased leptin expression in placental tissue in addition to hypoxia. However, an important biological role of this small increase in nonhypoxic conditions does not seem very likely.     

Selective cholesteryl ester uptake from high density lipoprotein by human first trimester and term villous trophoblast cells

As villous trophoblast does represent the contact zone between foetal and maternal tissues, the present in vitro study was aimed at investigating cholesterol supply from human high density lipoprotein subclass 3 (HDL(3)) to trophoblast cells isolated from human first trimester and term placenta. Binding of (125)I-HDL(3) was specific and saturable with similar K(d)-values for first trimester (54 microg HDL(3)-protein/ml) and term villous trophoblast cells (29 microg HDL(3)-protein/ml). The cell-association of (125)I-HDL(3) was 3-fold higher for term trophoblast cells while the specific cell-association of [(3)H]cholesterol ester(CE)-labelled HDL(3) was higher for first trimester trophoblast preparations. As a consequence, first trimester trophoblast cells have a pronounced capacity for selective CE-uptake from HDL(3). Competition experiments with native and oxidized low-density lipoprotein as well as cAMP-mediated stimulation of cell-association of [(3)H]CE-HDL(3) in both trophoblast preparations suggested the scavenger receptor class B, type I (SR-BI) as a likely receptor mediating this pathway. SR-BI m RNA could be identified by RT-PCR and Northern blot experiments in both trophoblast preparations. Western blot analysis and immunocytochemistry revealed high expression of SR-BI in first trimester trophoblast. A polyclonal antiserum raised against murine SR-BI significantly decreased cell-association of [(3)H]CE-HDL(3) in trophoblast cells. We conclude that human first trimester and term trophoblast cells express SR-BI which could serve as an efficient route for supplying cholesterol esters from maternal lipoproteins to foetal tissues.     

In vitro susceptibility of mouse placental trophoblast to cytotoxic effector cells

The susceptibility of mouse placental cells to hyperimmune cell killing directed against paternal combined H-2 and non-H-2 antigens was investigated using [3H]uridine as a terminal label in an in vitro cell-mediated microcytotoxicity test. The stage of development of the conceptus from which the short-term placental cell monolayers were prepared determined their susceptibility to immune cell lysis. Cultures from whole placentae taken at 9 days post-coitum (p.c.) were not significantly affected whereas similar monolayers prepared at 10.5 days p.c. or later underwent extensive destruction. Embryonic fibroblasts were susceptible at all stages examined from 9-16 days p.c. The onset of susceptibility correlates with the reported appearance of H-2 antigens on the placenta during ontogeny. All cultures of dissected populations of 13-day p.c. placental spongiotrophoblast were susceptible but only 70% of those of labyrinthine trophoblast. It is suggested that of the two major trophoblastic components of the mouse placenta the spongiotrophoblast expresses antigens that render it vulnerable to maternal immune attack whilst the labyrinthine trophoblast is only weakly or non-antigenic, with the observed killing being due largely to contamination of these cultures by antigenic foetal mesenchymal elements. Since failure to express appropriate target antigens cannot be the reason for the in vivo survival of the spongiotrophoblast it must be presumed that some form of immunoregulatory mechanism(s) is responsible for the maintenance of the foeto-placental allograft.     

Characterisation of the humoral immune response during murine pregnancy

Blood samples from female C57BL/10 mice mated with CBA/Ca males were obtained before, during and after both first and second pregnancies. A cellular enzyme-linked immunospecific assay (CELISA) was used to detect maternal antibodies against antigens on paternal splenocytes. Alloantibodies were detected in 48% of mice during or 9 days after a first pregnancy and in 82% of mice by the ninth day after the second pregnancy; these antibodies were first observed on day 10 of the first pregnancy. In two of four active multigravid sera tested, an increase in IgG1 concentration was detected; the level of all other isotypes remained within normal limits. Weak binding of alloantibody to an antigen of approximate molecular weight 44,000 was detected on CBA/Ca splenocytes by immunoblotting sera from multiparous animals. These sera also recognised an antigen of similar molecular weight on H-2b identical 129J splenocytes but not on splenocytes from the maternal strain. These results provide further information on the maternal humoral immune response during murine pregnancy.     

Expression of Fas-ligand in first trimester and term human placental villi

The expression of Fas-ligand (FasL) on trophoblast cells is thought to play a role in immune regulation during human pregnancy. However, there are some discrepancies in the published data concerning the cell types expressing FasL in the placental villi. Therefore, we examined the expression of FasL on cryosections of first trimester and term placental tissue with three different anti-sera against FasL, which are in common use. By immunohistochemistry, all three anti-sera principally gave the same staining result. In the first trimester of pregnancy, villous cytotrophoblast cells underlying the syncytium, as well as all extravillous trophoblast cells of cell columns and cell islands, gave a clear, mainly membrane-located staining, whereas the syncytiotrophoblast, which forms the borderline to the maternal blood flow, only gave a spot-like reaction in distinct areas. The same result was obtained with term placental villi; however, in this tissue, the staining of the villous cytotrophoblast cells was less pronounced. From our results, we suggest that in placental villi, an important role of FasL in immune regulation is not very conclusive because this molecule is mainly expressed on trophoblast with no access to maternal blood or tissue. This is in contrast to the uterine part of the placenta, where FasL expressing trophoblast cells are in close contact with apoptotic maternal leukocytes.     

HLA-DP and HLA-DQ antigen expression on trophoblasts of normal human pregnancy and trophoblastic diseases

A blocking antibody in pregnant sera seems essential in understanding why a fetus can survive as a semi-allograft in an immunologically competent mother. It has been demonstrated that blocking antibody is closely related to the paternal HLA-DR antigens. However, we previously reported that HLA-DR is not expressed on any trophoblast constituting the fetal frontier at the feto-maternal interface. The present study was then undertaken to clarify whether or not trophoblasts of normal pregnancy and trophoblastic diseases express HLA-DP or HLA-DQ antigens which are closely linked to HLA-DR as a haplotype. Materials were taken from ten pregnant uteri, ranging from 8 to 22 weeks of gestation, two uteri containing complete mole and two uteri each containing gestational choriocarcinoma. Curettage specimens obtained from three cases of complete mole and two cases of partial mole were also employed. Antigen expression was examined by an indirect immunoperoxidase technique using various monoclonal antibodies. From this immunohistochemical study, neither HLA-DP nor HLA-DQ seemed to be expressed on any trophoblast of normal pregnancy and trophoblastic diseases, which, together with the negative findings of HLA-DR on trophoblasts, may suggest that blocking antibody is not generated against HLA-D locus antigens on trophoblasts.     

Localization of vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 in bovine placentomes from implantation until term

Interactions of vascular endothelial growth factor (VEGF) with its receptors VEGFR-1 and VEGFR-2 promoting angiogenesis have been described in placentation of human, mink and pig. The bovine placenta is multiplex, villous and synepitheliochorial due to migratory trophoblast giant cells (TGC). To determine the role of VEGF in bovine implantation and placentation, placentomes and interplacentomal areas from 33 cows from early implantation until near term were evaluated by immunohistochemistry. VEGF immunoreactivity was detected in fetal and maternal blood vessel tissues during implantation and throughout gestation, and in preimplantatory trophoblast cells and uterine epithelium. After implantation the immunoreaction was confined to TGC and uterine epithelium. An antibody against bovine VEGF revealed a strong reactivity in the stroma of maternal caruncular septa in early and mid-gestation, which distinctly decreased near term. In interplacentomal areas, VEGF was found in luminal and glandular epithelia as well as in trophoblast, with distinctly higher reactivity in giant cells. VEGFR-1 was observed in trophoblast and uterine epithelium around implantation. Later, in definite placentomes, VEGFR-1 was localized in TGC near the chorionic plate and in maternal endothelial cells in the center of the placentome. VEGFR-1 and VEGFR-2 were co-localized in uterine epithelium and trophoblast as well as in blood vessel tissue and uterine glands. The presence of VEGF, VEGFR-1 and VEGFR-2 at the feto-maternal interface and in vasculature indicates that in the bovine VEGF may have (1) classic functions in angiogenesis and vascular permeability, (2) growth factor properties, facilitating feto-maternal exchange via paracrine action, (3) chemotactic activity on capillary endothelium, and (4) an autocrine influence on TGC migratory activity.     

Inhibition of lymphocyte proliferation and activation: a mechanism used by equine invasive trophoblast to escape the maternal immune response

At days 36-38 of gestation, the equine invasive trophoblast cells migrate into the endometrium of the pregnant mare to form the endometrial cups. During their migration, they become surrounded by maternal CD4+ and CD8+ T lymphocytes, and stimulate a cytotoxic antibody response to the paternal major histocompatibility complex class I antigens that they express. Nevertheless, endometrial cup cells remain viable at the site of uterine invasion up to days 80-100 of gestation, suggesting the participation of immunomodulatory mechanisms to the maternal cellular immune response. To determine the effects of the invasive trophoblast cells on lymphocyte proliferation, an in vitro co-culture system was developed using isolated equine invasive trophoblast cells and peripheral blood lymphocytes. Fetal fibroblast cells from the same conceptuses were used as controls. The presence of invasive trophoblast cells or their pre-conditioned medium inhibited 50% or more of lymphocyte proliferation, while fetal fibroblasts had no effect. The invasive trophoblast cell inhibitory factor needed to be present constantly to affect lymphocyte proliferation, and it was ineffective if lymphocytes had been previously stimulated to proliferate. The lymphoproliferative inhibitory mechanism affected lymphocyte subpopulations similarly. In addition, lymphocyte expression of cytokine mRNA including IFNgamma, IL-2, IL-4, and IL-10 was affected compared to controls. The implication of these observations in vivo may explain, in part, the apparent equine maternal immune acceptance of the presence and development of endometrial cup cells.     

Relationship of paternal factors to birth weight

OBJECTIVE: To investigate the relationship between paternal characteristics and birth weight. STUDY DESIGN: A total of 241 gravidas with uncomplicated, singleton, term pregnancies were studied. Maternal demographic and pregnancy-specific characteristics were used to calculate the expected birth weight for each fetus using a previously validated birth weight prediction equation. The additional independent predictive value of 4 paternal variables was assessed using multiple regression. RESULTS: Before adjustment for other variables, paternal height and weight significantly correlated with birth weight, but paternal age and body mass index did not. After controlling for maternal and pregnancy-specific factors that are known to influence fetal weight, only paternal height was significant as a predictive variable. The proportion of variance in birth weight that could be independently explained by paternal height was 2%. A 10-g gain in fetal weight was associated with each centimeter of increase in paternal height (P < .02). Using the resulting combination equation that included paternal height as a variable, 31% of the variance in term birth weight could be explained, and birth weights could be accurately predicted to within +/- 8.3% (+/- 288 g). Fathers with heights 2 SD above and below the mean had the term birth weight of their offspring increased and diminished by 125 g, respectively. CONCLUSION: Paternal height explains an independent portion of the variance in term birth weight among normal newborns of up to 250 g that cannot be explained by other maternal or pregnancy-specific factors. Paternal age, weight and body mass index do not independently influence birth weight.     

Autoantibodies against spermatozoal antigens detected by immunoblotting and agglutination. A longitudinal study of vasectomized males

Sera taken pre- and post-operatively at regular intervals within a year from 16 men undergoing vasectomy were analysed for autoantibodies against spermatozoal proteins by immunoblotting. The reaction patterns were compared with the results of sperm agglutination tests. Immunoblotting revealed the presence of autoantibodies against various spermatozoal polypeptides in all sera taken pre-operatively and post-operatively. On average, seven polypeptides showed reaction. During the post-operative period two patients developed spermatozoal agglutinins in moderate titers (greater than 16) but in immunoblotting no change in band reactivity was observed for these two patients. However, scanning of the immunoblotting results revealed that one of the patients, although without sperm agglutinins, during the post-operative period showed an increasing band colouring of a polypeptide of Mr 31,500, reflecting an increased level of the corresponding antibodies.     

Detection of fetal-specific DNA after enrichment for trophoblasts using the monoclonal antibody LK26 in model systems but failure to demonstrate fetal DNA in maternal peripheral blood

Trophoblast cells can be detected in maternal blood during normal human pregnancy and DNA from these cells may be used for non-invasive prenatal diagnosis of inherited diseases. The possibility of enriching trophoblast cells from maternal blood samples using a monoclonal antibody (LK26) against a folate-binding protein, which recognizes trophoblast in normal tissues, in conjunction with immunomagnetic cell sorting was investigated. Verification of the presence of fetal DNA in the sorted samples was done by detection of fetal/paternal-specific short tandem repeat (STR) alleles using polymerase chain reaction (PCR) and automated fluorescence-based genotyping. After successful initial experiments using retroplacental blood samples with a high number of trophoblast cells or an artificial mixture of trophoblast cells and blood, several versions of the enrichment method were attempted on peripheral maternal blood samples. However, it was not possible to detect fetal DNA sequences in these samples, most probably due to the extremely low number of trophoblast cells. Positive identification and retrieval of trophoblast cells in suspension or trophoblast nuclear material prepared on microscope slides after cell sorting procedures can be a solution to this problem.     

Review: Where is the maternofetal interface?

Ask where the maternofetal interface is and placental biologists will tell you, the syncytiotrophoblast and extravillous cytotrophoblasts. While correct, this is not full extent of the maternofetal interface. Trophoblast debris that is extruded into the maternal blood in all pregnancies expands the maternofetal interface to sites remote from the uterus. Trophoblast debris ranges from multinucleated syncytial nuclear aggregates to subcellular micro- and nano-vesicles. The origins of trophoblast debris are not clear. Some propose trophoblast debris is the end of the life-cycle of the trophoblast and that it results from an apoptosis-like cell death, but this is not universally accepted. Knowing whether trophoblast debris results from an apoptosis-like cell death is important because the nature of cell death that produced trophoblast debris will influence the maternal responses to it. Trophoblast debris is challenging to isolate from maternal blood making it difficult to study. However, by culturing placental explants in Netwells™ we can readily harvest trophoblast debris from beneath the Netwells™ which is very similar to debris that has been isolated from pregnant women. We have found that trophoblast debris from normal placentae shows markers of apoptosis and is phagocytosed by macrophages or endothelial cells, producing a tolerant phenotype in the phagocyte. Whereas, when we culture normal placental explants with factors such as antiphospholipid antibodies (a strong maternal risk factor for preeclampsia), or IL-6 (which is found at increased levels in the sera of preeclamptic women), the death process in the syncytiotrophoblast changes, such that the trophoblast debris becomes more necrotic. Phagocytosis of this necrotic debris leads to activation of endothelial cells. Trophoblast debris greatly expands the maternofetal interface and the nature of that debris is likely to strongly influence the responses of the maternal vascular and immune systems to the debris.     

Cytotoxic antibody against trophoblast and lymphocytes present in pregnancy with intrauterine fetal growth retardation and its relation to anti-phospholipid antibody

To elucidate immunological mechanisms involved in the genesis of intra-uterine fetal growth retardation (IUGR), an in vitro cytotoxicity assay against normal trophoblast and lymphocytes was performed. The data demonstrated the existence of cytotoxic antibody directed against trophoblast exclusively in the IgG fraction of the sera of 9 out of 15 mothers with IUGR, but in none of the sera from normal pregnant women. This antibody showed differential reactivity patterns that may be indicative women. This antibody showed differential reactivity patterns that may be trophoblast in common. Out of 9 IUGR mothers with this cytotoxic antibody, in 6 cases (66.7%) chronic villitis was evident upon microscopic examination of the placenta, the frequency being significantly higher than that in IUGR mothers without cytotoxicity or in control mothers (P less than 0.02). It is suggested that in situ inflammatory change triggered by this antibody might lead to IUGR through chronic villitis. This antibody showed cross-reactivity with anti-negatively charged phospholipid antibody, as confirmed by an absorption experiment, indicating that the trophoblast antigenic stimuli in pregnancy can induce the production of various autoantibodies including anti-phospholipid antibodies. These results are of interest in relation to the pathogenesis of autoimmune diseases.     

The gonadotropic hormones and their subunits in human maternal and fetal circulation at delivery.

The blood levels of HCG, LH, their alpha and beta subunits, and FSH were measured by double-antibody radioimmunoassays in 20 normal pregnanat women and in matched fetal cord arterial and venous samples at term. High levels of HCG, alpha subunit, and HCGbeta subunit, with low levels of LHbeta and FSH, were detected in maternal sera. In the fetal circulation the major detectable components were alpha subunits and presumably HCG. There was no significant arteriovenous difference in any of the hormones in the fetal circulation and no correlation between levels of hormones in maternal and fetal circulation. Column chromatography of sera confirmed that alpha subunits were present independent of intact hormones in both maternal and fetal sera.