Granulysin-mediated apoptosis of trophoblasts in blighted ovum and missed abortion.

PROBLEM: Granulysin (GNLY) is a cytotoxic molecule mostly present in decidual natural killer (NK) cells. Blighted ovum (BO) and missed abortion (MA) represent the early pathological pregnancies with hindered development of the embryoblast or a dead embryo. We investigated the GNLY-mediated apoptotic mechanism potentially responsible for delayed termination of pregnancy. METHOD OF STUDY: We performed immunohistological and immunofluorescence labeling of decidual tissues (GNLY, Apaf-1, NF-κB). NKG2A expression was analyzed by flow cytometry and GNLY mRNA by RT-qPCR. RESULTS: The GNLY labeling intensity (H score) was lower in the nuclei of trophoblast cells in BO and MA. GNLY gene levels were inversely detected in BO and MA. A decreased decidual NK cell percentage was found in MA. NK cells from pathological pregnancies expressed lower NKG2A levels. The highest frequency of Apaf-1 was found in trophoblast cells of MA. NF-kB was highly expressed in decidual cells of BO. CONCLUSION: The reduced activation of GNLY-mediated killing might be implicated in the slower rejection of trophoblast cells in BO and MA. A decreased authentic decidual NK cell number could be responsible for low cytotoxicity against trophoblast cells in MA. In BO, trophoblast cells have a higher survival potential due to increased NF-kB expression.

Trophoblast CD200 expression in successful human pregancies and missed abortions.

Presence of the CD200 immune check-point inhibitor at the feto-maternal interface is linked to prevention of spontaneous abortion in mice and humans. In human missed abortions (MA), absence of Th17-driven inflammation has been attributed to expression of villus trophoblast CD200 quantified using immunohistochemistry. While rapid aneuploidy (QF-PCR) testing linked low CD200 to pregnancy failure, data showing normal VT CD200 in first trimester normal pregnancy and in abortion of chromosomally abnormal embryos has not been demonstrated. The present report shows normal CD200 in a 7 week gestation termination with normal male QF-PCR and in a 10 week male trisomy 18 MA.

Changes in expression of the CD200 tolerance-signaling molecule and its receptor (CD200R) by villus trophoblasts during first trimester missed abortion and in chronic histiocytic intervillositis.

PROBLEM: Expression of CD200 at the feto-maternal interface is associated with successful murine and human pregnancy. CD200 binding to CD200 receptors on lymphomyeloid cells suppresses inflammation and induces Tregs. CD200 receptors are also expressed on mouse and human placental trophoblast cells. What is the expression of CD200 and CD200R in human missed abortions which have preserved Treg levels and in chronic histiocytic intervillositis (CHI) where maternal inflammatory cells cause IUGR? METHODS: Immunohistiochemistry for CD200, CD200R, and Ki67 using human placental sections from missed abortions, term placenta, and CHI. PCR testing was done for trisomy in missed abortion. RESULTS: CD200 and CD200R were expressed by human villus trophoblasts from 2 weeks post-implantation to term. Cytotrophoblast proliferation (Ki-67+ count) decreased at term. In first trimester missed abortion cases, CD200>CD200R villus trophoblasts accompanied missed abortion of non-trisomic male fetuses. CD200 and Ki67+ trophoblast proliferation was preserved in CHI with maternal inflammatory cell infiltration but CD200R was greatly decreased. CONCLUSION: Residual CD200 activity may prevent completion of abortions via induction of Treg cells. In CHI, infiltrating maternal effector T cells may block Treg induction. An autocrine role for CD200-CD200R interaction versus inhibition of soluble CD200 by soluble CD200R is discussed.

The structure of immunocompetent decidual cells in recurrent missed abortions.

Background/Aim: Recurrent or habitual missed abortions (RMA) are defined as three or more consecutive abortions. In the first trimester of pregnancy habitual missed abortions occur in about 1% of population. The aim of this immunohistochemical study of decidua in RMA of unknown etiology was to identify subpopulations of decidual lymphocytes in recurrent miscarriages and compare the distribution of immunocompetent cells in artificial abortions and RMA. Methods. The study included 30 women with at least 2 consecutive miscarriages in the first trimester of pregnancy. Curettements of the third missed abortion were immunohistochemically analyzed. The control group consisted of 20 women without loaded reproductive anamnesis, with the abortion for social reasons. Criteria for exclusion from the study were diagnosed uterine anomalies, positive screening for thrombophilia and women who suffered from diabetes mellitus and disorders in the function of the thyroid gland. Immunophenotyping was performed by immuno-alkaline phosphatase (APAAP) using monoclonal antibodies: CD 30, CD 45 RO, CD 56 and CD 57, CD 68. Methods: The number of missed abortions (1,223) was on the average 9.7% of all deliveriies during the test period. Among them RMA were registered in 52 (4.2%) patients and in 30 (57%) the exact etiology of abortions was not determined. RMA was most common in the 25-34 years of age group. The largest number of RMA showed the ultrasound characteristics of missed abortion in 60% of cases and was in nulliparous patients (76.7%). The number of NK CD56 positive cells did not differ significantly between the types of abortion. In the decidual tissue, a number of NK CD57 positive cells was significantly higher in missed abortions compared to artificial interruptions (p < 0.01). In artificial termination of pregnancy there was an absolute predominance of CD45RO lymphocyte subpopulations, whereas in the RMA group there was slightly greater predominance of CD30 positive cells. The completed analysis showed a significantly higher number of CD68 positive macrophages in a decidual tissue of RMA pregnancy (p < 0.01). Results: The number and phenotypic structure of NK cells are significantly different in normal pregnancy decidua and in RMA. The NK cell dominance is present in the RMA group, in favor of CD56+ and CD 57 of subpopulations with increased CD30 of T lymphocyte subpopulations. Macrophages are more numerous in the decidua of pregnancies ended in abortion, so the cause to RMA of unknown etiology in a number of cases could be disregulation of immunocompetent cells.

The role of Hofbauer cells on the pathogenesis of early pregnancy loss.

INTRODUCTION: Hofbauer cells (HC) are the placental macrophages that play a significant role in many important placental events. The aim of this retrospective study is to investigate the role of HC in the pathogenesis of early pregnancy loss (EPL). METHODS: The slides were obtained from archival blocks of missed abortion (MA, n = 15) and blighted ovum (BO, n = 15) cases. Unwanted pregnancies materials constituted the control group (n = 15). HC and endothelial cells were identified using immunohistochemical methods. HC were counted under light microscope. The extent of villous vasculature was evaluated using two methods; the Chalkey method and microvessel scoring. RESULTS: The mean number of villous HC was found to be significantly higher in both MA and BO groups in contrast to the control group. MA group also showed a higher number of HC in comparison with the BO group. Higher microvessel scoring was also found in MA group in contrast to other two groups. Chalkey method revealed no significant difference in the extent of villous vasculature for the control group in comparison with MA and BO. DISCUSSION: As we identified relatively low quantity of HC in BO associated with defective vasculature, we hypothesize that an inadequate microvessel formation after hypoxic insult can explain the pathogenesis of BO. We believe that HC are increased in MA due to their divergent roles on immunity and inflammation. CONCLUSION: We therefore conclude that HC may be of biological importance in the pathogenesis of EPL.

Maternal serum soluble HLA-G levels in missed abortions.

BACKGROUND AND OBJECTIVE. It is unclear how immune tolerance develops to a semiallograft fetus in pregnancy. Human leukocyte antigen G (HLA-G) expressed by extravascular trophoblasts plays an important role in the recognition of the gestational tissues as self and the development of immune tolerance against the gestational tissues by the maternal immune system. The soluble form of the HLA-G (sHLA-G) molecule in the maternal serum is also reported to contribute to the prevention of rejection during pregnancy. The aim of the study was to compare the maternal serum sHLA-G levels of the women with missed abortions and control subjects with uncomplicated pregnancies. MATERIAL AND METHODS. The prospective cross-sectional study involving 40 with missed abortions and 40 control women, matched by age, gestational age, and body mass index, was carried out. The study group consisted of the women with singleton pregnancies, who were diagnosed with a missed abortion. Only the patients who were confirmed to have an uncomplicated term delivery during follow-up were included in the control group. The serum sHLA-G level was compared between the groups. RESULTS. There was no significant difference in the mean serum sHLA-G levels in terms of gravidity (P=0.761) and a history of abortion (P=0.379) in the control group. The median serum sHLA-G level in the missed abortion group was significantly lower compared with the control group (16.8 [8.5-35.8] vs. 26 [11-135] U/mL, P<0.001). All the women in the control group had uncomplicated term deliveries. CONCLUSION. Our results showed that the women with missed abortions had significantly lower serum sHLA-G levels compared with the healthy pregnant controls, which may have potentially played a role in the impairment of physiological immunological tolerance during pregnancy. However, the determination of the exact role and the potential clinical utility of maternal serum sHLA-G for the detection/prediction of a missed abortion risk requires further detailed studies.

Serum interleukin-1ß, interleukin-8 and anti-heat shock 60 Chlamydia trachomatis antibodies as markers of ectopic pregnancy.

Anti-Chlamydial trachomatis (anti-CT) responses, particularly anti-heat shock 60 (Hsp60), antibodies confer a higher risk of ectopic pregnancy. With emerging evidence supporting the pivotal role of interleukin-1ß (IL-1ß) and IL-8 in the immunopathogenesis of CT-specific tubal obstruction, we determined anti-CT Hsp60 antibody reactivity and serum concentrations of IL-1ß and IL-8 in failed pregnancies consisting of 30 consecutive ectopic pregnancies and 30 missed abortions, with 32 viable intrauterine pregnancies tested as normal controls. ELISAs were utilised to measure IgA or IgG anti-CT major outer membrane outer protein (MOMP) antibodies, IgG anti-CT Hsp60 antibodies and IL-1ß and IL-8. IgG anti-CT Hsp60 antibodies were more prevalent in ectopic pregnancy cases (43.3%, 13/30) than in intrauterine pregnancies (16%, 5/32, p=0.016). All 13 ectopic pregnancy anti-CT Hsp60-positive cases had anti-CT MOMP antibodies. CT-specific antibodies were more frequent in merged ectopic pregnancy and missed abortions cases (35%, 21/60) than in intrauterine pregnancies (16%, p=0.049). The median (range) levels of IL-1ß in ectopic pregnancy, missed abortions and normal intrauterine pregnancies were 1.74 (0.2-8.7), 1.14 (0.2-16) and 1.22 (0.2-16.2) pg/ml, respectively (p>0.05, for all). Serum IL-8 levels were comparable amongst groups: ectopic pregnancy (median [range]: 25.1 [18.3-1000]); missed abortions (32.9 [15.39-1000]); and intrauterine pregnancies (25.11 [18.3-1000] pg/ml). Anti-CT antibody-positive ectopic pregnancy had significantly lower IL-1ß levels (1.29 [0.2-2.93]) pg/ml than sero-negative ectopic pregnancy cases (2.09 [1.10-8.70]) pg/ml, (p=0.022), but IL-8 did not differ. Our data demonstrate that anti-CT Hsp60 immunity is a predominant feature of ectopic pregnancy. We conclude that neither IL-1ß nor IL-8 can be considered markers of failed pregnancy, although lower levels of the former cytokine are associated with CT-related ectopic pregnancy.

Investigation of systemic inflammatory response in first trimester pregnancy failure.

BACKGROUND: The contribution of local and systemic inflammation to the pathophysiology of sporadic first trimester miscarriages remains unclear. The objective of this study was to investigate the inflammatory response in the circulation of women presenting with first trimester miscarriage. METHODS: Levels of tumour necrosis factor alpha (TNFα), TNF receptors 1 and 2, interferon gamma (IFNγ), interleukin (IL)-6 and IL-10 were assayed using cytometric bead arrays in plasma samples from 29 euploid and 21 aneuploid missed miscarriages, 35 normal pregnant controls and 31 non-pregnant women (NPW). Whole blood flow cytometry was carried out with samples from 17 euploid and 16 aneuploid miscarriages, 18 pregnant controls and 13 NPW. RESULTS: The plasma of women with euploid miscarriage contained significantly higher circulating levels of TNFα (P < 0.005), IFNγ (P < 0.005), IL-6 (P < 0.005) and IL-10 (P < 0.01) than that of pregnant controls, irrespective of gestational age. Significantly (P < 0.05) higher TNF-R1 levels at 6-9 weeks, and significantly higher TNFα/IL-6 (P < 0.001) and significantly lower TNFα/IL-10 (P < 0.001) and IFNγ/IL-10 (P < 0.001) ratios at 10-14 weeks, were also found in euploid miscarriage cases compared with pregnant controls. TNFα/IL-10 ratio in plasma was significantly (P < 0.05) lower in miscarriages with an abnormal karyotype than those with normal karyotype. Normal pregnant women had a significantly higher plasma level of IFNγ (P < 0.01) and IFNγ/IL-10 ratio (P < 0.005), a significantly (P < 0.005) lower TNF-R1 level, and a significant (P < 0.05) increase in stimulated TNFα in monocytes, compared with NPW. CONCLUSIONS: Our data confirm that there is an inflammatory reaction in normal pregnancy compared with the non-pregnant state, which may be disrupted during miscarriage.

How valuable is measurement of peripheral blood natural killer cells at the time of abortion?

PROBLEM: Increased peripheral blood natural killer (NK) cells are associated to immune-mediated abortion, but their diagnostic value when measured at the time of miscarriage is unknown. METHOD OF STUDY: In women with therapeutic (A = 79) or elective (C = 34) pregnancy termination, the NK-cell percentage was measured before and 5 days after curettage. Additionally, immune-mediated lesions (scored 0-3) and CD56(+) and CD16(+) decidual NKs (scored 1-3) were detected on the abortion material. RESULTS: Aborters differed from controls in histological scores (P = 0.000) and in NK percentage (>12%) only in the measurement 5 days after the operation (P = 0.038). In comparison to histological lesions, NK measurement was found to have sensitivity 70%, specificity 73.68%, positive prognostic value 89.39% and negative prognostic value 43.75%. CONCLUSION: An Increased NK-cell percentage 5 days after the pregnancy termination could be a marker of immune aetiology of miscarriage, as the probability of an aborter with NK >12% to have an immune-mediated abortion is almost 90%.

Maternal serum levels of pro-inflammatory cytokines in missed and threatened abortion.

PROBLEM: A study of association between pro-inflammatory cytokines, and missed and threatened abortions with good outcome has been performed. METHOD OF STUDY: The presence of pro-inflammatory cytokines, namely interleukin (IL)-8 and IL-12 and the soluble interleukin-2 receptor (SIL-2R) was investigated in maternal serum of 12 patients with threatened abortion twice (at admission and discharge), 14 patients with missed abortion, 14 women with healthy first-trimester pregnancy, and 14 normal non-pregnant women, using specific enzyme-linked immunosorbent assays. RESULTS: SIL-2R and, in particular, IL-12 was detected with significantly higher levels in missed abortion group compared with all other groups. IL-8 was detected with no significant difference among all the groups studied. CONCLUSIONS: In spite of caution due to the small sizes of the subject samples, these results support a role of the immune system in the first trimester pregnancy and hypothesize that missed abortion may be associated with an enhanced Th1 reactivity, whereas threatened abortion with good outcome resembles the normal pregnancy with a non-enhanced Th1 reactivity.

T-helper 2-cytokine levels in women with threatened abortion.

OBJECTIVE: Considering that successful embryo development has been immunologically attributed to a T-helper 2 phenomenon and that threatened abortion is a very frequent but pathogenetically not well-defined clinical entity, our purpose was to investigate serum levels of the main T-helper 2-type cytokines during the evolution of this condition. STUDY DESIGN: Three T-helper 2-type cytokines (interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-13 (IL-13)) were measured by an enzyme-linked immunosorbent assay (ELISA) in serum of 12 women with threatened abortion both at hospital admission and discharge time. Fourteen women with missed abortion, 14 normal pregnant women and 14 normal non-pregnant women represent study control groups. RESULTS: Serum concentrations of the selected T-helper 2-type cytokines showed no significant differences in women with threatened abortion with those of normal pregnant and non-pregnant women, whereas showed significantly lower values in women with missed abortion. CONCLUSION: Our data (a) confirm the concept that first-trimester normal pregnancy is a T-helper 2 phenomenon, (b) show that threatened abortion, when T-helper 2-biased, may tend to a positive evolution of the condition, (c) display that interleukin-10, particularly, may represent a useful diagnostic and prognostic marker for predicting the normal continuance of the pregnancy in threatened abortion, (d) confirm the existence of a T-helper 2-type pattern deficiency in missed abortion, and finally (e) may open the way to new T-helper 2-biased immune therapies in case of difficult first-trimester pregnancies.

Deficiency of decidual IL-10 in first trimester missed abortion: a lack of correlation with the decidual immune cell profile.

PROBLEM: To determine if first trimester missed abortion decidua is characterized by an altered immune cell profile and/or a modified interleukin (IL)-10 and interferon (IFN)-gamma production pattern compared with decidua from elective termination. METHOD OF STUDY: Flow cytometry and immunohistochemistry techniques were used to determine the decidual immune cell phenotypic profile and production pattern of IL-10 and IFN-gamma in cases of elective termination (n = 14) and missed abortion (n = 12). RESULTS: Both groups had a similar proportion of CD56+ CD16-, CD56+ CD16+, CD19+, CD3+, CD4+, CD8+, alphabeta T cells and gammadelta T cells. The majority of alphabeta and gammadelta positive T cells in both groups coexpressed the natural killer (NK) cell marker CD56, but lacked cell surface expression of CD3. Diminished decidual IL-10 staining was noted in 7/10 missed abortion cases compared with none of the elective termination cases (n = 12) (P = 0.007). A uniform decidual IFN-gamma staining pattern was observed in both groups. CONCLUSION: Decreased IL-10 production coupled with a sustained IFN-gamma presence noted in missed abortion compared with elective termination cases suggest that these cytokines may be important determinants in pregnancy outcome. In contrast, differences in the proportion of immune cells between both groups may not be a critical factor in early pregnancy loss. In normal pregnancy, decidual alphabeta and gammadelta positive T cells with reduced CD3 on their cell surface may be intrinsically restricted in T-cell receptor (TCR)-mediated activation.

Role of decidual natural killer (NK) cells in patients with missed abortion: differences between cases with normal and abnormal chromosome.

In order to study the mechanism of abortion, the proportions of NK cells in the peripheral blood and decidual lymphocytes were evaluated in both chromosomally normal and abnormal missed abortions. In normal pregnancy, CD56+16-3- NK cells are a major element of decidual lymphocytes. The percentages of CD56+16-3-NK cells of peripheral lymphocytes in normal pregnancies were not statistically significantly different from those of chromosomally normal and abnormal abortions. In the decidua, the percentages of CD56+16-3- NK cells of decidual lymphocytes showed no statistically significant differences between normal pregnancies and chromosomally abnormal abortions. However, the percentages of CD56+16-3-NK cells of chromosomally normal abortions were lower than those of chromosomally abnormal (P = 0.0025). Moreover, the percentages of CD56+16- NK cells in abortions with normal chromosomes were lower than those in normal pregnancies or abortions with abnormal chromosomes (P = 0.0037, P = 0.0025). However, when the proportion of CD56+ NK cells expressing CD16 was evaluated, there were no statistically significant differences in the percentages of CD56+16+ NK cells in normal pregnancies and missed abortions with normal chromosomes and abnormal chromosomes. We conclude that the expression of decidual CD56+16-3- NK cells in missed abortions with normal chromosomes is different from abortions with abnormal chromosomes and that this phenomenon may depend on an abnormal immune response of the maternal side.

[Successful therapeutic management of risk pregnancy in primary Sjögren syndrome with plasmapheresis and dexamethasone]. / Erfolgreiches therapeutisches Management einer Risikoschwangerschaft bei primärem Sjögren-Syndrom mit Plasmapherese und Dexamethason.

We describe a 28-year-old woman with primary Sjögren's syndrome who had a miscarriage in the 10th week of gestation in December 1993. In her second pregnancy plasma hyperviscosity and high antibody reactivities to 52/60 kD Ro(SS-A) and La(SS-B) represented elevated risk factors for abortion and fetal congenital heart block. Therefore, we performed plasmapheresis and administered dexamethason. At the end of 37th week of gestation a healthy boy was born by caesarean operation. Interestingly, antibody levels to the anti-Ro(SS-A)/La(SS-B) complex showed an increase during both pregnancies with a decline after the abortion as well as the partus. The combined therapy led to a decrease in both the autoantibody reactivities to 52 kD Ro(SS-A) and La(SS-B) and the plasmaviscosity. Plasmapheresis and dexamethason are safe treatment modalities in identified high risk pregnancies for the birth of a child with CHB.

Decidual-trophoblast interactions: decidual lymphoid cell function in normal, anembryonic, missed abortion and ectopic human pregnancy.

This study was designed to investigate the consequences of decidua-trophoblast interactions on the phenotype, spontaneous and induced proliferation and immunoregulatory potential of decidual leukocytes in normal pregnancies (NP), anembryonic pregnancies (AP), missed abortions (MA) and ectopic pregnancies (EP). Spontaneous proliferation of decidual non-adherent cells (NAD) from pregnancies with viable trophoblast inside the uterus is significantly higher than proliferation of peripheral blood lymphocytes (PBL) from the same groups (P < 0.001 for NP; P < 0.05 for AP). Spontaneous proliferation of decidual NAD cells from NP was higher (P < 0.001) when compared with AP and EP. The induced (PHA and Con A) responses of PBL from women with normal and pathological pregnancies were significantly higher than that of decidual NAD cells (P < 0.001). Higher proliferation of NAD decidual cells was obtained when Con A-stimulated NP were compared with MA and EP (P < 0.01). The interaction of viable trophoblast with intrauterine decidua appears to be a prerequisite for the activation of NAD suppressor cells, since NAD cells from MA produced stimulation instead of suppression, and NAD cells from EP had no suppressive effect. On the contrary, both NAD and adherent (AD) decidual leukocytes from NP and AP produced very strong suppression of PHA or alloantigen-induced PBL proliferation. The contact between trophoblast and AD decidual leukocytes is not necessary for their suppressive function, since even higher suppression is obtained with the cells from ectopic pregnancies.

A morphological and immunological study of human placental bed biopsies in miscarriage.

Placental bed biopsies were examined in three groups of pregnant women for maternal vascular response to placentation as well as for the presence of cells associated with local immunosuppression activity. In the group of women undergoing legal abortion, the histological appearance of trophoblastic invasion was normal in all but one, and the proportion of immunosuppressor cells was also normal. In the missed miscarriage group the histological appearances were abnormal except in one patient. In women with a history of recurrent miscarriage who had miscarried after immunization, placentation was normal in some and defective in others. Immunosuppressor cells appeared to be diminished in number, although there was no correlation between the cytotoxic status of their sera and their pregnancy outcome.

Histologic and immunologic study of uterine biopsy tissue of women with incipient abortion.

Biopsy specimens taken from the region of the placental bed were examined for the presence of phloxinophilic granulated mononuclear cells in women with a history of recurrent miscarriage and who would eventually miscarry a current pregnancy. They were compared with biopsy specimens from women with intact pregnancies presenting for elective termination of pregnancy and those with "missed abortion." Cells with large cytoplasmic granules (greater than or equal to 1 micron) were abundant in the group of ongoing pregnancies whereas cells with smaller granules (less than 1 micron) that were similar to large granular lymphocytes were more abundant relative to cells with large granules in the biopsy specimens from failing pregnancies. Immunosuppressive activity was tested in the supernatants of cultured biopsy samples of each group and found to be significantly lower in the incipient miscarriage group. These findings could represent alterations associated with the process of miscarriage, such as inflammation, or there may be deficient suppressor cell activity at the fetomaternal interface as the reason for "rejection" of the early embryo.

Preliminary characterization of two types of suppressor cells in the human uterus.

Survival of the mammalian fetus in an immunologically hostile host has been shown to be determined by the properties of the tissue at the maternal-fetal interface. Suppressor cells have been found in the decidua in both murine and human systems. This study was carried out to further characterize these cells and to determine whether they were present in abnormal early pregnancies and in the endometrium at the time of implantation. Suppressor cells appear in the endometrium after ovulation, and their activity is present in the decidua of successful pregnancies but is absent in missed abortions. Two types of cells have been identified. An early phase large cell appears to be hormone dependent, and later phase small cell appears to be trophoblast dependent. This would suggest that suppressor cells may play a role in protecting the fetal allograft, from the time of implantation, against maternal immunity, thereby allowing a pregnancy to succeed.