Different expression of NOD2 in decidual stromal cells between normal and unexplained recurrent spontaneous abortion women during first trimester gestation

The NOD2 gene, encoding intracellular paternal recognition receptor (PRR) also called caspase activation and recruitment domain 15 (CARD15), is mutated in Crohn’s disease, an autoimmune-disorder. Unexplained recurrent spontaneous abortion (URSA) involved in complex auto-immune disorder. However, little is known about the expression of NOD2 protein at maternal-fetal interface with URSA patients. Our aim was to compare the expression levels of NOD2 in the decidual stromal cells (DSCs) from patients with normal pregnancy to those with unexplained recurrent spontaneous abortion (URSA) during first trimester pregnancy. Tissues and DSCs were collected from 12 patients with URSA and 26 patients with normal pregnancies that required abortion. DSCs in the normal pregnancy group showed significantly higher mRNA and protein levels of NOD2 than those isolated from the URSA group using real time PCR and in cell western. The appropriate expression of NOD2 in normal DSCs suggests that this protein may be required to sustain normal pregnancy.     

人早孕期蜕膜基质细胞下调蜕膜NK细胞杀伤活性

通过分析人早孕期蜕膜基质细胞(decidual stromal cell,DSC)对蜕膜NK细胞(dNK)表面趋化因子受体CXCR4与细胞内颗粒酶B表达水平的影响,研究早孕蜕膜基质细胞对局部NK细胞的训导作用。收集早孕蜕膜组织,分离DSC及蜕膜免疫活性细胞,进一步通过磁珠分选蜕膜CD3-CD56bright NK细胞,将分离的蜕膜NK细胞与DSC按1∶1比例共培养24h,收集蜕膜NK细胞,流式细胞仪检测其表面趋化因子受体CXCR4和细胞内颗粒酶B(granzyme B)的表达水平。结果显示,与对照组相比,在与DSC细胞共培养之后,趋化因子受体CXCR4+NK细胞的百分率明显上升,而蜕膜NK细胞内颗粒酶B阳性率显著下降(P<0.05)。结果表明,人早孕母-胎界面DSC细胞上调蜕膜NK细胞表面趋化因子受体CXCR4的表达,下调NK细胞内颗粒酶B的表达水平,可能抑制其杀伤活性。     

Status of peripheral blood natural killer cells in women with recurrent spontaneous abortions and infertility of unknown aetiology

The aim of this study was to investigate the functional status and immunophenotypic characteristics of natural killer (NK) cells in women who suffer recurrent spontaneous abortions (RSA) or have infertility of unknown aetiology. Peripheral blood mononuclear cells (PBMC) were obtained from 40 study patients and 13 normal healthy multiparous controls. NK cells were identified using anti-CD56 and anti-CD16 monoclonal antibodies (mAb). The expression of CD69, CD25, CD122, CD30, CD154, CD128 and CD94 on NK cells was detected using specific mAb and analysed by flow cytometry. CD69 expression on NK cells after ED(27) human trophoblast cell line co-culture with PBMC was also investigated. A significant increase in CD69 expression on CD56(+) NK cells was demonstrated in women with RSA (P < 0.005) and infertility (P < 0.05) as compared with that of normal controls. Conversely, CD94 expression was significantly decreased in women with RSA (P < 0.005) and infertility (P < 0.05) in comparison with that of controls. Increased CD69 expression on NK cells was induced after 24 h co-culture with ED(27). In conclusion, peripheral blood NK cells of women with RSA and infertility of unknown aetiology have higher proportions of activated NK cells in vivo. Unbalanced CD69 and CD94 expression may explain the underlying pathology.     

42例孕妇孕早期绒毛组织细胞遗传学分析

目的评价早孕期B超引导下经腹绒毛取材组织样本的细胞遗传学分析在产前诊断中的应用价值。方法回顾分析2010年1月到2011年6月42例早孕期产前细胞遗传学诊断的病例资料。结果 42例取材时间9～13周,穿刺成功率100%,培养成功率为97.6%,平均培养时间为10天,发现异常核型4例。结论孕9周后行B超引导经腹绒毛取材是一项安全可行的早孕期介入性产前诊断方法。绒毛细胞长期培养法和染色体制备方法简单、技术稳定、结果可靠,可用于胎儿染色体疾病的产前诊断。     

300例孕早期绒毛细胞染色体核型分析

目的分析妊娠早期胎盘绒毛染色体核型,实行优生优育,避免遗传病胎儿的出生.方法对300例孕早期绒毛细胞直接制片法,进行染色体核型分析.结果发现异常核型18例,异常核型出现率为6.00%,其中三体型10例,占异常核型的55.56%,分别为21三体7例,18三体2例,13三体1例,其次为平衡易位7例,占异常核型的38.89%,再次为染色体缺失1例.高龄孕妇中21三体的检出率5.77%(3/52),非高龄组为3.16%(4/248),x2=1.6900,P=0.1931,两者差异无显著性.结论孕早期绒毛染色体直接制片法是预防性优生学的重要措施.     

Massive immunoglobulin treatment in women with four or more recurrent spontaneous primary abortions of unexplained aetiology

The aim of this trial was to investigate the efficacy of massive i.v. immunoglobulin (MIVIg) treatment for women with a history of recurrent spontaneous abortion (RSA) due to unexplained aetiology. The study included nine women (11 pregnancies) with a history of four or more consecutive RSA with unexplained aetiology and no live births. The mean number of fetal losses was 4.5 (range 4-6 abortions). Over the course of 5 days, immunoglobulin (20 g/day) was infused i.v. at gestational weeks 4-7. No additional infusions were carried out. Two pregnancies out of the 11 conceptions resulted in missed abortions at gestational weeks 6 and 7 respectively. Mosaicism (46XX/ 48XX, +16, +20), and tetraploidy (92XXXX) were found by chromosome analyses of the two aborti. Eight out of the other nine pregnancies resulted in full term deliveries of healthy neonates. One pregnancy developed intrauterine growth retardation and fetal distress, resulting in a premature delivery (30 gestational weeks) by Caesarean section. Thus, excluding the two abortions with chromosome aberrations, the MIVIg treatment was effective in all nine pregnancies of RSA women with unexplained aetiology. This MIVIg treatment (100 g administered in early gestation) may be a beneficial alternative to previous IVIg infusion methods, and should be further evaluated in a multicentric, placebo-controlled study, employing a larger number of homogeneous patients who fall into a high risk category of first trimester abortions.      

早孕期人蜕膜趋化因子CCL2及其受体CCR2的表达及意义

目的：分析人早孕蜕膜及蜕膜基质细胞趋化因子受体CCR2及其配体CCL2在人早孕蜕膜组织及蜕膜基质细胞的表达和分泌，以探讨CCR2／CCL2在母-胎界面的生物学作用。方法：收集早孕期蜕膜组织，分离蜕膜基质细胞，分别用半定量RT-PCR、免疫化学方法分析正常人早孕蜕膜组织和培养的人蜕膜基质细胞CCR2／CCL2表达；并且用流式细胞术和ELISA法分别检测蜕膜基质细胞表面CCR2的表达和培养的蜕膜基质细胞上清中CCL2的分泌。结果：人早孕蜕膜组织和蜕膜基质细胞均高水平转录和翻译CCR2／CCL2，培养的基质细胞能分泌大量的CCL2，其分泌量呈时间依赖性。结论：早孕蜕膜高表达和分泌CCR2／CCL2可能参与早孕期母一胎免疫调节。     

Decidual vascularization and the expression of angiogenic growth factors and proteases in first trimester spontaneous abortions

BACKGROUND: Decidual vascular development is important for implantation.This study analysed decidual vascular adaptation to implantationin correlation with miscarriage in decidual secretory endometrium(DSE), decidua parietalis (DP) and decidua basalis (DB) of miscarriagepatients and matched controls. METHODS: Decidua was obtained during first trimester termination of pregnancy(controls) and vacuum aspiration in case of missed abortion(cases). Vascularization and the expression of VEGF-A, placentalgrowth factor, Flt-1, KDR, angiopoietin (Ang)-1, Ang-2, TIE-2,and membrane-type matrix metalloproteinases MT1-, MT2-, MT3-and MT5-MMP were determined at mRNA and protein level. Uterinenatural killer cells (CD56), macrophages (CD68), proliferation(Ki67) and apoptosis (activated caspase-3) were evaluated inconsecutive sections. RESULTS: Decidual vascularization showed differences between cases andcontrols, i.e. fewer vessels with larger circumference in cases.This correlated with the differential expressions of variousfactors at mRNA/antigen level and with increased endothelialflt1, KDR, MT2- and MT5-MMP expression in miscarriage patients.The differences between cases and controls were probably notbased on altered proliferation and/or apoptosis, since Ki67and active Caspase-3 showed comparable expression levels inboth groups. Although DB of cases and controls showed similaramounts of CD56- and CD68-positive cells, the case group didshow elevated levels of CD56 in DSE (P < 0.05) and of CD68in DP compared with the control group (P < 0.05). CONCLUSIONS: The differences in vascularization and in the expression ofangiogenic factors and proteases between groups suggest a correlationbetween decidual vascularization and the occurrence of miscarriages.     

Analysis of human leukocyte antigen (HLA)-G polymorphism in normal women and in women with recurrent spontaneous abortions.

Human leukocyte antigen (HLA)-G is a non-classical class I antigen. It has limited expression, but is high at the foetomaternal interface. This unique expression pattern of HLA-G suggests that it might be important for survival of the foetus during pregnancy. In the present study, 120 women with recurrent spontaneous abortions (RSA) and 120 fertile control women were genotyped for the HLA-G locus. This is the first report describing HLA-G polymorphism in normal fertile and RSA women from India. The allele HLA-G*010103 was higher in women with recurrent foetal losses. Interestingly, the HLA-G*010105 and G*010108 alleles were totally absent in normal fertile women but present in RSA women with frequencies of 1.7% and 0.4%, respectively. Allele G*010107 was absent in both the groups. The frequency of the null allele G*0105N was high (13.8%) in our population as compared to other world populations. Our data support the hypothesis that HLA-G polymorphism may contribute to recurrent foetal loss.     

Serological study of parvovirus B19 infection in women with recurrent spontaneous abortions

The feto-pathogenic association of parvovirus B19 (B19) has been sparingly studied in women with abortion, but not in women with recurrent spontaneous abortions (RSA). Serum samples from 116 women with RSA who were pre-screened for S-TORCH, Chlamydia trachomatis infections, anatomical, chromosomal, endocrinal abnormality and Rh incompatibility and those who had no such known causes of abortion were included in the study. Sera were also collected from 136 normal pregnant women and 120 normal non-pregnant women as disease and normal control respectively. All sera were tested at 1:400 dilutions for B19 IgM by in-house ELISA using cloned and baculovirus expressed VP1 and VP2 antigens of B19. The frequency of anti-B19 IgM antibodies in women with RSA was 19.8%, in pregnant females it was 11% and in normal non-pregnant female was 5%. Sera of 23 women with RSA which were positive for B19 IgM tested negative for B19 DNA by PCR. Patients with RSA should be screened for B19 infection and guidelines for treatment should emerge.     

Risk factors associated with neural tube defects: exposure during the first trimester of gestation

The neural tube defects (NTD) are a group of malformations of multifactorial etiology. Their high incidence in Mexico and the etiologic heterogeneity observed in several studies, prompted the present investigation with the main objective of looking for risk factors associated to NTD. We analyzed maternal exposure during the first trimester of pregnancy to different environmental factors, such as acute or chronic illnesses, immunizations, smoking, alcoholism, maternal or paternal occupation and exposure to chemicals. The sample include 360 patients with anencephaly, 249 with spina bifida and 44 with encephalocele, ascertained from a total of 230 635 live births and 4,020 stillborns, studied in the Mexican program of Registro y Vigilancia Epidemiológica de Malformaciones Congénitas Externa. Of the risk factors considered, significant differences with the control group were found for anencephaly in relation to maternal viral upper respiratory infection, hyperthermia, ingestion of analgesics, antiemetics and paternal occupation. In the case of spina bifida, significant differences were found only for viral upper respiratory infections.     

Human embryonal tissues of all three germ layers can express the CD30 antigen. An immunohistochemical study of 30 fetuses coming after therapeutic abortions from week 8th to week 16th of gestation

Originally, expression of the CD30 antigen was shown to be typical of the tumor cells of Hodgkin disease and of anaplastic large cell lymphomas. In reactive lymphoid tissue, CD30 is expressed only in a small population of activated lymphoid blasts. Since then, several reports have been published describing CD30 expression in non lymphoid tissues and neoplasms, such as embryonal carcinomas, seminomas, cultivated macrophages, histiocytic neoplastic cells, deciduals cells, and mesothelioma cells. In order to gain insight into the functions of CD30, given that it can mediate signals for cell proliferation and apoptosis, we studied the distribution of the antigen in different fetal archival paraffin-embedded tissues from week 8th to 16th of gestation. We investigated the immunohistochemical expression of CD30 in 30 paraffin-embedded tissue samples representing all three germ layers, using the monoclonal antibody Ber-H2 CD30 is expressed early in human fetal development (8th-10th week) in a wide variety of tissues, with the exception of the skin and thymus in which it is expressed later on. This is consistent with the observation that these organs are not fully differentiated before 10th and 13th week, respectively. No expression was observed in the cardiovascular and respiratory systems. The finding of CD30 expression in the terminal period of organogenesis, period, which is highly hormone related, implies that the antigen has an important role in cell development, maturation, and pathway to terminal differentiation in almost all fetal tissues and structures.     

Inhibition of trophoblast adhesion to endothelial cells by the sera of women with recurrent spontaneous abortions.

PROBLEM: May anti-phospholipid or other autoantibodies interfere with trophoblast-endothelial cells interaction in women with unexplained pregnancy losses? METHODS OF STUDY: The sera of 72 women with recurrent spontaneous abortions (RSA) containing antibodies to endothelial cells (28), trophoblast (14), and cardiolipin (10) or lacking antibodies (25), and 26 controls were examined in an inhibition assay of trophoblast adhesion to endothelial cells using an ELISA based on the recognition of trophoblast by antibodies to cytokeratin. RESULTS: Adhesion of trophoblast to endothelial cells was time- and dose-dependent. Patients and control sera inhibited trophoblast adhesion with mean values of 37% and 7%, respectively. Inhibition above 2SD of the mean control value was still observed in 58% of the patients sera and 8% of the control sera. Sera containing antibodies to endothelial cells had higher inhibitory effect (38%) than those with antibodies to trophoblast (23%) and cardiolipin (28%) or lacking antibodies (26%). CONCLUSIONS: Antibodies and other undefined factors in the sera of women with RSA inhibit adhesion of trophoblast to endothelial cells.     

Characteristics of spontaneous bioelectrical activity of the brain in women with normal gestation and gestosis

Pregnant women were examined during I, II, and III trimesters in order to detect EEG correlations of gestation dominant in health and threatened abortion. Single uncomplicated pregnancy was characterized by predominant activation in the central and temporal areas contralateral to the location of the placenta. Threatened abortion was associated with inversion of interhemispheric asymmetry of activation with respect to the placenta location.     

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.     

An immunohistochemical study of the secretory immune system in human fetal membranes and decidua of the first trimester of pregnancy

PROBLEM: We analyzed the presence and distribution of components of the secretory immune system (SIS) in human fetal membranes (amnion, yolk sac, chorion) and decidua from the first trimester of pregnancy. MATERIALS AND METHODS: Specimens from 17 embryos (4-8 weeks of pregnancy) and nine fetuses (9-12 weeks) were divided into those that had not been exposed to massive foreign antigenic effects (Group I, n = 18) and those that had suffered acute chorioamnionitis (Group II, n = 8). RESULTS: Positive immunostaining for secretory component (SC), joining (J) chain, IgG, IgA, and macrophages was seen from 4 to 5 weeks of development and then during the whole first trimester of pregnancy in the syncytio- and cytotrophoblasts, the amniotic epithelium, the yolk sac endoderm and decidual cells. Macrophages with J chain, IgG and IgA were found in embryonic tissues on week 4, whereas lymphocytes, including those synthesizing IgA and IgM, appeared only at the end of the first trimester of pregnancy. In the decidua, lymphocytes and macrophages were recognized during the whole period of study. In cases with chorioamnionitis (Group II), reactivity of IgG and IgA in the mentioned cells of fetuses decreased sharply while the rate of immunoreactivity of SC and J chain as well as the number of T and B lymphocytes did not change. In the decidua, the number of immune reactive cells sharply increased with the appearance of plasma cells. CONCLUSIONS: In the fetal membranes and decidua all the SIS components are present. We suggest that two SIS, maternal and fetal, participate in the formation of the barrier between a mother and the fetus. Both these systems have different origin and cellular content as well as different immune reactions.     

复发性流产患者妊娠早期滋养细胞中E-cadherin的表达及其与HPV感染的相关性

目的:研究E-钙粘素(E-cadherin)在复发性流产患者妊娠早期滋养层细胞中的表达,探讨复发性流产与HPV感染的相关性。方法:采用免疫组织化学与PCR法分别对34例复发性流产患者(实验组)与41例正常早孕女性(对照组)妊娠早期滋养层细胞中E-cadherin的表达水平与HPV-DNA进行检测。结果:实验组滋养细胞中E-cadherin的表达明显低于对照组(P<0.001),HPV感染率则高于正常早孕组(P=0.039);HPV感染阳性的标本中,实验组E-cadherin的表达水平要低于正常早孕组,其差异有显著性(P=0.015)。结论:妊娠早期滋养层细胞E-cadherin的异常表达可能与复发性流产的发生有关,而人乳头瘤病毒(HPV)可能参与这一过程。     

Ultrasonographic detection of a live fetus in recurrent spontaneous abortion during the first trimester

To examine whether recurrent spontaneous abortion (RSA) canbe distinguished from repeated sporadic spontaneous abortion,the clinical course of 38 cases with three or more consecutiveand unexplained first trimester RSAs were retrospectively investigatedin this study. For comparison with controls, the clinical coursewas examined of 38 fertile females, who had had sporadic abortions.In 19 (50%) RSAs and 6 (16%) controls, fetal cardiac activitywas demonstrated by ultrasound during the course of pregnancy.The rate of detection of live fetus during pregnancy or at 8weeks ± 7 days gestation, was significantly greater inthe RSA group compared to the control. The rate of vaginal bleedingbefore spontaneous abortion was significantly less in the RSAgroup than in the control group. There was no difference betweenthe two groups in age or gestational age at spontaneous abortion.The patients with RSA were all examined for antiphospholipidantibodies in their sera and these were detected in eight ofthem. However, there was no difference in the rate of positivefetal cardiac activity between the RSA patients who tested positiveor negative for antibody. These results reveal that the clinicalcourse of RSA is very different from the course of sporadicabortion. Although sporadic abortion is a common complicationof pregnancy, RSA is not a random repeated abortion, but rathera separate disease from sporadic abortion in normal fertilefemales.     

Striated muscle cells in the leptomeninges. An immunohistochemical study

Striated muscle cells coexisting with neuroglial tissue were found in the leptomeninges of a 3-day-old neonate. An immunohistochemical study demonstrated the presence of myoglobin in the striated muscle cells, glial fibrillary acidic protein, and S-100 protein in the heterotopic glia, S-100 protein in the Schwann cells of the heterotopic nerve bundles, and neuron-specific enolase in some striated muscle cells as well as the neurons. A possible explanation for this may be that the striated muscle cells originated from neuroectoderm.     

Analysis of human leukocyte antigen (HLA)‐G polymorphism in normal women and in women with recurrent spontaneous abortions

Human leukocyte antigen (HLA)‐G is a non‐classical class I antigen. It has limited expression, but is high at the foetomaternal interface. This unique expression pattern of HLA‐G suggests that it might be important for survival of the foetus during pregnancy. In the present study, 120 women with recurrent spontaneous abortions (RSA) and 120 fertile control women were genotyped for the HLA‐G locus. This is the first report describing HLA‐G polymorphism in normal fertile and RSA women from India. The allele HLA‐G*010103 was higher in women with recurrent foetal losses. Interestingly, the HLA‐G*010105 and G*010108 alleles were totally absent in normal fertile women but present in RSA women with frequencies of 1.7% and 0.4%, respectively. Allele G*010107 was absent in both the groups. The frequency of the null allele G*0105N was high (13.8%) in our population as compared to other world populations. Our data support the hypothesis that HLA‐G polymorphism may contribute to recurrent foetal loss.