Stimulation of hCG protein and mRNA in first trimester villous cytotrophoblast cells in vitro by glycodelin A

AIM: Human chorionic gonadotropin (hCG) is produced by fetal trophoblast cells and secreted into maternal circulation mainly in the first trimester of pregnancy. Another glycoprotein, glycodelin A, is one of the main products of the maternal decidua during this period. The purpose of this study was to investigate the effect of glycodelin A on hCG release by isolated cytotrophoblast cells in vitro. METHODS: Cytotrophoblast cells were prepared from human first trimester placenta and incubated with varying concentrations of glycodelin A. Supernatants were assayed for hCG protein concentrations, and quantification of beta hCG mRNA was carried out by RT-PCR. Expression of hCG was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. RESULTS: Glycodelin A induces a dose-dependent increase of hCG production. An increase of hCG expression was measured at 100 and 200 microg/mL glycodelin-A treatment in trophoblast cell culture by TaqMan assay on mRNA level. We found a moderate staining of hCG in control trophoblast cells, whereas a strong hCG staining was seen in glycodelin A-treated trophoblast cells. CONCLUSIONS: HCG is a marker for the differentiation process of trophoblast cells. Our results suggest that glycodelin A secreted by the decidualized endometrium is involved in the regulation of hormones produced by the trophoblast.     

Detection of gamma-globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization

OBJECTIVES: Fetal nucleated red blood cells (NRBC) that enter the peripheral blood of the mother are suitable for non-invasive prenatal diagnosis. The application of peptide nucleic acid (PNA) probes for tyramide amplified flow fluorescence in situ hybridization (FISH) detection of gamma-globin mRNA in fixed fetal NRBC is investigated. METHODS: Hemin-induced K562 cells or nucleated blood cells (NBC) from male cord blood were mixed with NBC from non-pregnant women and analysed using both slide and flow FISH protocols. Post-chorionic villus sampling (CVS) blood samples from pregnant females carrying male fetuses were flow-sorted (2 x 10(6) NBC/sample). Y chromosome-specific PNA FISH was used to confirm that the identified gamma-globin mRNA stained cells were of fetal origin. RESULTS: Flow FISH isolated gamma-globin mRNA positive NBCs showing characteristic cytoplasmic staining were all Y positive. The amplification system generated a population of false positive cells that were, however, easy to distinguish from the NRBCs in the microscope. CONCLUSION: The gamma-globin mRNA specific PNA probes can be used for detection and isolation of fetal NRBCs from maternal blood. The method has additional potential for the study of gamma-globin mRNA levels or the frequency of adult NRBC (F cells) in patients with hemoglobinopathies.     

Cardiotonic steroids induce anti-angiogenic and anti-proliferative profiles in first trimester extravillous cytotrophoblast cells

ObjectivePreeclampsia (preE), is characterized by abnormal placental invasion and function. Marinobufagenin (MBG), a cardiotonic steroid (CTS), inhibits cytotrophoblast (CTB) cell functions that are critical for normal placental development. This study tests the hypothesis that CTSs induce anti-angiogenic and anti-proliferative effects in CTB cells.MethodsHuman extravillous CTB cells of the line Sw-71, derived from first trimester chorionic villus tissue, were incubated with 0, 0.1, 1, 10, and 100 nM of each of three CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. Thereafter, levels of pro-angiogenic (vascular endothelial growth factor (VEGF165), placental growth factor (PlGF)) and anti-angiogenic (soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng)) factors were measured in culture media using ELISA kits. Expression of three receptors (VEGF receptor 1 (VEGFR1), angiogenic angiotensin type 1 receptor (AT₁) and anti-angiogenic angiotensin type 2 receptor (AT₂)) were assayed using immunoblotting (western blots) in cell lysates.ResultssFlt-1 and sEng secretion were increased while VEGF165 and PIGF were decreased in the culture media of CTB cells treated with 1 nM or more of each CTSs (p < 0.01 for each). The AT₂ receptor expression was up-regulated (p < 0.05) in CTB cells treated with 1 nM or more of MBG and CINO and with 100 nM OUB, while AT₁ and VEGFR1 expressions decreased (p < 0.05) with 1 nM or more of MBG and 10 nM or more of CINO and OUB.ConclusionsCTSs influence extravillous CTB cells to induce an anti-angiogenic and anti-proliferative profile.     

Secretion of cytokines by villous cytotrophoblast and extravillous trophoblast in the first trimester of human pregnancy

Cytokines are proposed to play roles in regulation of trophoblast invasion, spiral artery remodeling and immunoregulation during early pregnancy. Secretion of 12 cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, IFNγ, GM-CSF, MCP-1 and RANTES) by first trimester extravillous trophoblast and villous cytotrophoblast cells was examined using multiplex cytokine array technology. Seven (IL-1β, IL-8, IL-12p70, IL-13, GM-CSF, MCP-1 and RANTES) of the 12 cytokines examined were detectable in the samples studied (n=10 each group). Villous cytotrophoblast production of IL-1β and IL-8 increased with gestational age. Extravillous trophoblast production of IL-8, IL-13 and RANTES increased with gestational age. At 12-14 weeks gestation extravillous trophoblast cells secreted higher levels of IL-8, IL-13 and RANTES than villous cytotrophoblast cells.     

Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization

Purpose: The purpose of the study was to investigate the reliability of the fluorescent in situ hybridization (FISH) analysis of the first polar body (IPB) for cytogenetic evaluation of human oocytes as a method of choice in preimplantation diagnosis of chromosomal aneuploidies. Design: Human unfertilized oocytes and their extruded IPB were analyzed using the directly labeled fluorescence alpha-satellite DNA probes to chromosomes X and 18. Results: Paired signals for chromosomes X and 18 were observed in the second meiotic prophase (MII) of unfertilized oocytes and their extruded IPB. In the series of 156 unfertilized oocytes in which the number of X chromosome-and chromosome 18-specific signals were analyzed in both MII and IPB, five nondisjunction events have been detected, with corresponding signals in MII and their IPB: missing signals in MII corresponded to extra signals in their IPB and extra signals in MII corresponded to missing signals in IPB. In one oocyte chromosome 18 nondisjunction was detected, with both chromosome 18 signals in MII and no chromosome 18 signal in IPB. In four oocytes chromatid malsegregations for chromosome X or chromosome 18 were detected: in two oocytes, three of four chromosome 18 signals were present in MII, with only one in IPB, and in the other two oocytes, three of four chromosome signals were present in MII, with only one left in IPB. Conclusions: The data suggest the possibility of detecting chromosomal aneuploidy in oocytes through cytogenetic analysis of their corresponding IPB by FISH as a possible approach for preimplantation diagnosis of major chromosomal trisomies.Presented in part at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, Austria, April 3–7, 1995.     

Detection of kappa- and lambda-expressing cells in the endometrium by in situ hybridization.

The detection of kappa- and lambda-expressing cells in endometrial biopsies using in situ hybridization was correlated with the histologic findings. Forty endometrial biopsies were examined in conjunction with kappa and lambda expression in serial sections, recorded as the number of positive cells per 10 x100 fields. Cells expressing kappa or lambda were found in 39/40 (98%) biopsies with the average total number per 10 x100 fields as follows: proliferative (n = 13) 13; secretory (n = 6) 16; endometritis (n = 6) 623; polyp (n = 4) 72; adenocarcinoma (n = 6) 677; oral contraceptive effect (n = 5) 8. Many of the B lymphocytes expressing kappa and lambda did not have the cytologic features of plasma cells. The diagnosis of chronic endometritis can be made when the histologic findings of out-of-phase endometrial glands and focal fibrosis are seen with increased plasma cells; in cases where the latter is equivocal, in situ hybridization testing for light chain expression can be useful. Cells expressing kappa and lambda mRNA are relatively common in normally cycling endometrium, implying that mild chronic antigenic stimulation is present in most endometrial tissues. In situ hybridization for light chain expression can be helpful in endometria where only very rare plasma cells are seen; a baseline result would rule out chronic endometritis.     

过氧化物酶体增殖物激活受体γ及其配体对早孕期绒毛组织及细胞滋养细胞浸润能力的影响

目的探讨过氧化物酶体增殖物激活受体γ（PPARγ）及其配体对早孕期绒毛组织及细胞滋养细胞浸润能力的影响。方法采用免疫组化方法、免疫荧光细胞化学染色法、蛋白印迹法和RT-PCR技术检测20例孕6～8周（早孕早期组）及20例孕11～12周（早孕晚期组）绒毛组织及细胞滋养细胞中的PPARγ蛋白及其mRNA的表达;并检测不同浓度PPARγ激动配体——15-脱氧-前列腺素J2（15-d-PGJ2）和曲格列酮,以及不同浓度拮抗配体——双酚丙烷二环氧甘油醚（BADGE）对原代无血清培养的细胞滋养细胞浸润能力的影响。结果（1）PPARγ／蛋白在早孕早期组和早孕晚期组绒毛组织中均有表达,主要定位在细胞滋养细胞核中,合体滋养细胞及绒毛间质细胞中无表达。（2）早孕早期组绒毛组织和培养的细胞滋养细胞中,PPARγ／蛋白表达水平分别为1．35±0．08、1．13±0．11,PPARγ／mRNA表达水平分别为36．0±5．1、13．4±3．1;早孕晚期组绒毛组织和培养的细胞滋养细胞中,PPARγ／蛋白表达水平分别为1．17±0．03、0．86±0．05,PPARγ mRNA表达水平分别为23．3±5．5、6．1±1．3,早孕晚期组PPARγ蛋白及其mRNA表达水平明显低于早孕早期组,两组分别比较,差异均有统计学意义（P〈0．05）。（3）PPARγ／激动配体15-d-PGJ2和曲格列酮均有抑制细胞滋养细胞的浸润的作用。15-d-PGJ2浓度为1、10μmol／L,曲格列酮浓度为10μmol／L时,早孕早期组细胞滋养细胞浸润指数分别为0．57±0．03、0．43±0．02、0．50±0．06,早孕晚期组分别为0．69±0．02、0．59±0．03、0．66±0．05,两组分别比较,差异均有统计学意义（P〈0．05）。（4）PPARγ拮抗配体BADGE浓度为20、50μmol／L时,早孕早期组细胞滋养细胞浸润指数分别为1．23±0．07和1．58±0．04;早孕晚期组分别为1．05±0．03和1．38±0．08,两组分别比较,差异均有统计学意义（P〈0．05）。结论PPARγ／在调节滋养细胞浸润过程中起重要作用;在早孕期胎盘绒毛组织,PPARγ／激动配体可抑制滋养细胞浸润;PPARγ／拮抗配体可促进滋养细胞浸润,且能部分逆转激动配体的作用。     

A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity

We developed a method for isolating highly pure villous cytotrophoblast cells from first trimester and term placenta that excludes extravillous trophoblast and syncytiotrophoblast fragments. The method is based on positive immunoselection using an antibody (mAb C76/18) reacting with hepatocyte growth factor activator inhibitor 1, HAI-1, a membrane antigen on villous cytotrophoblast. As a comparison, we also immunopurified cells using an antibody against CD105, present on syncytiotrophoblast and some extravillous trophoblast cells. The isolates were characterized by flow cytometry. HAI-1-positive cells from first trimester and term placentae were highly pure (>98 per cent cytokeratin 7-positive) mononuclear trophoblast cells. These isolations were contaminated with only very small percentages of vimentin and CD45-positive cells. HAI-1-positive trophoblast cells lacked CD105 and also HLA class I, a marker for extravillous trophoblast. In culture HAI-1-positive cells adhered, displayed an epithelial morphology, and survived for more than three days. In contrast, CD105-positive cell fractions from first trimester placenta were a heterogeneous mixture of mononuclear and multinuclear elements consisting of syncytiotrophoblast fragments, extravillous trophoblast cells, as well as around 5 per cent non-trophoblastic contaminants. In conclusion, the positive immunoselection method using antibody C76/18 yielded highly pure villous cytotrophoblast cells devoid of elements derived from syncytiotrophoblast or extravillous trophoblast.     

MHC class I antigen expression by molar trophoblast

It is an issue for debate why molar tissues are not rejected by an immunologically potent host, since all genes in complete moles and 2/3 genes in partial moles are considered to be paternally derived. Molar trophoblasts are in direct contact with host cells, and therefore HLA expression by these cells may hold the key to the elucidation of the immunological reaction between molar tissues and the host. It has been reported that villous trophoblasts are negative and extravillous trophoblasts are positive for HLA-A, B,C, but the expressed HLA-A,B,C molecule has been noted to lack their polymorphic determinants. We analyzed the reactivity of two monoclonal antibodies to a monomorphic determinant of HLA-A,B,C (W6/32 and Cappel anti-HLA-A,B,C) with molar trophoblasts, using three uteri containing complete moles and two containing partial moles. The reactivity was examined by an indirect immunoperoxidase method. The staining patterns were almost identical in complete moles and partial moles. Villous trophoblasts showed a negative reaction with both antibodies. On the other hand, extravillous trophoblasts exhibited intense staining for W6/32 and negative staining for Cappel anti-HLA-A,B,C, which may suggest that the expression of a constant region as well as a variant region of HLA-A,B,C molecule is incomplete.     

Trisomy 13 or 18 (mosaicism) in first trimester cytotrophoblast cells: false-positive results in 11 out of 51 cases

OBJECTIVE: The finding of full or mosaic trisomy 13 or 18 in first trimester chorionic villus sampling (CVS) may be a false-positive result. This report provides incidence and outcome information that may be helpful in counselling individual patients and in choosing adequate follow-up. STUDY DESIGN: From a series of 6820 CVS cases, we retrospectively collected data on all patients (n=51) with full (n=30) or mosaic (n=5) trisomy 18, and full (n=13) or mosaic (n=3) trisomy 13 in cytotrophoblast cells. RESULTS: Five false-positives were seen in patients with full trisomy 18 and three in the mosaic cases. One false-positive result was observed in full trisomy 13 and two false-positives in cases of mosaicism. No false-negative results were reported. CONCLUSION: The diagnosis of trisomy 13 or 18 in cytotrophoblasts should be confirmed in other tissues, unless fetal abnormalities are seen at ultrasound. In case of mosaicism, follow-up amniocentesis is advised.     

Detection of fetal cells in intrauterine lavage samples collected in the first trimester of pregnancy.

OBJECTIVES: The aim of the present study was first to evaluate the presence of fetal cells in transcervical cell (TCC) samples collected by intrauterine lavage in the first trimester of pregnancy, and then to compare different methods for the detection of these cells. METHODS: TCC samples were collected by intrauterine lavage before termination of pregnancy (TOP) from 81 pregnant women between 7 and 12 weeks of gestation. Samples of placental tissue were collected from each patient at TOP, whereas maternal peripheral blood samples were obtained in 57 cases. DNA extracted from 81 lavage and the corresponding placental samples was amplified by a polymerase chain reaction (PCR) assay using primers for SRY and HUMARA genes. All 81 lavage samples were also analysed by fluorescent in situ hybridisation (FISH) using direct-labelled probes for X chromosome alpha-satellite (DXZ1, Xp11.1-q11.1) and Y chromosome alpha-satellite (DYZ3, Yp11.1-q11.1) regions. In 57 cases, a quantitative fluorescent (QF) PCR assay, involving the use of two small tandem repeat (STR) markers (D21S11, D21S14.11) specific to chromosome 21 was employed to analyse DNA extracted from placental tissue, lavage and maternal blood samples. RESULTS: PCR analysis revealed that 40/81 placental samples were from male pregnancies. Correct sexing was achieved with the PCR technique in 30/40 (75%) lavage samples retrieved from pregnant women with male conceptuses and in all 41 (100%) samples collected from pregnancies with female fetuses. With the FISH analysis, nuclei bearing X and Y signals were observed in 32/40 cases (80%) from known male pregnancies, the rate of fetal cells ranging between 2% and 95%, whereas nuclei showing X and Y signals were not detected in any of the 41 lavage samples from known female pregnancies. Paternal peaks were present in 30/57 (52.6%) lavage samples tested by QF-PCR. CONCLUSION: The results suggest that fetal cells can be found, at a significant rate, in a very high proportion of intrauterine lavage samples. Therefore, this sampling technique can be regarded as a promising tool towards minimally invasive prenatal diagnosis. The FISH and PCR methods showed a similar efficiency in detecting fetal cells.     

The role of tapasin in MHC class I protein trafficking in embryos and T cells

Preimplantation mouse embryos express both classical (class Ia) and nonclassical (class Ib) MHC class I proteins, and yet are not rejected by the maternal immune system. Although the function of the embryonic MHC class Ia proteins is unknown, one MHC class Ib protein, Qa-2, the product of the preimplantation embryo development (Ped) gene, actually enhances reproductive success. Similar in structure to MHC class Ia proteins, Qa-2 protein is a trimer of the alpha (heavy) chain, β₂ microglobulin and a bound peptide. Studies on the folding, assembly and trafficking of MHC class Ia molecules to the cell surface have revealed this process to be dependent on multiple protein chaperone molecules, but information on the role of chaperone molecules in Qa-2 expression is incomplete. Here, we report the detection of mRNA for four chaperone molecules (TAP1, TAP2, calnexin and tapasin) in preimplantation embryos. We then focused on the role of the MHC-dedicated chaperone, tapasin, on Qa-2 protein expression. First, we demonstrated that tapasin protein is expressed by preimplantation embryos. Then, we used tapasin knockout mice to evaluate the role of tapasin in Qa-2 protein expression on both T cells and preimplantation embryos. We report here that optimal cell surface expression of Qa-2 is dependent on tapasin in both T cells and preimplantation embryos. Identification of the molecules involved in regulation of MHC class I protein expression in early embryos is an important first step in gaining insight into mechanisms of escape of embryos from destruction by the maternal immune system.     

Susceptibility of MHC class I expressing extravillous trophoblast cell lines to killing by natural killer cells.

Purified human first trimester extravillous trophoblast (EVT) cell lines HTR-8 and HT-116 were examined for susceptibility to natural killer (NK) cell-mediated lysis. Based upon nucleic acid sequencing of an amplified fragment of cDNA, Western blot analysis and immunostaining of fixed and live cells, it was shown that both EVT cell lines expressed HLA-G mRNA and protein within the cytoplasm when cultured on laminin-coated plates. Very weak HLA-G expression was detectable on the cell surface under these conditions. However, strong cell surface expression of a classical MHC class I molecule (most likely HLA-C) was exhibited by these EVT cell lines when grown on laminin, as indicated by W6/32 FACS analysis (Ab recognizing pan MHC class I), and Western immunoblotting with HC10 (Ab recognizing HLA-B/C). When these EVT cells, cultured on laminin, were used as targets for peripheral blood natural killer (NK) cells in a standard chromium release assay, both HTR-8 and HT-116 cells were lysed by NK cells in a dose-dependent manner. The respective percentage specific lysis at an effector to target (E/T) ratio of 100 was 28+/-7, and 48+/-14. The choriocarcinoma cell lines JAR and JEG-3 which were respectively MHC class I negative and HLA-G positive were resistant to NK cell lysis. Thus, there was no clear relationship between the MHC class I expression and NK cell resistance or susceptibility among the EVT cell lines and choriocarcinoma cells. These findings raise the possibility that NK cells may take part in the surveillance of the invasive EVT cells during normal placentation.     

In situ hybridization identifies renin mRNA in the rat corpus luteum

All components of the renin-angiotensin system (RAS), including angiotensinogen messenger RNA (mRNA), renin-like activity (RLA), renin mRNA, angiotensin II (A II) immunoreactivity and angiotensin converting enzyme, have been found in the whole rat ovary and A II receptors have recently been localized to ovarian follicles. Our study was designed to test further which part of the ovary can produce renin.     

Detection of numerical chromosome abnormalities in human spermatozoa by three-color fluorescence in situ hybridization

Three-color fluorescence in situ hybridization (FISH) was used to detect numerical X, Y, and 17 chromosomal aberrations in human sperm nuclei. Digoxigeninlabeled alpha satellite chromosome X-specific probe DXZ1, biotin-labeled classical satellite chromosome Y-specific probe DYZ1, and biotin plus digoxigenin-labeled alpha satellite chromosome 17-specific probe D17Z1 were simultaneously hybridized to sperm preparations from donors with normal semen (group A) and abnormal semen (group B) characteristics. The proportions of haploid X, Y, 17 and disomy, diploidy of them before and after swim-up were determined. At least 3,000 sperm were analyzed for each sample. Overall, up to 98% of sperm were labeled with the probes, and all statistical analyses were performed using chi 2 tests. A significant difference was observed between group A and group B in frequency of sex chromosome disomy (p < 0.05). In group B, there were significant differences in frequencies of sex chromosomes disomy (p < 0.05) and diploidy (p < 0.01) before to after swim-up. There was no significant difference in frequency of disomy 17 between the 2 groups. In group A and B, the ratios of X- to Y-bearing sperm were 1:1 (neat semen), but in both groups there was a significant increase in Y-bearing sperm after swim-up. The results of this study demonstrated that abnormal semen has sex chromosome disomy more frequently than does normal semen and that portion of sex chromosome disomic and diploid sperm is removed by swim-up, especially for abnormal semen. These findings suggest that we should be careful in using abnormal semen for IVF, especially for ICSI, and should perform swim-up if possible.     

In situ hybridization identifies renin mRNA in the rat corpus luteum

All components of the renin-angiotensin system (RAS), including angiotensinogen messenger RNA (mRNA), renin-like activity (RLA), renin mRNA, angiotensin II (A II) immunoreactivity and angiotensin converting enzyme, have been found in the whole rat ovary and A II receptors have recently been localized to ovarian follicles. Our study was designed to test further which part of the ovary can produce renin. Renin mRNA was detected in the gonadotropin-stimulated rat's corpus luteum using in situ hybridization histochemistry techniques. No positive signal was detected in theca or granulosa cells. These results indicate ovarian renin gene expression in rat luteal cells, where the renin molecule is probably produced, and support our concept of an intrinsic ovarian RAS.