Implantation and placental development in somatic cell clone recipient cows

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.     

Human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis

Human cytomegalovirus (CMV) is the leading cause of prenatal viral infection. Affected infants may suffer intrauterine growth retardation and serious neurologic impairment. Analysis of spontaneously aborted conceptuses shows that CMV infects the placenta before the embryo or fetus. In the human hemochorial placenta, maternal blood directly contacts syncytiotrophoblasts that cover chorionic villi and cytotrophoblasts that invade uterine vessels, suggesting possible routes for CMV transmission. To test this hypothesis, we exposed first-trimester chorionic villi and isolated cytotrophoblasts to CMV in vitro. In chorionic villi, syncytiotrophoblasts did not become infected, although clusters of underlying cytotrophoblasts expressed viral proteins. In chorionic villi that were infected with CMV in utero, syncytiotrophoblasts were often spared, whereas cytotrophoblasts and other cells of the villous core expressed viral proteins. Isolated cytotrophoblasts were also permissive for CMV replication in vitro; significantly, infection subsequently impaired the cytotrophoblasts' ability to differentiate and invade. These results suggest two possible routes of CMV transmission to the fetus: (i) across syncytiotrophoblasts with subsequent infection of the underlying cytotrophoblasts and (ii) via invasive cytotrophoblasts within the uterine wall. Furthermore, the observation that CMV infection impairs critical aspects of cytotrophoblast function offers testable hypotheses for explaining the deleterious effects of this virus on pregnancy outcome.     

Mesenchymally-derived Insulin-like growth factor 1 provides a paracrine stimulus for trophoblast migration

Background  

Trophoblast migration into maternal decidua is essential for normal pregnancy. It occurs in a defined time window, is spatially highly restricted, and is aberrant in some pathological pregnancies, but the control mechanisms are as yet ill-defined. At the periphery of the placenta, chorionic villi make contact with decidua to form specialised anchoring sites that feed interstitially migrating cytotrophoblast into the placental bed.     

First trimester prenatal diagnosis of adrenoleukodystrophy by determination of very long chain fatty acid levels and by linkage analysis to a DNA probe

Summary A first trimester prenatal diagnosis of adrenoleukodystrophy has been done on chorionic villi biopsy in the pregnancy of a carrier woman. Two different approaches allowed one to determine that the male fetus was affected: the linkage analysis of DNA from chorionic villi using the highly polymorphic probe St 14 and the determination of very long chain fatty acid levels in cultured chorionic villi.     

Trisomy 16 confined to chorionic villi and unfavourable outcome of pregnancy.

Two cases of trisomy 16 confined to placental tissue associated with an unfavourable outcome of the pregnancy are reported. In the first case, after a diagnosis of an apparent non-mosaic trisomy 16 at chorionic villi sample (CVS), an intrauterine fetal death occurred at the 22nd week. In the second case a mosaic with trisomy 16 was found in chorionic villi and the fetus was still-born at 38 weeks. From a comparison of their cases with those of the literature, the authors conclude that a trisomy 16 confined to placental tissue has a negative effect on fetal growth and pregnancy outcome.     

Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta

Heme oxygenase (HO) catalyzes the oxidation of heme to carbon monoxide (CO), biliverdin, and iron and is thought to play a role in protecting tissues from oxidative damage. There are three isoforms of HO: HO-1 (inducible), HO-2 (constitutive), and HO-3 (unknown function). Preeclampsia is characterized by an inadequately perfused placenta and areas of tissue damage. We hypothesized that damaged areas of placentas from women with PE and uncomplicated pregnancies are associated with an alteration in HO expression. Compared with microsomes isolated from morphologically normal and peri-infarct chorionic villi of pathological placentas, microsomes from infarcted chorionic villi from the same placentas had decreased HO activity measured under optimized assay conditions. There was no correlation between microsomal HO levels and activity and tissue damage in uncomplicated pregnancies. Whereas there was no significant difference in HO-1 protein levels across all regions of uncomplicated and mildly preeclamptic pregnancies, HO-2 protein levels were decreased (P < 0.05) in peri-infarct regions and infarcted chorionic villi of mildly preeclamptic pregnancies. Immunohistochemical analysis revealed an apparent decrease in both HO-1 and HO-2 protein expression in damaged tissues. HO-1 and HO-2 were immunolocalized in the syncytiotrophoblast layer of the chorionic villi, the underlying cytotrophoblast, and in the vascular endothelium. This study suggests that the ability of the chorionic villi to oxidize heme to CO, biliverdin, and iron may be compromised in areas of tissue damage in the placenta of women with preeclampsia.     

Expression patterns of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in the rat placenta

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and is known to act as a tropic factor in various cells. Recent report revealed the expression of PACAP and the PACAP type I (PAC(1)) receptor in human and rat placentas at term. Placenta is a critical organ that synthesizes several growth and angiogenic factors for its own growth as well as fetal development. However, there is little information regarding the expression pattern and cellular localization of PACAP and PAC(1) during pregnancy. The aim of this study was to define the expression and distribution of PACAP and PAC(1) receptor mRNAs in the rat placenta during pregnancy. PACAP and PAC(1) receptor mRNAs were expressed in decidual cells, chorionic vessels, and stromal cells of the chorionic villi. Interestingly, the expression of these genes varied with the day of gestation. For example, PACAP and PAC(1) receptor mRNAs expressed in decidual cells on day 13.5 and 15.5, their expression was strong in chorionic vessels and stromal cells of the chorionic villi within the labyrinth zone on day 17.5, 19.5, and 21.5. In fact, as gestation advanced, the expression of PACAP and PAC(1) receptor mRNAs in the decidua cells disappeared, as their high expression became evident in the chorionic vessels and stromal cells of the chorionic villi. Our finding that PACAP and the PAC(1) receptor are co-localized and their genes seemingly co-regulated within specific placental areas, strongly suggest that this peptide may play an important role, as an autoregulator or pararegulator via its PAC(1) receptor, in physiological functioning of the placenta for gestational maintenance.     

229例流产绒毛染色体核型分析及两种长期培养方法比较

目的:探讨早期自然流产绒毛染色体核型分析在自然流产病因检测中的应用价值,并比较两种长期培养方法的差别。方法:选择孕早期自然流产的孕妇229例,在无菌条件下,从宫腔内取出绒毛,同时或单独经胰酶消化法与切碎贴壁法进行细胞培养,传代之后常规进行G显带,在显微镜下做核型分析。结果:229例流产胎儿绒毛,培养成功206例,成功率为89.96%。异常核型105例,异常率为50.97%,数目异常者101例,占异常核型的96.19%,以16三体最为多见。胰酶消化法的培养成功率及收获时间都显著优于直接贴壁法,差异具有统计学意义(P0.05)。结论:自然流产绒毛染色体核型分析对流产查因具有实用价值。胰酶消化法较切碎贴壁法对流产绒毛长期培养及染色体核型分析更具实用性。     

Prenatal diagnosis of a true fetal tetraploidy in direct and cultured chorionic villi

Prenatal diagnosis of a true fetal tetraploidy in direct and cultured chorionic villi: Tetraploidy is characterized by four complete sets of chromosomes (4n= 92). Although it has been frequently reported in spontaneous abortions, tetraploidy is extremely rare in term pregnancy. Most of late surviving patients are diploid/tetraploid mosaics and present severe mental and physical impairment. Up to date, only five tetraploidies were ascertained in the prenatal stage in amniocytes and/or fetal blood lymphocytes. No one has been reported in chorionic villi probably because tetraploidy is generally considered in this tissue as a false positive result due to confined placental mosaicism (CPM) or placental culture artefacts. We report here on a case of tetraploidy detected in chorionic villi because of fetal cystic hygroma. We discuss the reliability of this diagnosis and propose guidelines in the follow-up of tetraploidies detected after chorionic villus sampling (CVS). Thus a misdiagnosis of this poor condition will be avoided at best and an appropriate genetic counseling will be given to the parents.     

Chorionic DNA analysis for the prenatal diagnosis of familial hypercholesterolaemia

Prenatal diagnosis for familial hypercholesterolaemia (FH) was performed by using restriction fragment length polymorphisms (RFLPs) of the LDL receptor gene on chorionic villi DNA taken during the 10th week of pregnancy. Both parents were FH heterozygotes and had previously had a healthy son and an FH homozygous son. Two RFLPs were informative in this family and revealed that the fetus was unaffected by FH. At birth the child was found to have an LDL cholesterol level of 30 mg/dl and a normal LDL receptor activity in cultured umbilical cord fibroblasts. RFLP analysis on chorionic villi DNA is highly recommended for all heterozygous FH couples in whom the LDL receptor gene mutation/s is/are still to be characterized.     

Chorionic villi sampling: cytogenetic and clinical findings in 500 pregnancies.

The cytogenetic findings were analysed in a series of 500 pregnancies in which chorionic villi sampling was performed. In all cases a direct method was used, karyotyping being successful in 481 cases (96.2%). The main indication for sampling was maternal age over 36 (412 cases; 82.4%). Abnormal laboratory findings resulted in 24 terminations of pregnancy (4.8%); in addition five unexpected balanced chromosome rearrangements were detected. Twelve of 15 cytogenetic discrepancies were detected at amniocentesis, two after termination, and one at spontaneous abortion. Complete follow up data were available for the first 250 patients, among whom nine pregnancies (3.6%) ended in spontaneous abortion before the 20th week. There were no false negative findings. Seventy additional chromosome studies were performed because of failure of chorionic villi sampling or equivocal results, or for confirmation. Counselling before chorionic villi sampling should include the possibility that subsequent amniocentesis may be needed should mosaicism or other unexpected abnormalities be found. The success rate and accuracy of karyotyping chorionic villi samples by the direct method are acceptable but distinctly less than those of karyotyping cultured amniotic fluid cells.     

Heterogeneous histochemical reaction pattern of the lectin Bandeiraea (Griffonia) simplicifolia with blood vessels of human full-term placenta

Bandeiraea simplicifolia lectin (BS-I) stains vascular endothelium in various species. In humans, less than 10% of the specimens studied exhibit a reaction with BS-I. In the present histochemical study, the reactivity of BS-I with placental blood vessels and its correlation with the blood group from mother and newborn child was investigated. Acetone-fixed cryosections of representative tissue segments of human full-term placenta and umbilical cord were stained with BS-I. The staining pattern of tissues from patients with different blood groups was identical, although the reaction of BS-I in the placenta was heterogeneous. BS-I did not react with the umbilical cord. Vascular smooth muscle cells at the insertion site of the umbilical cord into the chorionic plate, and endothelium deeper in the chorionic plate, became progressively stained. The endothelial cells and tunica muscularis of smaller arteries and veins in stem villi lost their reactivity in parallel with decreasing vessel size. Arterioles and venules reacted heterogeneously. Capillaries, trophoblastic basement membranes, especially epithelial plates, and sometimes the syncytiotrophoblast were labelled in several terminal villi. The data indicate that 1) the placenta binds BS-I to fetal endothelium independent of the blood group, 2) cell-surface antigens on placental endothelial cells are expressed heterogeneously and 3) cell-surface glycans are constituted in an organ-specific manner on human endothelial cells.     

Beta-mannosidase of trophoblast and humana skin fibroblasts]

Conditions for assay of beta-mannosidase activity in human chorionic villi were studied using the fluorogenic substrate 4-methylumbelliferyl-beta-D-mannopyranoside. Comparison of the biochemical properties of the chorionic villi beta-mannosidase with those of the enzyme from human cultured fibroblasts showed their similarity. Like the enzyme from skin fibroblasts, the chorionic villi beta-mannosidase had rather high activity. Both enzymes had virtually the same pH optimum (4.2-4.7) and Km value. The data presented suggest that chorion biopsy specimens can be used for prenatal determination of beta-mannosidase activity at the early stage of development.     

Molecular and morphologic analyses of expression of ESX1L in different stages of human placental development

The mRNA expression of the ESX1L gene was analyzed by RT-PCR and in situ hybridization in human normal cytogenetically placentas, of different gestational ages. Our RT-PCR analysis showed that ESX1L mRNA is expressed from 5 weeks of gestation until term, suggesting a role not only in trophoblast differentiation but also in the maintenance of the villi and microvasculature. We also observed, by in situ hybridization, that ESX1L mRNA is expressed by cytotrophoblast from chorionic plate, syncytiotrophoblast and stromal cells of all terminal, intermediate and stem villi of term placentas. ESX1L mRNA expression was more pronounced in trophoblast cells of terminal villi than in intermediate and stem villi. In conclusion, ESX1L is expressed during all stages of placental development and is localized to sparse areas of trophoblast in terminal villi in association with cytotrophoblastic cells.     

Expression of trophinin, tastin, and bystin by trophoblast and endometrial cells in human placenta

Trophinin, tastin, and bystin comprise a complex mediating a unique homophilic cell adhesion between trophoblast and endometrial epithelial cells at their respective apical cell surfaces. In this study, we prepared mouse monoclonal antibodies specific to each of these molecules. The expression of these molecules in the human placenta was examined immunohistochemically using the antibodies. In placenta from the 6th week of pregnancy, trophinin and bystin were found in the cytoplasm of the syncytiotrophoblast in the chorionic villi, and in endometrial decidual cells at the utero placental interface. Tastin was exclusively present on the apical side of the syncytiotrophoblast. Tissue sections were also examined by in situ hybridization using RNA probes specific to each of these molecules. This analysis showed that trophoblast and endometrial epithelial cells at the utero placental interface express trophinin, tastin, and bystin. In wk 10 placenta, trophinin and bystin were found in the intravillous cytotrophoblast, while tastin was not found in the villi. After wk 10, levels of all three proteins decreased and then disappeared from placental villi.     

Long dwell-time exposure of human chorionic villi to transvaginal ultrasound in the first trimester of pregnancy induces activation of caspase-3 and cytochrome C release

Bioeffects after exposure to ultrasound are correlated to its duration. Although diagnostic ultrasound has been suggested to induce apoptosis, the underlying signal transduction pathway remains elusive. In this study, women in the first trimester of pregnancy were exposed to transvaginal diagnostic ultrasound with 5.0-MHz frequency for 0, 10, 20, or 30 min. The chorionic villi were obtained 4 h after exposure and activation of caspase-3 and cytochrome c release were analyzed by Western blotting. In contrast with the 0- and 10-min groups, cleavage products of active caspase-3 and cytochrome c release significantly increased in 20- and 30-min groups in a time-dependent manner. We show that long-duration exposure to transvaginal ultrasound activates effector caspase-3-mediated apoptotic cascade of chorionic villi in the first trimester of pregnancy. This occurs through the intrinsic death pathway involved in cytochrome c release. Our findings provide a molecular rationale for discriminant use of transvaginal ultrasound at the early stage of pregnancy.     

Murine serum cytokines throughout the estrous cycle, pregnancy and post partum period

Cytokines are pleiotropic glycoproteins participating in many aspects of mammalian reproductive physiology. Although murine models have been established to study normal and pathological pregnancy, the small volume of retrievable sample has hampered investigations into the role of cytokines in these processes. These problems were overcome by using fluid-phase multiplex immunoassays to monitor the serum profiles of 18 cytokines in single animals throughout normal murine reproduction: estrus, diestrus, post coitum, preimplantation, implantation, mid-pregnancy, late pregnancy and post partum. Most cytokines were detectable throughout all stages studied. Modest changes in profile were associated with estrous cyclicity and early pregnancy while virtually all cytokine levels increased markedly in mid- to late pregnancy and either fell slightly or levelled off post partum. The functional interrelationships between the various cytokines and the hormonal milieu are discussed with respect to gestational stage. Although certain profiles supported the 'conventional' Th1:Th2 cytokine paradigm of pregnancy, many of the changes recorded were orchestrated around IL-12 (p40) and (p70). The present findings suggest that the traditional cytokine dichotomy poorly describes complex immunological processes like pregnancy.     

Isolated symplasts and stromal syncytial buds of the normal human placenta at 6 to 14 weeks of pregnancy

Formation of isolated symplasts (IS) ("deported trophoblast", according to English nomenclature) and stromal syncytial buds (SSB) has been studied in 10 normal patients at pregnancy of 6-14 weeks by means of the indirect immunoperoxidase method. Monoclonal antibodies against beta-subunit of the human chorionic gonadotropin (beta-HCG), serial paraffin slices, stained with hematoxylin-eosin, semithin slices, stained with toluidin blue-pyronine and scanning electron microscopy have been used. A great amount of syncytiotrophoblastic processes and IS, analogous by their structure to syncytiotrophoblast are revealed. Presence of beta-HCG in the syncytiotrophoblast and its absence in the cytotrophoblast are proved. Concentration of beta-HCG in the IS is higher than in the syncytiotrophoblast of the villi. Maximal concentration of beta-HCG in the SSB is described for the first time. The functional role of the IS and SSB is discussed in the system mother-placenta-fetus.     

米非司酮对早孕绒毛Oct4、Sox2、Nanog 表达影响*

目的:探讨米非司酮对早孕绒毛组织Oct4、Sox2、Nanog mRNA和蛋白水平表达的影响。方法:要求终止妊娠的正常早孕妇女60例,分为:负压吸宫组及服用米非司酮150mg及米索前列醇联合行药物流产组。运用Real-time PCR方法检测两组早孕绒毛组织中Oct4、Sox2、Nanog mRNA的表达;采用免疫组织化学方法检测两组早孕绒毛组织Oct4、Sox2、Nanog蛋白的定位及半定量表达情况,比较两组差异。结果:Oct4、Nanog、Sox2 mRNA在药物流产组早孕绒毛中相对表达量明显低于负压吸宫组,差异有统计学意义(相对表达量分别为:0.15±0.045;0.37±0.053;0.23±0.040,P值均<0.05);Oct4、Nanog、Sox2在药物流产组早孕绒毛蛋白表达量亦明显低于负压吸宫组,差异有统计学意义(药物流产组蛋白表达量分别为13869±541、19251±1503、139492±918明显低于负压吸宫组22017±235、30543±729、37237±710)。结论:米非司酮可以通过抑制早孕绒毛中Oct4、Sox2、Nanog表达,发挥抗早孕作用。