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[The value of placental histology for the detection of genetically-induced abortions].

The majority of early spontaneous abortions is caused by genetic disorders. Chromosomal aberrations do not only imply characteristical morphologic alterations in embryo/fetus, but also in chorionic villi. Its possible to draw conclusions through the combination of a small amount of histological findings on chorionic villi to explain the kind of aneuploidy. Among 968 early spontaneous abortions 18.3% were defined as genetically caused by histological examination. 87.6% are probably trisomies, 7.3% polyploidies. 5.1% were diagnosed as complete hydatidiform moles with a diploid, androgenetic chromosome set. The value of histological examination of early spontaneous abortion tissue are discussed and it is pointed out to remaining problems. In a different percentage it is possible to establish the type of aneuploid by microscopic examination alone. Frequently there are sings to allow the conclusion there is a developmental disorder like genetic aberration.

Abortion, Incomplete↗

Aspiration and microscopy in the diagnosis of ectopic pregnancy.

OBJECTIVE: To compare Karman cannula aspiration followed by dissecting microscopy with suction curettage and permanent histology in obtaining and identifying chorionic villi. METHODS: Karman cannula aspiration was performed before standard curettage for failed intrauterine pregnancies (N=22) or possible ectopic gestation (N=24). Dissection microscopy for chorionic villi was performed on aspirates before submission for permanent histology. Sensitivity, specificity, positive and negative predictive value of each method in obtaining and identifying villi was determined. RESULTS: Overall, all methods were only moderately sensitive in detecting chorionic villi (50-76%). If failed intrauterine pregnancies were excluded, all methods had poor sensitivity (25-64%). However, if villi were detected, the positive predictive value of all methods was high (> 80%). CONCLUSIONS: Karman cannula aspiration followed by dissecting microscope examination or permanent histology may offer an alternative to traditional curettage in the diagnosis of ectopic gestations. A larger trial to validate these findings seems justified.

Biopsy, Needle↗

Microinjection of human placenta. I: Techniques.

An apparatus is described which has been used successfully to microinject the syncytiotrophoblast of chorionic villi, the mesenchymal core of chorionic villi, and cultured cells grown from first-trimester human trophoblast. It will accurately and repeatedly pulse picolitre quantities of injectate from the micropipette.

Female↗

Prenatal diagnosis of purine nucleoside phosphorylase deficiency in the first and second trimesters of pregnancy.

Prenatal diagnosis was performed in two successive pregnancies of a mother with a previous child with purine nucleoside phosphorylase (PNP) deficiency. In one pregnancy, an affected fetus was diagnosed in the 18th week of gestation after the demonstration of PNP deficiency in cultured amniotic fluid cells. Also an abnormal purine nucleoside profile was found in the amniotic fluid. The diagnosis of an affected fetus was confirmed by the analysis of cultured fetal skin fibroblasts and placental villi. The complete deficiency of PNP activity in placental villi confirms that the prenatal diagnosis of this disorder is possible by the direct investigation of chorionic villi. In the subsequent pregnancy, a heterozygous fetus was predicted in the tenth week of pregnancy by using chorionic villi.

Adenosine Deaminase↗

[Increase of Hofbauer cells in human placentas cocultured in vitro with Trypanosoma cruzi].

To analyze the interaction between normal human placentas with Trypanosoma cruzi, optical and electron microscopy of chorionic villi stroma cocultured in vitro with 1.5 x 106 Tulahuen strain Trypomastigotes of the T. cruzi for 1 h, 3 hs and 12 hs in Eagle minimal essential medium were done. An agglutination of chorionic villi in experimental cultures (with T. cruzi) from 1 h cultures was observed that was not present in control ones. this phenomenon resisted soft mechanical agitation to separate the isolated villi. Microscopical observations of stromal villi showed edema, separated structures and increment of Hofbauer cells as found by qualitative analysis. The chorionic villi agglutination could be caused by glycoproteins' modifications of the trophoblast, which in turn could be caused by secreted products of T. cruzi, as other authors have postulated in various cells' types. The increment of Hofbauer cells could represent a regulator mechanism of the placenta to equilibrate the intracellular water of the villi stroma.

Animals↗

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

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.

Adrenoleukodystrophy↗

Chorionic villus cDNA library displays expression of butyrylcholinesterase: putative genetic disposition for ecological danger.

Gene expression in chorionic villi may be particularly vulnerable to environmental exposure to poisonous substances. To reveal villus gene products which are thus subject to poisoning, molecular cloning was employed. A single sample of apparently normal chorionic villi (approximately 40 mg, from 9 weeks' gestation) was microscopically dissected to ensure purity of fetal tissue. Total RNA was extracted by isothiocyanate and directly employed for reverse transcription. A chorionic villus cDNA library was constructed from this preparation in the phage vector lambda gt10 and contained 60,000 independent recombinants. In the present study, this cDNA library was screened with labelled cDNA probes encoding human butyrylcholinesterase (BCHE) and acetylcholinesterase (ACHE). Nine BCHEcDNA clones were isolated out of 1.6 x 10(6) phages (5.7 x 10(-6) of screened recombinants) and exhibited similar restriction patterns to those observed for BCHEcDNA from other human tissues. In contrast, no ACHEcDNA clones could be found in 4.0 x 10(6) screened phages (less than 2.5 x 10(-6) of recombinants). These findings demonstrate efficient transcription (similar to fetal brain levels) from the BCHE gene but not from the ACHE gene in chorionic villi, and support the notion that BCHE is involved in chorionic villus growth and development.

Acetylcholinesterase↗

The effects of mifepristone (RU486) on prostaglandin dehydrogenase in decidual and chorionic tissue in early pregnancy.

Prostaglandin dehydrogenase is the main inactivating enzyme for prostaglandins and therefore controls local levels of prostaglandins. Since there is some evidence that the expression of this enzyme is under progesterone control it is reasonable that one of the effects of antiprogestin is to reduce the concentration of this enzyme and thus increase the effective concentration of prostaglandin within tissue. We have investigated the amount of enzyme activity within decidua and chorionic villi from women receiving the antigestagen mifepristone (RU486) 12, 24 and 36 h prior to surgical abortion, and examined the effect on tissue concentrations of prostaglandin dehydrogenase. Women receiving mifepristone in all groups had a significant reduction in concentration of prostaglandin dehydrogenase enzyme in decidual tissue. There was also a marked reduction in prostaglandin dehydrogenase in decidual cells following RU486, as demonstrated by immunochemical methods. At this stage of pregnancy, prostaglandin dehydrogenase was present in abundance in cytotrophoblast cells of chorionic villi but virtually absent from syncytiotrophoblast. In chorionic villi after RU486 administration in vivo, there were no obvious differences in prostaglandin dehydrogenase distribution or reactivity in the majority of cases.

Abortion, Induced↗

[Fluorescence in situ hybridization in prenatal diagnosis. First experiences].

The authors examined the potential use of non-radioactive in situ hybridization in prenatal screening. Probes for chromosomes 18, 13/21, 21 and X were applied on fourteen samples of peripheral lymphocytes and nine samples of chorionic villi. The aim of the study was to compare the analyzability of the two different DNA probes for chromosome 21 on six samples of chorionic villi. Six of the nine samples of chorionic villi were hybridized with probes specific for chromosome 21 and all six cases were diagnosed properly. However, we need more data to establish a screening protocol for routine prenatal cytogenetics.

Chromosomes, Human, Pair 13↗

Advances in the prenatal diagnosis of hematologic diseases.

Prenatal diagnosis of hematologic diseases can now be performed with fetal blood, fetal amniotic fluid cell DNA, and fetal chorionic villi DNA. Some hemoglobinopathies can be detected by all three methods, and the choice will depend on the available obstetric and laboratory techniques, as well as the time of presentation of the pregnancy. Hopefully, further development of molecular probes and techniques will soon expand these options to all of the globin disorders. Detection of coagulation disorders in utero currently requires samples of pure fetal blood. Gene cloning is accomplished for some (factor IX and antithrombin III) and is underway for others (factor VIII), and further investigation is necessary to determine whether deficiencies in these gene products are due to gene deletion or to mutant genes linked to polymorphic restriction enzyme sites of diagnostic use. Thus, molecular biology may be applied to prenatal diagnosis of the clotting problems, but this has not yet been accomplished. Disorders affecting the number and/or function of erythrocytes, leukocytes, and platelets can be diagnosed by analysis of fetal blood. Blood samples will continue to be required until more is known about the molecular biology of hematopoiesis. Syndromes that can be diagnosed by chromosome studies should be revealed in cultures of amniotic fluid cells, fetal blood lymphocytes, and chorionic villi cells. Cultured cells can be examined for karyotypes, Y-chromatin, spontaneous or induced chromosome breakage, DNA repair, SCEs, and translocations. The techniques for culturing amniotic cells and fetal blood white cells are established, and those for growing cells from chorionic villi are improving rapidly. Direct preparations of cells from villi only may suffice for some of the above analyses. The study of hematologic disease in utero has thus come full circle, from the use of amniotic cells to determine the sex in X-linked disorders, to fetal blood sampling for the analysis of gene products, then back to amniocentesis for DNA, and now earlier in gestation to chorionic villi. All of this has occurred in less than ten years, and it is anticipated that developments in the next ten years will be equally dramatic. The future should bring all prenatal testing into the first trimester, use molecular probes, and provide for both early diagnosis and early treatment of genetic hematologic disease.

Amniocentesis↗

[Early fetal karyotyping and its role in prenatal diagnosis].

OBJECTIVE: Shift of indicated invasive examination in prenatal diagnostics towards the earlier phases of pregnancy with preservation of quality of cytogenetic detection. DESIGN: Cytogenetic and molecular-cytogenetic analysis of the chorionic villi after long term culture. SETTING: Department of Medical Genetics and Foetal Medicine, Faculty of Medicine, Palacky University Olomouc, Faculty Hospital in Olomouc. METHODS: Cultivation of fibroblasts developing from chorionic villi after enzymatic or mechanical disintegration and their karyotyping. Using fluorescent in situ hybridisation to identify the most common chromosomal aneuploidies and to determine gonosomes in indicated cases. RESULTS: Testing and optimisation of long term culture method and its routine use. Method was utilised so far in 12 patients and successfulness was 83%. Additional fluorescent in situ hybridisation was performed in 6 cases. CONCLUSION: Using long term culture method of chorionic villi as reliable and routine tool in prenatal diagnostics.

Chorionic Villi Sampling↗

Expression of placenta growth factor mRNA in the rat placenta during mid-late pregnancy.

The placenta is an essential organ that synthesizes several growth and angiogenic factors for its own growth as well as fetal development. It is known that the placenta growth factor (PlGF)is a member of the vascular endothelial growth factor family and is critical for placental growth and fetal development. However, there is little information regarding the expression pattern and cellular localization of PlGF mRNA in rat placenta during pregnancy. The aim of this study was to define the distribution of PlGF mRNA in rat placenta at various gestations. RT-PCR analysis showed that the expression level of PlGF mRNA increased as gestation advanced. Using in situ hybridization histochemistry, positive cells of PlGF mRNA were detected in chorionic villi. PlGF mRNA was expressed in the trophoblast cells and stroma cells surrounding the blood vessels within chorionic villi on day 13 and 15. Also, positive signals of PlGF mRNA were strongly detected in stroma cells of chorionic villi on day 17, 19, and 21. In particular, the density and number of positive signals of PlGF mRNA was significantly increased as gestation advanced. The expression pattern of PlGF mRNA in rat placenta during pregnancy demonstrates that PlGF plays a functional role for placental growth and fetal development during mid-late pregnancy.

Animals↗

Improved technique for the expression of fragile-X in cultured amniotic fluid cells.

An improved technique for inducing fra(X) expression in cultured cells was obtained by using diazepam for mitotic arrest and 5-fluorodeoxyuridine (FUdR) for the induction of fra(X) expression. The method was developed using cultured fibroblast and urinary cells from fra(X) patients. Prenatal studies were performed on cultured amniotic fluid cells in five pregnancies at risk for fra(X). In two cases the cultured cells showed a 46,XY, fra(X) karyotype. One of the pregnancies was terminated and the diagnosis was confirmed by chromosome studies on several fetal tissues including chorionic villi and by histopathologic changes in the lymphatic vessels of the fetal testes. The fra(X) was also demonstrated in chorionic villi in a case in which amniotic fluid cells were not studied. Chorionic villi were isolated after a spontaneous abortion, the cultured cells had a 45,X karyotype and in addition 5% of the cells were fra(X) positive.

Adult↗

Chorionic villus sampling: diagnostic uses and limitations of enzyme assays.

Control ranges for enzymes in uncultured chorionic villi were established, based on: (1) 21 of 22 enzymes (mainly lysosomal) in villi had similar properties to the enzyme in cultured fibroblasts; (2) isoenzyme patterns in villi were similar to those in fibroblasts for five lysosomal enzymes but different for aryl sulphatases; (3) control ranges were determined for 12 enzymes in abortion villi and for 21 enzymes in biopsy villi, values tending to be higher in the latter for those enzymes studied in both types of sample; (4) storage of samples under various conditions revealed no major changes in activity of seven lysosomal enzymes. A number of potential pitfalls in the use of chorionic villus samples for diagnosis of metabolic disorders by enzyme assay are described: (1) the presence of aryl sulphatase C in chorionic villi, an isoenzyme which may interfere in assays of aryl sulphatase A; (2) the presence of maternal enzyme in chorionic villus material illustrated by the detection of the A isoenzyme of B-hexosaminidase in chorionic villus from a pregnancy affected with Sandhoff's disease; (3) the finding of falsely normal levels of alpha-iduronidase in chorionic villus samples from a pregnancy affected with Hurler's disease, probably due to contamination with maternal tissue which has relatively high levels of this enzyme compared with fetal chorionic material: (4) the inadequacy of indirect assays of incorporation of radiolabel into macromolecules using chorionic villi, for example [14C]propionate incorporation for prenatal diagnosis of methylmalonic aciduria. Provided that such pitfalls are recognized and great care is taken in selection of villus samples and interpretation of results, chorionic villus sampling allows reliable prenatal diagnosis of a large number of disorders using enzyme assays.

Chorionic Villi Sampling↗

Prenatal diagnosis of I-cell disease in the first and second trimesters.

First trimester prenatal diagnosis of I-cell disease (1 case) was based on demonstration of profound deficiency of N-acetylglucosamine 1-phosphotransferase in chorionic villi and in cultured trophoblasts derived from the chorionic villus specimen. Deficiency of this enzyme in cultured amniotic fluid cells obtained via amniocentesis was the basis for prenatal diagnosis of I-cell disease in the second trimester (2 cases). In both procedures, the diagnosis was corroborated by the finding of intracellular deficiency and extracellular elevation of multiple lysosomal enzymes in the fetal cell cultures (trophoblasts and amniotic fluid cells), as well as a significant increase in several lysosomal enzyme activities in the maternal serum.

Amniocentesis↗

Chorionic villus sampling: cytogenetic experience in preliminary investigations and in 50 diagnostic cases.

The results of preliminary cytogenetic investigations of chorionic villus sampling (CVS) and its diagnostic application in 50 cases are reported. Preliminary investigations included the effect of short delays between biopsy and processing, levels of mitotic activity related to variations in processing, the incidence of tetraploidy, correlation of the karyotype of the chorionic villi with that of the fetus, and the results of banding studies. A protocol was then designed for application to diagnostic CVS. The 50 diagnostic cases included 4 with male karyotypes which were terminated due to a family history of an X-linked disorder, 1 trisomy 21, 1 trisomy 18, the finding of 1 aberrant cell out of 50 in an otherwise normal analysis, a 45, XY, t (13;21) karyotype, 2 fragile X negative karyotypes and in one case the presence of mosaicism was detected in chorionic villi and subsequently not confirmed at amniocentesis. The karyotype on the chorionic villi was 46XY/47XY +3.

Adult↗

Changed expression of heat shock proteins in various pathological findings in placentas with intrauterine fetal growth restriction.

Heat shock proteins (HSPs) are activated in the cells of most organisms in response to sublethal heat shock and other stressors. It has been reported that HSP27, HSP60, HSP70, and HSP90 are expressed in normal human placenta, and it was thought that these HSPs play a role in the demonstration of cell viability and function. In this study, we performed an immunohistochemical (IHC) study of these HSPs for 27 placentas that had complicated intrauterine fetal growth restriction (IUGR) and compared the IHC findings with the pathological findings. To quantify HSP27, HSP60, HSP70, and HSP90, immunoreacted cells in the chorionic villi, syncytiotrophoblasts (ST), and cytotrophoblasts (CT) were counted. In thrombus, excessive syncytial knots, and avascular villi, the expression of HSPs was higher in the pathological sections compared to control in both ST and CT. In contrast, all HSPs decreased in both ST and CT around the infarction region. The data suggested that chorionic villi cells locally responded to some stresses, e.g., hypoxia and increase or decrease in the expression of HSPs. Although the villous cells around the infarction histologically appear viable, they may have received lethal damage, and as a result the expression of HSPs was decreased. These results are expected to improve our understanding of the pathological findings of IUGR in placentas, including the quality, damage, and function of the chorionic villi.

Chorionic Villi↗

[Value of chorionic biopsy in routine clinical diagnosis].

To test the advantages and risks of chorionic villi sampling, a method still new in prenatal diagnosis, 100 patients were examined. Most of the biopsies were taken in the 8th week of pregnancy. A soft catheter with a flexible mandrin was introduced transcervically to the chorion frondosum guided by ultrasound. The mandrin was then removed and trophoblastic tissue obtained by aspiration. The risk of abortion within 10-14 days after biopsy could be reduced to 4%. In the last series of chorionic villi samplings 92% of these could be evaluated cytogenetically. The use of quinacrine fluorescence technique showed that female mitoses were never found in male tissue. Because of the relatively low risk of abortion and the high diagnostic accuracy achieved in a very short period of time chorionic villi sampling can be considered as an alternative to amniocentesis.

Abortion, Spontaneous↗