Clinical findings of multiple pregnancy with a complete hydatidiform mole and coexisting fetus.

OBJECTIVE: The aim of this series was to evaluate the clinical features, management, and outcomes of multiple pregnancy with a complete hydatidiform mole and coexisting fetus (CHMCF). METHODS: Between 1998 and 2008, we investigated 6 women with a diagnosis of a CHMCF. The gestational age at diagnosis, symptoms, serum b-human chorionic gonadotropin levels, cytogenetic and molecular analysis findings, complications, routes of delivery, and pregnancy outcomes were assessed. RESULTS: All cases were diagnosed before 14 weeks' gestation by sonography. Only 1 ended with the delivery of a live-born neonate, whereas the other 5 cases required termination of pregnancy (TOP) before 21 weeks' gestation because of severe maternal complications (eg, preeclampsia, thyrotoxicosis, lung metastasis, and heavy bleeding) or intrauterine fetal death. The pathologic diagnosis of a complete hydatidiform mole was confirmed in all cases. Two patients required methotrexate for treatment of persistent trophoblastic disease (PTD). CONCLUSIONS: On the basis of our experience, in cases with a normal karyotype and no gross fetal abnormalities on sonography, we carefully recommend continuation of pregnancy as long as maternal complications are absent or controllable. However, updated treatment criteria are still needed, and intensive maternal follow-up is necessary in the postpartum period because maternal complications during pregnancy and PTD after TOP are not uncommon.

Microscopía electrónica de barrido de la hiperplasia en la vellosidad molar / Scanning electron microscopy of placental hyperplasia

Objetivo: Estudio de la superficie externa del sincitiotrofoblasto de vesículas de mola hidatidiforme, fue rastreada utilizando la microscopía electrónica de barrido. Método: Especimenes de material molar de 21 semanas de embarazo se fjaron en 2 por ciento de glutaraldehido 0,1 M a 4ºC en sala de parto y posteriormente post fjados en 1 por ciento de tetraoxido de osmio siguiendo los procedimientos convencionales de la microscopía electrónica de barrido como deshidratación, desecado de punto crítico, cubrimiento iónico y observación con el microscopio electrónico de barrido. Resultados: Los resultados revelan cambios morfológicos en la membrana plasmática sincitial, desde superfcie aplanadas con pequeños gránulos o promontorios, hasta la formación de numerosas prolongaciones de membrana que originan pliegues, bandas o columnas, las cuales se ramifcan intensamente, tomando contacto entre sí para conformar una complicada trama, que deja un retículo de espacios como cavernas, las cuales se abren hacia el espacio intervelloso siendo la expresión de una intensa proliferación de membranas en hiperplasia. Conclusión: La observación de esta trama permite un mejor entendimiento de la estructura de la mola comparada con las imágenes de microscopía de luz.

Objective: Study of the external surface of the syncytiotrophoblast in vesicles of hydatidiform mole was examined and analysed using scanning electron microscopy. Method: Vesicles of molar material were taken at 21 weeks of pregnancy and fxed in 2 percent of glutaraldehyde 0.1 M at 4 °C in delivery room and furtherly post-fxed in 1 percent of tetroxide of osmium according with conventional procedures of the scanning electron microscopy as dehydration, critical point drying, surface coating and examination using scanning electron microscope. Result: The fndings reveal the morphological changes in the syncytial membrane from smooth surface with small bridges or granules to the formation of numerous prolongations of plasma membrane, which produce folds, bands or columns with ramifcations, that get in touch organizing a complex net that contain spaces opened to the intervillous space. This is an expression of the proliferation of syncytial plasma membrane during the hyperplasia. Conclusion: The tridimensional observation of this net complex permit a better understanding of the structure of the molar vesicle when these images are compared with those of light microscopy.

Numerous vessels detected by CD34 in the villous stroma of complete hydatidiform moles.

It has been considered that the villous stroma of complete hydatidiform moles (CHMs) is usually avascular. Vessels would normally form if embryogenesis had occurred. But judging by the structural maturity of molar villi, it was suspected that they must contain a considerable number of blood vessels and an attempt was made to demonstrate their presence. It has been shown previously that the vascular endothelial cell markers factor VIII related antigen (FVIII-RAg), Ulex europaeus 1 agglutinin (UEA-1) and CD31 were of no use in demonstrating the vessels in molar villi. A monoclonal antibody, QBEND/10, raised against the CD34 antigen in human endothelial cell membranes and hemopoietic progenitor cells, was selected to test its use as a marker of villous vascular endothelial cells in CHMs. On immunohistochemical analysis, numerous blood vessels were found using CD34 antibody in the stroma of CHMs, and the numbers corresponded to those found in normal villi of gestational age 8-12 weeks. Moreover, the luminal free surface of all vascular endothelial cells was so specifically delineated that it permitted the identification of vessels not apparent on hematoxylin- and eosin-stained sections; other stromal cells and trophoblast were not labeled.

[The ultrastructure of intermediate type trophoblast cell].

Transmission electron microscopy was used to clarify the detailed morphology of the "intermediate type" trophoblast cell in normal and tumor issue. 67 normal placental villi specimen, 10 placental bed specimens and 10 malignant mole, 10 hydatidiform mole, 5 choriocarcinoma specimen (the last three types taken before chemotherapy) were examined. Results showed that the transitional type trophoblasts of the placenta were developed from cytotrophoblasts through differentiation and fusion to syncytiotrophoblasts which showed features of maturation and aging, having features of cytotrophoblast nuclei and syncytiotrophoblast cytoplasm. The transitional trophoblast of placental bed showed similar morphology as that of transitional type cells of villi. The morphology of transitional type cells of villi. The morphology of transitional type cells of trophoblastic tumors had both normal morphology and cellular hyperplasia, atypia and features of tumor ultrastructure. The prominent feature was the high electron density of the granules and polymorphic cysts crowded in villi, demonstrating that the morphology of "intermediate type" trophoblasts in placental and tumor tissue are similar, whereas heterotype cellular morphology is present in varying degrees in tumor tissue.

[Mitochondrial heredity in hydatidiform moles]. / Herencia mitocondrial en las molas hidatidiformes.

A mitochondrial DNA study of seven hydatidiform moles and seven full term placentas as controls was carried out to determine the role played by mitochondrial DNA as the only maternal genome participating in the pathogenesis of these trophoblastic growths. Mitochondrial DNA was digested by restriction enzymes Eco R1 and Hind III, processed by electrophoresis and stained by ethidium bromide. Molar mitochondrial DNA showed two restriction bands at 9416 and 2322 kbs with Eco R1 and one band at 2322 kbs with Hind III, whereas the controls showed three bands of 9416, 4361 and 2322 kbs with Eco 1, and two bands at 4361 and 2322 kbs with Hind III. The results were interpreted as a DNA alteration consistent with a mutation at level of tARN genes, initiating the reading of gen ND2 of Complex I, NADH dehydrogenase and affecting Complex CO III that transcribe cytochrome c and oxidoreductase genes. The alterations are considered as mutations probably resulted from folic acid deficiency at threshold levels during nuclear and mitochondrial DNA synthesis in oogenesis and meiosis that renders anucleated ova (cytoplasts), fertilized, and further accelerated development of a zygote bearing an entire androgenic genome.

[AgNOR of gastational trophoblastic tumors and its clinical significance].

A total of 120 paraffin-embedded gestational trophoblastic tumor tissue blocks was selected and divided into 5 groups: (1) 20 cases of normal chorionic villi. (2) 40 cases of hydatidiform mole with no malignant change during a following-up period of at least two years. (3) 40 cases of hydatidiform mole which developed into invasive mole or choriocarcinoma. (4) 10 cases of invasive mole. (5) 10 cases of choriocarcinoma. Nucleolar organizer regions (NORs) was Ag-stained and AgNOR dots were counted using the Plotion's method. The result showed that there was significant difference between group 1 and group 2 (P < 0.005), group 2 and group 3 (P < 0.001), group 3 and group 4 (P < 0.05). Taking the AgNOR count 4.00 as a standard, 75% of the cases in group 2 (mean = 2.730) was below this standard. The study suggested that with the increase of malignancy of trophoblastic tumor, the AgNOR count increased correspondingly. A quantitative study of AgNOR might be a useful measure to detect the early malignant change of hydatidiform mole.

[Immunoelectron microscopic localization of human chorionic gonadotropin in the syncytiotrophoblast of hydatidiform moles].

The immunocytochemical localization of hCG in the syncytiotrophoblast of hydatidiform moles was observed by means of the immunoglobulin gold technique with rabbit anti-human hCG serum. Immunoreactive gold particles of hCG are preferentially localized in the distal portion of the Golgi complexes, Golgi-derived secretory granules 200-300 nm in diameter, and large electron dense bodies 500-1,000 nm in diameter. The secretory granules exist throughout the cytoplasm above the nuclear region and are occasionally released by exocytosis from the syncytiotrophoblast. On the other hand, the large electron dense granules seem to be cytolysosomes in nature and are involved in uptake and storage of hCG.

Protein-secretory patterns of normal and abnormal human placentas with special reference to human chorionic gonadotropin.

[35S]Methionine-labeled protein-secretory patterns resolved by two-dimensional polyacrylamide gel electrophoresis in abnormal hydatidiform-mole placentas were compared with those in normal full-term placentas with special reference to human chorionic gonadotropin (hCG) by means of immunoblotting and immunoelectron-microscopic techniques. Although basic protein-secretory patterns of both placentas were similar to each other, four polypeptide spots appeared and one spot disappeared in the hydatidiform-mole samples. Among four newly synthesized and secreted spots, three were immunoreacted with anti-hCG serum by an immunoblotting experiment. Ultrastructural localization of hCG showed that the labeling intensity of anti-hCG serum in hydatidiform-mole placentas was much heavier than that in full-terms ones. Particularly, the Golgi apparatus, middle-sized granules and large bodies were highly immunoreactive. The present study reveals that hydatidiform-mole placentas have different protein-secretory functions especially in hCG synthesis and secretion from those of normal pregnancy.

Application of gene amplification by polymerase chain reaction to genetic analysis of molar mitochondrial DNA: the detection of anuclear empty ovum as the cause of complete mole.

To investigate the pathogenesis of complete hydatidiform mole (complete mole), we employed a newly developed gene amplification method by polymerase chain reaction (PCR) for the restriction fragment length polymorphism (RFLPs) analysis of extranuclear DNA (mitochondrial DNA) of complete mole. Whole cellular DNA was extracted from six molar tissues and from peripheral blood mononuclear cells of parents. Two hyperpolymorphic regions of mitochondrial DNA, a 1.5-kb-long fragment and a 1.9-kb-long fragment, were selectively amplified from the extracted DNA by the PCR method. The amplification products amounted to over 10 micrograms after 30 cycles of PCR. The PCR products were digested with endonucleases (HaeIII, HinfI, AluI, and TaqI) and then electrophoresed on agarose gel. The electrophoretic pattern of digested DNA showed that the RFLPs of molar mitochondrial DNA coincided with those of the patient, indicating that the mitochondrial DNA of complete mole was inherited from the ovum. As it has been identified that the intranuclear DNA of complete mole is transmitted only from the spermatozoa, our results verify that complete mole results from the fertilization of an anuclear "empty" ovum with normal sperm at the molecular level.

Hydatidiform mole: an ultrastructural analysis of syncytiotrophoblast surface organization.

The scanning ultrastructural examination of a series of 31 hydatidiform mole and 12 healthy placental specimens of similar gestational age has revealed a variety of surface architectures more common in molar tissue. Characteristic paddle-shaped sprouts, ridging of the syncytial maternal oriented surface and microgibbosities are described. These structures are explicable in terms of organellar hyperplasia of cortical cytoskeletal elements found in healthy tissue. Specific morphological evidence of involvement of these elements in a condition where aberrant growth control leads to the characteristic trophoblastic hyperplasia is a further indication that cytoskeletal elements may mediate transformation. An increase in resolution obtained over previous scanning electron microscope studies has allowed the description of detailed features such as 'caveolar collars' on the maternal oriented healthy and molar trophoblast surfaces. These observations are of relevance to understanding the mechanisms of several cell physiological processes, including transepithelial transport. New observations of a reticular organization in the surface layer of molar trophoblast indicate that a syncytioskeletal layer, with organization resembling that previously described in healthy chorionic villi, is also present in molar villi.

The behavior of heterochromatin in mouse and human nuclei.

The arrangement of heterochromatin in various human and mouse nuclei has been analyzed with C banding. In most nuclei of 7-day mouse trophoblast, the heterochromatin consists of twin dots, or bigger clumps, apparently attached to the nuclear membrane. This finding agrees with the observation that most of these nuclei, which range from diploid to highly polyploid, show endomitotic stages. No polarization of heterochromatin in a Rabl orientation is seen in the trophoblast nuclei. Neither is a Rabl orientation found in the interphases of cultured human lymphocytes or fibroblasts. From their telophase arrangement, the chromosomes have obviously spread rapidly around the nuclear membrane. In many of the giant mouse trophoblast cells in vivo and in vitro, heterochromatin is apparently underreplicated. The same is true of giant cells in human hydatidiform moles and cervical cancer. Of the 82 cervical cancers analyzed, 46 showed chromocenters, and each tumor was characterized by its own pattern of heterochromatin.

Direct chromosomal preparation for studying hydatidiform moles.

This report describes a simple direct method to obtain chromosomes from hydatidiform moles. Of 24 moles, 20 have been successfully karyotyped by this method. Of the 20 cases, 14 were complete and six were partial. The karyotype of complete moles was invariably diploid. Three of the partial moles were triploid (69,XXX), but three showed diploid/tetraploid mosaicism.

Hydatidiform mole: cytogenetic marker analysis in twin gestation. Report of two cases.

A hydatidiform mole associated with a fetus proved to be the result of twin gestation. On microscopic examination of the placenta the case was classified as a partial hydatidiform mole. Chromosomal markers were, however, consistent with a normal conception and a mole of diploid androgenetic origin. Chromosome analysis of a morphologic complete molar specimen yielded two cell lines, one consistent with a normal conception and one with diploid androgenesis. Twinning in molar specimens must therefore be considered, regardless of macroscopic appearance. The prenatal diagnosis of a coexisting fetus and molar placenta poses a real clinical problem; analyses must distinguish between a partial mole plus a triploid fetus and a normal fetus occurring with a partial or a complete mole. The distinction is important for decisions made during pregnancy and may be of prognostic significance after termination. The usefulness of chromosome marker analysis in distinguishing between the various origins is pointed out, and it is suggested that twin pregnancy with hydatidiform mole is more frequent than its description in the literature would suggest.

Identification of triploidy by DA/DAPI staining of trophoblastic interphase nuclei.

Combined staining of non-cultured interphase nuclei from hydatidiform moles with distamycin A and DAPI resulted in the presence of specific stained interphase bodies. A mean number of six interphase bodies was observed in moles with a diploid karyotype and a mean number of nine interphase bodies was observed in triploid moles. It is concluded that the interphase bodies are primarily due to specific staining of the large heterochromatic areas on chromosomes 1, 9 and 16. The method may permit a rapid measurement of the ploidy of non-cultured hydatidiform moles and identify partial, triploid moles.

Localization of transferrin receptor in the chorionic villi of complete molar pregnancy.

Transferrin receptors have been identified on the villous trophoblast of normal chorionic villi by immunohistochemical techniques. The current study investigated the expression of transferrin receptors on molar chorionic villi by using a monoclonal antibody in immunofluorescence assays. The villous trophoblast of molar chorionic villi was brightly positive for transferrin receptor in the immunofluorescence assay. The presence of transferrin receptors on molar villous trophoblast may influence the immunologic relationship between molar and host tissues.

Mechanisms of growth in hydatidiform moles.

Nuclear morphology and DNA synthesis were analyzed to determine the mechanism through which hydatidiform moles proliferate. Hydatidiform moles are characterized by a great variation in nuclear morphology and size. There are cells with small nuclei of variable size that have chromocenters and Barr bodies which do not undergo DNA synthesis or mitosis, as well as cells in the diploid range that have evenly stained nuclei that display numerous mitoses and a high proportion of interphase nuclei in the process of DNA synthesis. Nuclei in the medium range show classical endomitotic stages. Endomitotic nuclei in endometaphase do not label with tritiated thymidine; endoanaphase nuclei may have one or a few chromosomes labeled, and endotelophase nuclei are heavily labeled. Nuclei that are evenly stained and are in the medium- to giant-size range label differently, depending upon their size. Many of the medium-sized nuclei are labeled, indicating DNA synthesis; the large nuclei are rarely labeled, and the giant nuclei are never labeled. The growth of a hydatidiform mole appears to be the result of normal mitosis and cytokinesis, as well as polyploidization and accompanying cell enlargement achieved through endomitosis and endoreduplication.

New concepts and questions in gestational trophoblastic disease.

Hydatidiform moles can be divided into two distinct syndromes: partial, or transitional, and classic. Both classic and partial moles appear to result from abnormal fertilization but differ in the type of abnormal fertilization, karyotype, histology, epidemiology and malignant potential. Using chromosomal banding polymorphism, classic hydatidiform moles have been shown to be androgenetic in origin, developing from a sperm with the egg nucleus either absent or inactivated. No maternal chromosomes are transmitted to the classic mole. Studies using HLA and enzyme heterozygosity have suggested that fertilization occurs by a haploid sperm with duplication of its chromosomes and without cell division, giving the 46XX karyotype found in classic moles. About 4% of classic moles are 46XY and are also androgenetic but result from dispermic fertilization. The partial mole consists of hydropic villi, but some normal villi also are present. An embryo, cord and fetal membranes generally can also be found, and the karyotype frequently is aneuploid (usually triploid) and not the 46XX or 46XY of the classic mole. In contrast to the classic mole, the partial mole has a maternal chromosomal contribution. Preliminary data suggest that many partial moles arise from dispermic fertilization, with participation of the maternal genome giving a triploid karyotype. The malignant potential of the partial mole is still controversial, but preliminary data indicate that 2.1% of partial moles require treatment as compared to 10% of classic moles.

Microgibbosities in hydatidiform mole.

Ultrastructural analysis of the surface of the syncytial absorptive epithelium of the aberrant form of human placenta known as hydatidiform mole reveals modifications of the microvillous apical surface. We have described these features as microgibbosities. They are apparently groups or rings of microvilli greater than the usual length in the surrounding cytoplasm. The existence of such microvilli and the probable consequences for the control of the dynamics of microvillar cytoskeletal elements should be accommodated by future theories of microvillar biogenesis.