The arylsulphatases of chorionic villi: potential problems in the first-trimester diagnosis of metachromatic leucodystrophy and Maroteaux-Lamy disease

Three pregnancies at risk for late infantile metachromatic leucodystrophy have been monitored using chorionic villus biopsies. In the first of these a false negative diagnosis was made following assay of arylsulphatase A in villi. Subsequent studies have shown that this error was probably due to interference from another sulphatase in the villi, although the possibility that maternal contamination was also partly responsible could not be excluded. For reliable prenatal diagnosis of metachromatic leucodystrophy using chorionic villi it is advisable that studies with the nitrocatechol substrate are carried out on fractionated homogenates, or that the natural substrate is used. Problems may also occur when chorionic villi are used for assay of arylsulphatase B for first trimester diagnosis of Maroteaux-Lamy disease.     

诊断剂量超声波对绒毛影响的研究

本文采用诊断剂量超声波经腹部辐照宫内胚胎后，取绒毛进行生化、酶和遗传学研究，以探讨超声剂量对胚胎的安全性。结果表明：诊断剂量的超声波对绒毛染色体无致畸作用，亦不改变绒毛组织的酶代谢变化，但可引起超氧化物歧化酶（SOD）活性降低。     

First trimester prenatal diagnosis of lysosomal storage disease. Study of alpha-L-fucosidase isoenzyme patterns in fetal and maternal tissue

The normal range of activities of 6 lysosomal enzymes was determined in extracts of chorionic villi samples obtained by a rigid forceps in the first trimester of pregnancy. These activities were compared to those in villi obtained after abortion and in cultured amniotic fluid cells and fibroblasts. For five of the six enzymes tested, the data suggest that first trimester prenatal diagnosis should be possible and reliable. For the sixth enzyme, alpha-L-fucosidase, where on occasion very low activities were found, the results obtained on fresh chorionic villi have to be interpreted with extreme caution. Considerable lysosomal enzyme activities were also found in maternal decidua. Therefore, extreme care must be taken in the preparation of chorionic villi for prenatal diagnosis of lysosomal disorders since even small amounts of maternal tissue could lead to misdiagnosis. This study has allowed us to monitor 2 pregnancies at risk for lysosomal storage disease. Differences in the isoenzyme pattern of alpha-L-fucosidase were found in chorionic villi and maternal decidua. Although further studies are required, this observation could lead to the development of immuno-biochemical methods to evaluate the purity of chorionic villi used for prenatal diagnosis.     

The development of the baboon (Papio sp.) placenta during the fetal period of gestation

Baboon placentae ranging in age from 45 to 175 days were studied for progressive changes in their morphology. The baboon embryo implants superficially and develops a single, discoid, villous hemochorial placenta. The remainder of the chorion is membranous and a true decidua capsularis is not formed. A lobular structure is developed during the fetal period. At 45 days the amnion only partially fills the chorionic cavity but by 60 days has expanded and the two membranes are directly contiguous. Trophoblastic tissue is of two types: cytotrophoblast and syncytiotrophoblast. Cytotrophoblast is found primarily in the chorionic plate and cytotrophoblastic shell. The chorionic villi are originally composed of a double layer of trophoblast; cytotrophoblast internally and syncytiotrophoblast externally. The cellular layer gradually disappears so that by full-term the villous walls are formed by a single layer of syncytiotrophoblast. Hofbauer cells are common within the villous cores, diminishing in number toward term. Large amounts of collagenous connective tissue develop in the chorionic plate and in the villi, and fibrin and fibrinoid materials accumulate in the basal plate and anchoring villi. The endometrium is decidualized to its greatest extent by the beginning of the fetal period and undergoes only minor cytological alteration throughout the remainder of gestation.     

The fine structure of the placenta and chorionic vesicles of the bush baby,Galago crassicaudata

The fine structure of the placental villi and the chorionic vesicles of the bush baby, Galago crassicaudata, were examined. The placenta was of the diffuse epitheliochorial type. The trophoblastic epithelium of the placental villi consisted of cells joined by tight junctions and desmosomes. The apical borders of these cells interdigitated with corresponding processes on the uterine epithelial cells. The fetal capillaries indented the trophoblastic cells later in gestation, and the trophoblast over the capillaries was correspondingly thinner with advancing gestation. The trophoblast of the villi was characterized by numerous lipid droplets and moderate amounts of granular endoplasmic reticulum (ER). The apical cytoplasm often had many mitochondria as well as small electron-dense vesicles and tubules. Uterine epithelial cells contained agranular ER and a well-developed Golgi apparatus. The apical cytoplasm contained numerous granules often with an electron-dense content. The uterine epithelial cells appeared to be secretory, contributing to the electron-dense material found between the fetal and maternal cells. Fetal capillaries were of the continuous type, whereas occasional fenestrae were observed in maternal capillaries. Recesses in the trophoblastic epithelium occurred at the tips, sides, and bases of the villi. These areas had taller trophoblastic cells that appeared to be more active in phagocytosing uterine content. The chorionic vesicles are invaginations of the chorion opposite the mouths of uterine glands. A layer of columnar trophoblastic cells covered the villi of the vesicles. These cells had abundant vacuoles and coated pits and vesicles. The mesodermal component of the chorionic vesicles included a capillary network and a layer of smooth muscle cells in the wall of the vesicle. It was concluded that the trophoblast of the placental villi is engaged in both hemotrophic and histotrophic nourishment of the embryo. The specialized chorionic vesicles are particularly important in providing histotrophic nutrients to the embryo, especially the secretions of the uterine glands. The similarity of the chorionic vesicles to the areolae of other species suggests they may be the pathway by which iron is transported from mother to fetus, perhaps by receptor-mediated endocytosis of macromolecules derived from uterine gland secretions.     

几种孕妇禁忌中药对早孕绒毛的组织化学及超微结构的影响

本文从形态学及组织化学角度,观察了孕妇禁忌中药商陆、附子及蒲黄对器官培养的早孕绒毛的影响。结果表明:商陆对绒毛的损伤最为严重,光镜观察可见绒毛萎缩变小,滋养层与间质分离,细胞核固缩、溶解、碎裂;胞质减少且嗜酸性增强;电镜下可见,核染色质及核仁溶解,核膜皱缩不规则;各种细胞器损伤较为严重,形态结构难以分辨;胞浆中可见大量的自噬泡和髓样体;组织化学显示,ACP反应增强;ALP反应减弱,3β-HSD、SDH及TPPase反应极弱,甚至阴性。附子对绒毛也有一定程度的损伤,但较商陆为轻。蒲黄对绒毛未见明显损害。     

Anaphase lag as the most likely mechanism for monosomy X in direct cytotrophoblasts but not in mesenchymal core cells from the same villi.

A 36 year old white female was referred for chorionic villus sampling for advanced maternal age. Direct (cytotrophoblast) preparations of chorionic villi were 45,X, but cultured mesenchymal core cells from the same villi were 46,XX. Study of embryonic and extraembryonic tissues showed the aneuploidy to be limited to cytotrophoblasts from specific placental sites. In aggregate, the cytogenetic findings can best be explained by anaphase lag during development of the cytotrophoblast, suggesting that this cytological mechanism and not non-disjunction is responsible for the common occurrence of monosomy X in villi.     

Trisomy/tetrasomy 21 mosaicism in CVS: interpretation of cytogenetic discrepancies between placental and fetal chromosome complements

Trisomy/tetrasomy 21 mosaicism was found in chorionic villi (semidirect preparation) obtained from a 40 year old pregnant woman. Since both cell lines were abnormal, the couple elected for pregnancy termination. Placenta and fetal tissue samples were obtained for cytogenetic study. Long term cultured villi showed a non-mosaic trisomy 21 karyotype, while other tissues showed either a normal karyotype or normal/trisomy21 mosaicism. These discrepancies could be explained by a modified "bottle neck" embryogenic model with a trisomic zygote and a non-disjunction event taking place in one of the first divisions. Our case emphasises the need for confirmatory studies in other tissues when mosaicism is encountered in chorionic villi, even if all cell lines are abnormal.     

The size of chorionic villi in the placenta of diabetics (author's transl)

25 placentas in maternal diabetes and 25 placentas from normal pregnancies (39- 41 th week) were investigated by histoplanimetry. In each placenta we have measured in 2 areas out of the center and the periphery the plain of villi. The total sum of villi measured comes to 10302. The investigation has shown, in the average the plain of villi in diabetic placentas is higher than in normal placentas significantly. Besides, the number of small villi decreases and the number of villi with a plain over the average rises in cases of diabetes nellitus. These differences in the distribution of the chorionic ville in some classes of size between placentas of diabetic and such of normal pregnancies are significantly too.     

Glyceryl trinitrate inhibits hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of the human placenta: therapeutic implications for preeclampsia

Damage of the placenta resulting from ischemia-reperfusion is important to the pathophysiology of preeclampsia. Here we investigated whether low concentrations of glyceryl trinitrate (GTN), a nitric oxide mimetic with anti-apoptotic properties, inhibit hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of chorionic villous explants from human placentas. Compared with villi analyzed immediately after delivery or maintained under normoxic conditions, villi exposed to a 6-hour cycle of hypoxia/reoxygenation exhibited greater numbers of syncytiotrophoblasts with terminal dUTP nick-end labeling (TUNEL)-positive nuclei in the syncytiotrophoblast. This increased number of TUNEL-positive nuclei was paralleled by higher levels of 4-hydroxynonenal (marker of lipid peroxidation), nitrotyrosine residues, and active caspase-3 and polyADP-ribose polymerase expression. Morphological analysis of explants exposed to hypoxia/reoxygenation revealed apoptotic and aponecrotic features similar to those of chorionic villi from preeclamptic pregnancies. Treatment with GTN during the hy-poxia/reoxygenation cycle blocked the increases in the number of TUNEL-positive nuclei and in the levels of 4-hydroxynonenal, nitrotyrosine, and active caspase-3. Incubation with GTN also attenuated the hypoxia/reoxygenation-induced polyADP-ribose polymerase expression and the apoptotic and aponecrotic morphological alterations. These results suggest that small concentrations of nitric oxide protect chorionic villi from hypoxia/reoxygenation-induced damage and provide a rationale for the use of low doses of nitric oxide mimetics in the treatment and/or prevention of preeclampsia.     

The CpG island of the FMR-1 gene is methylated differently among embryonic tissues: implication for prenatal diagnosis

We studied the methylation status of the CpG island of the FMR-1(fragile X syndrome) gene to recognize the possibility of itsprenatal diagnosis with early pregnant subjects. Southern hybridizationusing EcoRI/BssHII restriction enzymes double digestion wasperformed in the brain and chorionic villi of 8th week embryos,and the placenta and cord blood of newborns. No methylationof the FMR-1 gene occurred in both of the tissues examined inmales, while 50% of the cells in females were methylated inboth the brain and the cord blood, indicating that methylationoccurs with inactivation of the X-chromosome in accordance withthe literature. However, there was no methylation in eitherthe chorionic villi or placenta in female as well as in males.Some extra-embryonic tissues such as the chorionic villi andthe placenta escape X-chromosome FMR-1 gene inactivation andit can be the exception in the lyonization. To assess the methylationstatus in prenatal diagnosis, precautions are needed and theyare not suitable for prenatal diagnosis.     

Analysis of full fragile X mutations in fetal tissues and monozygotic twins indicate that abnormal methylation and somatic heterogeneity are established early in development.

The fragile X syndrome, the most common cause of inherited mental retardation, is characterized by unique genetic mechanisms, which include amplification of a CGG repeat and abnormal DNA methylation. We have proposed that 2 main types of mutations exist. Premutations do not cause mental retardation, and are characterized by an elongation of 70 to 500 bp, with little or no somatic heterogeneity and without abnormal methylation. Full mutations are associated with high risk of mental retardation, and consist of an amplification of 600 bp or more, with often extensive somatic heterogeneity, and with abnormal DNA methylation. To analyze whether the latter pattern is already established during fetal life, we have studied chorionic villi from 10 fetuses with a full mutation. In some cases we have compared them to corresponding fetal tissues. Our results indicate that somatic heterogeneity of the full mutation is established during (and possibly limited to) the very early stages of embryogenesis. This is supported by the extraordinary concordance in mutation patterns found in 2 sets of monozygotic twins (9 and 30 years old). While the methylation pattern specific of the inactive X chromosome appears rarely present on chorionic villi of normal females, the abnormal methylation characteristic of the full mutation was present in 8 of 9 male or female chorionic villi analyzed. This suggests that the methylation mechanisms responsible for establishing the inactive X chromosome pattern and the full mutation pattern are, at least in part, distinct. Our results validate the analysis of chorionic villi for direct prenatal diagnosis of the fragile X syndrome.     

Pregnancy with concomitant chorangioma and placental vascular malformation with mesenchymal hyperplasia

We present two pregnancies associated with normal live births and the unusual concomitance of chorangioma and placental vascular malformation with mesenchymal hyperplasia. The enlarged placenta had the characteristic findings of chorangioma, dilated and varicose chorionic vessels and multiple vesicle-like villi containing hyaluronic acid. The vesicle-like villi showed diploid cellular DNA contents. Molecular genetic analysis using the polymerase chain reaction amplification of polymorphic microsatellite markers confirmed genetic identity among the chorangioma, the vesicle-like villi and the fetus. Both pregnancies were complicated by polyhydramnios, pre-term labour and prematurity. One neonate suffered from anaemia and thrombocytopenia. Another neonate suffered from haemangiomatosis. Our cases demonstrate that concomitant chorangioma and placental mesenchymal hyperplasia are genetically identical to the fetus and can coexist with a normal viable fetus. Since haemangiomas, chorangiomas, chorionic vessels and villi mesenchymal cells are all derived from the mesoderm, a combination of fetal haemangiomas, placental vascular malformation, chorangiomas and placental mesenchymal hyperplasia may represent a mixed form of congenital malformation of the mesoderm.      

Developmental changes in rhesus monkey placental villi and cell columns

Summary Developing rhesus monkey placentas were analyzed by scanning electron microscopy with special attention directed toward defining stages in the development of the villus branches. The initial phase in formation of villi was the conversion of reticulated trabeculae of syncytial trophoblast into chorionic villi by growth and proliferation of cell columns of cytotrophoblast. These villi were stout and unbranched. The second phase of development appeared to be the longitudinal splitting of the villi and cell columns to form groups of parallel branches but there was a common insertion of these into the basal plate. The third phase in formation of villi, which appeared to begin at about the same time as the longitudinal splitting occurred, was the outgrowth of largediameter side branches in a zone nearer the chorionic plate. At about 38–40 days of gestation the next stage in villus formation occurred, characterized by the emergence of numerous, small syncytial sprouts. Continued proliferation of villi at later stages of gestation resulted in a decreased diameter of the terminal villi and an increasing complexity in the course of fetal capillaries.     

Immunohistochemical demonstration of placental hormones in the diagnosis of uterine versus ectopic pregnancy

The authors find that immunohistochemical demonstration of placental hormones in endometrium is useful in the identification of trophoblast independent of the presence of chorionic villi. Human chorionic gonadotropin (HCG) and human placental lactogen (HPL) are markers for trophoblastic cells. The markers were studied in 21 cases of gestational endometrium without villi in which the clinical differential diagnosis was ectopic pregnancy versus missed or incomplete uterine abortion. Trophoblastic cells were identified in four cases by routine microscopy and in an additional seven cases using the markers. In none of these cases was there a subsequent demonstration of ectopic pregnancy. In six of the ten negative cases, ectopic pregnancies subsequently were removed. Thus, the use of these hormone markers in endometrial specimens increases precision in the diagnosis of uterine versus ectopic pregnancy.     

A reproducible modified method for direct preparation of chorionic villi cytogenetic analysis

Chorionic villi are finger like projections, which surrounds the embryonic sac in early pregnancy and will later form the placenta. The outer layer of the chorionic villi consists of trophoblast cells. These cells are actively dividing and can be used for direct preparation or short-term culture for prenatal diagnosis of chromosomal abnormalities. We herein describe a 100% reproducible modified technique for one day direct culturing (direct prepara-tion) of trophoblast cells. Direct preparation of chorionic villi provides the physician with additional diagnostic information during prenatal diagnosis and genetic counseling. Direct preparation also eliminates the risk of maternal cell contamination. This method was 100% reproducible provided all the precautions mentioned herewith were taken into consideration and can be completed in 45 minutes upon arrival of the samples in the laboratories.     

Transabdominal chorionic villus sampling in a multiple pregnancy at risk of argininosuccinic aciduria: a case report

We report on a 32-year-old G3P2 woman at increased risk of argininosuccinic aciduria in her offspring. In her first infant, the disease was diagnosed at age 3 months. In the second pregnancy, the disorder was excluded following incorporation of radio-labeled 14C-citrulline in intact chorionic villi obtained through transcervical chorionic villus sampling at 10 weeks gestation. Twins were diagnosed in the third pregnancy. Separate transabdominal chorionic villus sampling of both fetuses was carried out at 10 weeks gestation. Chromosome analysis demonstrated a normal male and female karyotype. Incorporation of radio-labeled 14C-citrulline in intact villi was deficient in both villus samples, establishing the diagnosis of argininosuccinic aciduria in both fetuses. The present results confirm the reliability of direct analysis of villi for the diagnosis of argininosuccinic aciduria.     

细胞凋亡基因及相关蛋白在早期自然流产胎盘绒毛和蜕膜组织中的表达

目的探讨自然流产和正常人流患者胎盘绒毛和蜕膜组织细胞增殖与凋亡的变化规律,及相关细胞因子在胎盘组织生长发育中的表达意义。方法应用免疫组织化学、末端脱氧核糖核酸转移酶介导的缺口末端标记法（terminal de-oxynucleotidy transferase mediated dUTP-biotin nick end labing,TUNEL）检测第40-90天自然流产和正常人流患者胎盘组织中细胞凋亡的表达状况。结果第40-90天的正常人流患者的绒毛和蜕膜组织细胞的增殖占主导地位,大部分细胞增殖活跃,细胞的增殖与凋亡始终处于一种动态平衡;而自然流产患者的绒毛和蜕膜组织中,凋亡细胞的比例明显增大,细胞增殖与凋亡的动态平衡被打破。结论妊娠妇女胎盘组织内细胞凋亡比例增高,细胞增殖与凋亡的动态平衡被打破是自然流产发生的基础。     

A reproducible modified method for direct preparation of chorionic villi cytogenetic analysis.

Chorionic villi are finger like projections, which surrounds the embryonic sac in early pregnancy and will later form the placenta. The outer layer of the chorionic villi consists of trophoblast cells. These cells are actively dividing and can be used for direct preparation or short-term culture for prenatal diagnosis of chromosomal abnormalities. We herein describe a 100% reproducible modified technique for one day direct culturing (direct preparation) of trophoblast cells. Direct preparation of chorionic villi provides the physician with additional diagnostic information during prenatal diagnosis and genetic counseling. Direct preparation also eliminates the risk of maternal cell contamination. This method was 100% reproducible provided all the precautions mentioned herewith were taken into consideration and can be completed in 45 minutes upon arrival of the samples in the laboratories.