Ultrastructure of the early human feto-maternal interface co-cultured in vitro

BACKGROUND: The study was designed to investigate the ultrastructural features of the early human feto-maternal interface when generated by in-vitro co-culture, and compare these with findings reported previously from human pregnancies. METHODS: Placental villi and decidua parietalis tissues from 8-12 week pregnancies were co-cultured in vitro over a 4-day period. The co-incubations were ended at 24 h intervals and processed for electron microscopical studies, and for immunocytochemistry using anti-cytokeratin antibody (CAM 5.2) for trophoblast. RESULTS: Loss of the syncytium at points of contact with the decidual stroma, cytotrophoblast column formation, differentiation and invasion of extravillous trophoblast (EVT) cells into the decidual stroma over the 4-day period of co-culture were observed. Cellular components, such as actin filaments, microtubules, glycogen granules and lamellipodic processes found in EVT cells were consistent with active cellular locomotion. CONCLUSIONS: These ultrastructural studies emphasize the usefulness of this model in investigating the formation of the feto-maternal interface of human pregnancy. The recruitment of cytotrophoblast to the syncytium by a process involving fusion of the intervening plasma membranes, and the migration of EVT cells causing little or no damage to the surrounding decidual cells, resemble in-vivo data.     

An in vitro model for the study of human implantation

Citation Holmberg JC, Haddad S, Wünsche V, Yang Y, Aldo PB, Gnainsky Y, Granot I, Dekel N, Mor G. An In vitro model for the study of human implantation. Am J Reprod Immunol 2012; 67: 169–178 Problem Implantation remains the rate‐limiting step for the success of in vitro fertilization. Appropriate models to study the molecular aspects of human implantation are necessary in order to improve fertility. Methods First trimester trophoblast cells are differentiated into blastocyst‐like spheroids (BLS) by culturing them in low attachment plates. Immortalized human endometrial stromal cells and epithelial cells (ECC‐1) were stably transfected with GFP or tdTomato. Co‐culture experiments were monitored using Volocity imaging analysis system. Results This method demonstrates attachment and invasion of BLS, formed by trophoblast cells, into stromal cells, but not to uterine epithelial cells. Conclusion We have developed an in vitro model of uterine implantation. The manipulation of this system allows for dual color monitoring of the cells over time. Additionally, specific compounds can be added to the culture media to test how this may affect implantation and invasion. This model is a helpful tool in understanding the complexity of human implantation.     

Ultrastructure of human Leydig cells at early gonadal embryogenesis

The ultrastructure of human Leydig cells at different stages of the testicular prenatal development is described by means of transmission and scanning electron microscopy. Between 5 and 7 weeks of gestation (w.g.) the interstitial tissue of the gonad is filled with small undifferentiated mesenchymal cells, migrating primordial germ cells and blood vessels. When the embryo is 7 to 8 weeks-old Leydig cells (LC) appear in basically two morphological patterns, light and dark cells. Their most significative feature is the development of the smooth endoplasmic reticulum (SER) as a dense tubulo-vesicular network and the presence of numerous pleomorphic mitochondria with mainly lamellar cristae. At 14 and 16 w.g. the testicular interstitium reaches the maximum development; the cytoplasm of the LC shows a widespread network of anastomosing tubules of the SER and mitochondria with tubular cristae. Fetal LC show a partial cell coat, lack the crystals of Reinke, have few lipid droplets and show no signs of massive cell degeneration, at least until 16 w.g. These ultrastructural modifications in fetal LC are in accordance with the changes in both steroidogenic activity and hCG levels reported by the literature to occur at this stage of development. Junctional complexes were often observed among LC from 7 to 8 w.g. onwards.     

A model for implantation of the human blastocyst and early placentation

We describe here a molecular model of blastocyst implantationwhich is based on two assumptions: (i) that implantation ofthe human blastocyst into the endometrium is a process whichis very similar to tumour invasion of a host tissue; and (ii)that the cytotrophoblastic cells of a first trimester pregnancyretain almost all the properties of the trophectodermal cellsof the blastocyst and can thus be used as surrogates to studythe implantation process in vitro. Our model considers thatthe trophectodermal cells, once they reach the endometrial basementmembrane, express integrins (6ß4) which anchor theminto the basement membrane and induce their secretion of gelatinases.These proteases digest the basement membrane, allowing the embryoto make contact with the endometrial extracellular matrix (ECM).Integrins (5ß1) anchor the embryo into the ECM andinduce its secretion of collagenases. These enzymes digest theECM, allowing the embryo to burrow into the endometrium. Thisprocess is under the paracrine control of endometrial cytokinesand ECM glycoproteins.     

Presence of uterine pinopodes at the embryo-endometrial interface during human implantation in vitro

In order to study changes occurring on the surfaces of human endometrial epithelial cells in the presence of an implanted blastocyst, we used scanning electron microscopy for investigation of five endometrial biopsies and three human implantation sites obtained in vitro. All specimens showed areas with endometrial pinopodes, separated by cells displaying microvilli or cilia at the apical surface. Pinopode formation was more pronounced in endometrial biopsies than in cell cultures. All blastocysts adhered to pinopode presenting cells. Endometrial surface changes were not seen around the blastocysts. The results of this study demonstrate that cultured endometrial epithelial cells are capable of pinopode formation. Furthermore, endometrial epithelial pinopodes, generally considered as a marker of endometrial receptivity, seem to be directly involved in the adhesion of the blastocyst to the endometrial surface.     

Ultrastructure of the invasion of human hair in vitro by the keratinophilic fungus Microsporum gypseum.

The pattern of invasion of human hair in vitro by the dermatophyte Microsporum gypseum was studied by transmission and scanning electron microscopy. Mycelia that invaded the hair cortex through the edge of cuticles showed a flattened "frond" growth in contrast to the filamentous form seen on ordinary laboratory media. The frond cells were characterized by the presence of vesicles formed by invaginations of plasmalemma, and lomasomes were prominent in the region adjacent to the hard keratinized tissue of the hair cortex being degraded as well. The initial perforating organ, which originated from the frond mycelium, appeared as an enlarged spherical cell which integrated with the laterally branched hyphae, as revealed by analysis of a three-dimensional model reconstructed from a series of sections. The fully developed perforating organ consisted of a column of wide and short cells which penetrated perpendicularly through the hair cortex. Through the medulla the filamentous hyphae had grown profusely in a longitudinal direction. Our studies confirm earlier light microscope observations and provide new ultrastructural details on the development of the eroding frond and the perforating organ.     

The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation

In a human in-vitro fertilization (IVF) programme, the effect of co- culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.      

Ultrastructure of the cortex in the human egg

Two distinct types of cortical granules were discerned in thehuman oocyte. The first type, Gl granules, had a mean diameterof 350 nm, contained a uniformly compacted, electron-dense substance;these granules were probably synthesized even before the oocytewas aspirated from mature follicles and their contents werenever found to be secreted in any of the preovulatory (immature),unfertilized and fertilized eggs examined. The G2 granules measured450 nm (mean diameter), had a granular content and were foundto be synthesized and secreted at all the stages of egg developmentstudied. EndocytosLs was also evident in the unfertilized andfertilized eggs. The secretion of the Gl cortical granules evenbefore fertilization suggests that they may have an additionalrole, rather than merely contributing to the blockade of polyspermy.     

Ultrastructure of early changes in circumscribed cryonecrosis of the liver

Introduction: The changes occurring during the initial phase of a circumscribed cryonecrosis of the liver were systematically observed.Material and Methods: Circumscribed necroses were produced in the liver with a copper cryoprobe (Grünenthal, Model H 29, Φ 3,2 mm, operating at ?196°C), applied for 30 sec. For light- and electronmicroscopic investiagtions, tissue samples from the frozen area and its surroundings were taken immediately after freezing and at certain time intervals until 24 hours later.Results and Discussion: No important changes were observed immediately after freezing. After 2 to 3 minutes, the frozen tissue had thawed. Considerable stasis occurred within the sinusoids and veins of the affected area. After 2 minutes, the sinusoidal endothelial cells showed a shift towards the lumen and ruptures of their plasma membranes; after 30 minutes, these cells were almost completely fragmented and surrounded by fibrin precipitates. After 30 minutes, hepatocytes showed “optically empty” vacuoles in the cytoplam, swelling of mitochondria (matrix type), disorganization, fragmentation, vesiculation and degranulation of the rough endoplasmic reticulum and pyknosis and karyorrhexis of the nuclei. After 2 hours, the sinusoidal endothelial cells and the hepatocytes bore marks of irreversible lesions and necrosis. At the periphery of the necrotic area there was a narrow zone of hepatocytes with well-preserved nuclear structure, some fatty change, and slight swelling of mitochondria. Simultaneously, a leukocytic infiltrate was present around the damaged tissue, from which leukocytes had penetrated into the necrotic area. After 18 and 24 hours, the peripheral zone with degenerative changes in the hepatocytes showed single cell necroses. The ultrastructural changes described are not specific in themselves; collectively, however — i.e. by the pattern of lesion — they are characteristic and differ from other lethal liver injuries, mainly by the very early necrosis of sinusoidal endothelial cells and the uniformity in degree and extent of the hepatocellular lesions. It is assumed that the most important pathogenetic factor is the formation of relatively large ice crystals within cells and the extracellular area due to recrystallization during thawing. Hypoxemia due to stasis appears to be of minor importance.     

Chromosome studies on early human embryos fertilized in vitro

The majority of early spontaneous abortions carry a lethal chromosomalanomaly. While it is recognized that several factors would beresponsible for some IVF failures, it is important to determinethe contribution of chromosomal aberrations to the preimplantationloss of embryos produced in vitro. Chromosome analysis of embryosnot destined for replacement in the uterus could help to elucidatethis phenomenon of early embryonic loss. Fifty-five out of 239embryos fertilized in vitro were successfully karyotyped andamongst these the overall rate of diploidy was 25.5% in thisstudy, which mainly comprised rejected embryos. Embryos withoutcleavage had mostly a chromosomal defect (20/38) and only aminority (9/38) were unfertilized. Numerical abnormalities werefound in a total of 33/46 (71.7%) morphologically normal embryos.In contrast a diploid chromosomal complement was found in only11.1% (1/9) of morphologically abnormal embryos.     

Ultrastructure of early plexogenic pulmonary arteriopathy

A lung biopsy specimen from a young woman with the clinical features of primary pulmonary hypertension showed grade 2 plexogenic pulmonary arteriopathy. Electron microscopy revealed 'dark', electron-dense smooth muscle cells in the inner part of the media of muscular pulmonary arteries. Many of these transformed myocytes had migrated into the lumens of pulmonary arteries and arterioles which they occluded. This migration of smooth muscle cells was associated with a substantial increase in the number of pulmonary endocrine cells in the bronchioles containing bombesin and calcitonin.     

Ultrastructure of the early ovary and testis in pig embryos

Pig embryos aged 26–27 days were used for an ultrastructural study of the early ovary and testis. Sex was identified by both chromosomal analysis and gonadal histology, with consistent results. The gonads occupied their original site in the medial coelomic angles in both sexes. The female gonad was composed of three tissues: the surface epithelium, the gonadal blastema and the mesenchyme. The gonadal structure was similar to that seen earlier at the age of 24 days. At 26 days the testis had distinctly differentiated into four tissues. The new components were the testicular cords and the interstitium, both derived from the gonadal blastema. The testicular cords resembled anastomosing sheets more than cords. The ultrastructure of the tissues and their cell types are described and compared to the previous indifferent stage at the age of 24 days. The cells of the surface epithelium, of the primitive cords, of the mesenchyme, and the primordial germ cells had an ultrastructure that was similar in both sexes. The sustentacular cells of the testicular cords resembled the primitive cord cells and the spermatogonia were similar to the primordial germ cells. No Leydig cells were present yet. The process of testicular differentiation is described on the basis of the present and a previous study, and a new hypothesis, based on the vascular organization, is presented.     

Ultrastructure of early cleavage stages and preimplantation in the rabbit

Summary Fertilized rabbit ova were studied in the period from 25 to 144 hr after insemination. Eggs were recovered by flushing the uterine horns and oviducts with Hank's solution. The cells were morphologically alike in the 24 and 48 hr ova. At 661/2 hr blastomeres were differentiated into an inner mass of cells with dense cytoplasm and an outer trophoblastic layer with less dense cytoplasm. Otherwise no morphological differences were seen. Whether the 661/2 hr ova were morulas or blastocysts is discussed. The 96 hr ova were clearly blastocysts. Inner cells and trophoblastic cells at this stage bad the same cytoplasmic density. Mitochondria were increased in number and crystal-like figures were present for the first time. In the 120 and 144 hr ova the cytoplasm of the trophoblastic cells was denser than that of the inner cells. Trophoblastic cells were characterized by their density, crystal-like figures, elongated mitochondria with transverse cristae and many single ribosomes and they were interconnected with well developed junctional complexes. In a few cases a continuity seemed to exist between trophoblastic cells and inner cells. The latter were characterized by cytoplasm of less density than that of the trophoblastic cells, rounded mitochondria and fewer ribosomes. The fine structure of the crystal-like figures, their possible origin and differentiation of the mitochondria are discussed.This investigation was supported by the U.S. Public Health Service NIH International Postdoctoral Fellowship No. 4 FO5 TW 1365-02.     

人子宫内膜蜕膜化间质细胞与囊胚体外共培养建立人胚胎着床模型

目的建立有效的体外人胚胎着床模型,为体外研究人胚胎着床过程提供条件。方法将人子宫内膜蜕膜化的间质细胞与囊胚共同培养,光镜下观察囊胚在细胞上的定位、粘附及侵入过程;免疫荧光法测定共同培养系统中的角蛋白,以确定着床模型的建立。结果与蜕膜细胞共培养5—10h后,囊胚开始黏附在细胞层上,48h后侵入蜕膜细胞间;共培养48h后,胚胎及周围的内膜细胞表达角蛋白阳性。结论囊胚与蜕膜化细胞共同培养,可以成功建立体外胚胎着床模型,更好地模拟体内着床时状态,是较理想的体外研究模型。