[The intensity of expression of matrix metalloproteinases type 2 and type 9 by invasive trophoblast cells in uncomplicated pregnancy and preeclampsia].

OBJECTIVE: To estimate the intensity of immunoexpression of matrix metalloproteinases type 2 and type 9 (MMP-2 and MMP-9) during cytotrophoblast invasion (CTI) in the uteroplacental region. MATERIALS AND METHODS: There were 6 groups: 1) medical abortions at 7 to 8 weeks' gestation; 2) amputated uteri at 9 to 12 weeks; 3) late medical abortions at 18 to 24 weeks' gestation and amputated uteri at 25 weeks; 4) amputated uteri and biopsies of the placental bed, performed during cesarean section at 38-40 weeks. Two more groups included biopsies from women with preeclampsia: biopsy at 28-33 weeks (Group 5) and 34-38 weeks (Group 6). A total of 49 women were examined. An immunohistochemical method performed using the standard procedure revealed the expression of MMP-2, MMP-9, and cytokeratin in three invasive elements: interstitial cytotrophoblast (IC), multinucleated giant cells (MGCs), and intravascular cytotrophoblast (IVC). RESULTS: The expression of MMP-2 is intensive in all the invasive cells in the first and second trimesters and then decreases in IC and MGCs. The immunoexpression of MMP-9 with its minimal values in the first trimester successively increases in IVC and IC by a full-term gestation. CONCLUSION: MMP-2 hyperproduction in IVC and IC is of the greatest value for the first wave of CTI and high MMP-9 immunoexpression in all the invasive cells is more important for the second wave. In cases of preeclampsia, the least expression of MMP-2 and MMP-9 is recorded in IC and MGCs in the complete absence of IVC.

[Maternal-placental interactions and fetal programming]. / Mütterlich-plazentare Interaktionen und fetale Programmierung.

Pregnancy-related complications not only represent a risk for maternal and fetal morbidity and mortality, but are also a risk for several diseases later in life. Many epidemiological studies have shown clear associations between an adverse intrauterine environment and an increased risk of diabetes, hypertension, cardiovascular disease, depression, obesity, and other chronic diseases in the adult. Some of these syndromes could be prevented by avoiding adverse stimuli or insults including psychological stress during pregnancy, intake of drugs, insufficient diet and substandard working conditions. Hence, all of these stimuli have the potential to alter health later in life. The placenta plays a key role in regulating the nutrient supply to the fetus and producing hormones that control the fetal as well as the maternal metabolism. Thus, any factor or stimulus that alters the function of the hormone producing placental trophoblast will provoke critical alterations of placental function and hence could induce programming of the fetus. The factors that change placental development may interfere with nutrient and oxygen supply to the fetus. This may be achieved by a direct disturbance of the placental barrier or more indirectly by, e. g., disturbing trophoblast invasion. For both path-ways, the respective pathologies are known: while preeclampsia is caused by alterations of the villous trophoblast, intra-uterine growth restriction is caused by insufficient invasion of the extravillous trophoblast. In both cases the effect can be undernutrition and/or fetal hypoxia, both of which adversely affect organ development, especially of brain and heart. However, the mechanisms responsible for disturbances of trophoblast differentiation and function remain elusive.

[Morphofunctional analysis of the efficiency of keratoplasty in the treatment of keratomycosis]. / Morphofunktionelle Untersuchungen der Wirksamkeit der Keratoplastik bei der Behandlung von Keratomykosen.

BACKGROUND: Keratoplastic surgery is an efficient treatment for serious keratomycosis, therefore these should be applied more often. The keratomycosis (mycotical keratitis) is a rare, but severe illness of the eye, since it is discovered often in a late stage and is challenging to cure. In these cases the accurately timed keratoplasty is very helpful. In this morphofunctional analysis the procedure of keratoplastic treatment and the efficiency of treatment of the keratomycosis were evaluated. METHODS: From January until November 2010 eight patients with microbiologically evident mycoses were treated operatively and conservatively by surgeons. The removed tissues were fixated in buffered formalin and embedded in paraffin. About 0.5 µm thin paraffin sections were analysed histologically and histochemically and evaluated semiquantitatively. Postoperatively all patients have been treated antimycotically for 7 until 10 days. RESULTS: The most important step for keratoplastic treatment was to remove the affected tissue completely and to treat with radical surgery in all cases. In this process the cornea was replaced by 3-mm and 5-mm pieces of sclera. The histological analysis of the eye after evisceration showed a complete destruction under the epithelium caused by the connective tissue-cellular proliferation. Postoperatively the topical, systemic and antimycotic therapy was continued for 30 days. In three cases the cortisone therapy was used for three to six days after surgery. CONCLUSIONS: After successful keratoplastic treatment an improvement of the acuteness and either an improvement of vision about 60 to 70 percent of the transparent regeneration were observed in all cases. To avoid recurrences for therapeutically keratoplastic treatment, radical removal of the injured tissue is necessary. Suppression of inflammations inside the treated eye was accelerated by systemic and topical application of corticosteroids, which influenced the condition of the transplants in a positive way.

PP015. Differential expression of Nrf2 and VEGF in human placental beds from normal and pregnancies complicated with preeclampsia and IUGR.

INTRODUCTION: Impaired trophoblast invasion into the uteroplacental arteries is accompanied with an evidence of oxidative stress in the extravillous trophoblast in preeclampsia complicated with IUGR. OBJECTIVES: Preeclampsia is characterised by increased lipid oxidation and diminished antioxidant capacity; recently, we have shown that PE is associated with an increased expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) in villous cytotrophoblast. A possible relationship between the vascular endothelial growth factor (VEGF) and Nrf2 was established in vitro and the activation of Nrf2 pathway could lead to upregulation of VEGF synthesis through the induction of Nrf2-dependent Heme oxygenase-1 (HO-1). In this study the expression of Nrf2 and VEGF was determined in the interstitial and intramural extravillous trophoblast in normal pregnancies and those complicated by preeclampsia and intra-uterine growth restriction (IUGR). METHODS: Full-thickness uterine tissues were obtained from caesarean hysterectomies performed in 5 healthy normotensive women delivering term infants and from 5 women with severe early-onset preeclampsia and IUGR (29-34 week's gestation). The interstitial and intramural trophoblasts were studied by immunohistochemical analysis of paraffin sections stained with anti VEGF and anti Nrf2. RESULTS: Cases suffering from preeclampsia with IUGR were characterised by reduced invasion of extravillous trophoblast into uteroplacental arteries in the endometrial and myometrial segments. In addition, these cells showed an increased expression of Nrf2 in the pathological sections. The overexpression of Nrf2 in cases with preeclampsia was associated with restricted expression of VEGF in these cells compared to controls. CONCLUSION: Our data suggest that besides villous cytotrophoblast, also the extravillous trophoblast is a source of Nrf2-dependent genes. VEGF deficiency may cause higher oxidative stress in extravillous trophoblast in cases with preeclampsia with IUGR. The resulting reduced basal defence against oxidative stress and the higher vulnerability to oxidative damage may play a role in the limited trophoblast invasion into uteroplacental arteries in cases suffering from early onset preeclampsia and IUGR.

PP119. The role of the nuclear factor erythroid 2-related factor 2 (Nrf2) in preeclampsia.

INTRODUCTION: Preeclampsia is a multi-organ syndrome characterized by maternal endothelial damage, is an independent long-term risk factor for hypertension and cardiovascular disease. OBJECTIVES: In animal models the administration of the Vascular Endothelial Growth Factor (VEGF) could reverse the hypertensive signs accompanying this disease. In addition VEGF is implicated in placental oxidative stress during preeclampsia. One of the major cellular defence mechanisms against oxidative stress is the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, the activation of Nrf2 up regulates the HO-1/CO system. The principal aim of this work is to investigate whether the activation of Nrf2 raises VEGF levels by up regulation of CO release. METHODS: This study took place in vitro, the choriocarcinoma cell line BeWo cells and the primary human umbilical vein endothelial cells (HUVECs) were used to study the relationship between VEGF and an Nrf2 inducer Sulforaphane, a naturally occurring compound derived from broccoli. ELISA, Western blot assay and the Dual Luciferase Assay were both mainly applied for protein and VEGF activity analysis. RESULTS: It was found that activation of HO-1 expression via Nrf2/ARE pathway augmented the production of CO, which in turn up-regulated the gene expression of VEGF, and down regulated the production of the antiangiogenic protein, the VEGF antagonist sFlt-1. CONCLUSION: Nrf2 driven HO-1 expression elevates the levels of VEGF via CO production. In particular, activating of Nrf2 via sulforaphane, may have therapeutic potential in preeclampsia.

Caviomorph placentation as a model for trophoblast invasion.

The guinea pig and its relatives are promising candidates as animal models for studying trophoblast invasion. The origin, migration routes and kinetics of invasive trophoblast cells were examined in two caviomorph species. Histology and immunohistochemistry were done on placentas from 38 guinea pigs of days 20-47 and 13 degus of days 25-51 of gestation. BrdU was used as an in vivo marker for proliferation and for tracing of migration routes in the placenta; it was injected 24h to 15 days before collecting the material. In both species extravillous-like trophoblast cells are derived from proliferating stem cell aggregations in the subplacenta, which are comparable to the cell columns in humans. Migration routes and kinetics under in vivo conditions revealed a mean invasive depth of 300-350 microm/day and a mean life span of the extravillous-like trophoblast of 30 days. The patterns of trophoblast invasion in caviomorphs are analogous to the situation in humans, suggesting that these rodents are appropriate animal models for the study of the dynamics of trophoblast invasion.

Cytokeratin antibodies as differential markers of trophoblast and fetomaternal syncytial plaques in the sheep placentome.

The sheep placenta is an often used model in placental research. Uterine epithelium and trophoblast of this synepitheliochorial placenta form a complex, intensely interdigitating epithelial barrier separating maternal and fetal organisms. The close topographical relation and additionally the presence of hybrid syncytia formed by focal fusion of both epithelia hamper identification of the various cellular constituents. Therefore we aimed to find a specific immunohistochemical marker differentiating between the fetomaternal syncytial plaques and trophoblast. A monoclonal antibody directed against type II cytokeratins strongly stained unicellular trophoblast. The syncytial plaques were only weakly stained while binucleate trophoblast remained unstained. This antibody proved to be a useful tool for easy histological orientation in the sheep placenta. In combination with other antibodies in double immunohistochemistry it facilitates exact localization of antigens.

Aberrations of early trophoblast differentiation predispose to pregnancy failure: lessons from the anti-phospholipid syndrome.

OBJECTIVES: The epithelium of the human placenta comprises an inner cytotrophoblast (CT) which proliferates and fuses with the outer differentiated syncytiotrophoblast (ST). Turnover has been studied focussing on second and third trimester placentas but with a paucity of data describing the normal first trimester trophoblast. The aim of this study was to compare the nuclear CT:ST ratio in normal and pathological pregnancy and thus establish the relationship between cytotrophoblast and syncytiotrophoblast nuclear number during early gestation. METHODS: Archival first trimester material from placentas from healthy pregnancy and recurrent miscarriage (anti-phospholipid syndrome) was stained with H&E, cytokeratin-7 and Mib-1. The area of trophoblast as a fraction of total villous area was calculated and the number of sectioned cytotrophoblast and syncytiotrophoblast nuclei as well as the number of proliferating cytotrophoblast was evaluated. RESULTS: Normal features of trophoblast development during the first trimester (rise in trophoblast area, increase in number of syncytiotrophoblast nuclei, increase in number of proliferating cytotrophoblast, decrease in the nuclear CT:ST ratio) are absent/reversed in tissues from recurrent miscarriage (decreasing trophoblast area, constant number of syncytiotrophoblast nuclei, decreasing number of proliferating trophoblast, constant nuclear CT:ST ratio). CONCLUSIONS: Proliferation of cytotrophoblast in early gestation provides a pool of trophoblast stem cells critical for ongoing placental development. Premature cytotrophoblast differentiation in favour of syncytial fusion results in deficiencies of cytotrophoblast and rarification of villous trophoblast. Abnormal trophoblast differentiation in early gestation may be due to a premature onset of maternal perfusion of the placenta and may be a likely antecedent for conditions associated with failure of placentation such as recurrent miscarriage.

Syncytial fusion of human trophoblast depends on caspase 8.

Differentiation of human placental villous trophoblast includes syncytial fusion of cytotrophoblast forming syncytiotrophoblast. Early stages of the apoptosis cascade were described to be involved in this differentiation process. We investigated the role of the initiator caspase 8 in syncytial fusion in vitro, cultivating placental villous explants with or without caspase 8 antisense oligonucleotides or peptide inhibitors for up to 120 h. Trophoblast fusion and differentiation were assessed by confocal microscopy, immunohistochemistry and Western blot analysis. Culture with caspase 8 antisense oligonucleotides or peptide inhibitors reduced the fusion of cytotrophoblast with the syncytiotrophoblast, and resulted in multilayered cytotrophoblast. Caspase 8 expression was suppressed by antisense oligonucleotides and caspase 8 activities were reduced by peptide inhibitors. The organic anion-transporter hOAT-4 normally expressed in the cytotrophoblast and transferred into the syncytiotrophoblast by syncytial fusion was retained in the cytotrophoblast due to lack of fusion. We conclude that expression and activity of caspase 8 is a prerequisite for differentiation and syncytial fusion of cytotrophoblast cells.

Pre-eclampsia and maternal anaemia display reduced apoptosis and opposite invasive phenotypes of extravillous trophoblast.

During pregnancy extravillous trophoblast invades maternal uterine tissues and remodels spiral arteries. Maternal anaemia and early onset pre-eclampsia are associated with perturbed trophoblast biology. We systematically compared numerical density, invasive depth and apoptosis rates of extravillous trophoblast in uterine tissues taken from hysterectomies following Caesarean section after normal pregnancies (n=4) or pregnancies complicated by pre-eclampsia (n=5) or anaemia (n=6). Full thickness sections of the placental bed were studied by immunohistochemistry using anti-active caspase 3, anti-cytokeratin 7, anti-lamin B, M30, Mib-1, anti-PARP, and by the TUNEL assay. In normal pregnancy extravillous trophoblast invaded 2.04+/-0.19 mm (mean+/-SEM ) from the endometrial-myometrial border into the myometrium; in pre-eclampsia 0.67+/-0.14 mm (P< 0.01), and in anaemia 3.84+/-0.21 mm (P< 0.001). The endometrial trophoblast density in normal pregnancy was 2.44+/-0.37 cells per 60,000 microm(3), in pre-eclampsia was 1.04+/-0.15 (P< 0.01), and in anaemia was 3.10+/-0.32. The rate of apoptotic extravillous trophoblast (M30-positive) in the endometrium in normal pregnancy was 7.17+/-1.46 per cent, in pre-eclampsia 4.4+/-0.71, and in anaemia 2.1+/-0.42 (P< 0.01). Maternal anaemia leads to general tissue hypoxia throughout gestation. Increased invasive depth could be explained by hypoxia-stimulated mitosis and decreased apoptosis of extravillous trophoblast. Reduced trophoblast invasion in pre-eclampsia cannot be explained by higher rates of apoptosis.

Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. I. Dynamics of polyploidization.

Polyploidization of the extravillous trophoblast (EVT) cells at different stages of differentiation and invasion into the uterine wall in human placenta has been studied. An increase in the ploidy level of EVT cells in the course of their differentiation within cell columns (CC) was shown. Stem cells were mainly diploid (86.2%); incidence of polyploid nuclei of highly proliferative cells of the proximal part of CC increased progressively. In the distal part of CC, where EVT cells did not divide mitotically, polyploid cells prevailed, with 58.0 and 3.5% nuclei being 4c and 8c, respectively. The highest percentage of polyploid cells was found in the population of EVT cells attached directly to the surface of the decidualized endometrium: percentage of tetraploid cells turned out to be 74.7% and the share of octaploid nuclei rose up to 4.9%; however, there appeared a few (0.3%) 16c cells. The majority of EVT cells invading the decidualized endometrium were polyploid, the share of octaploid and hexadecaploid cells rose up to 9.7 and 1.4%, respectively. On the other hand, the percentage of diploid cells also increased up to 29.2% as compared to EVT cells attached to decidua (20.0%). The same tendency proved to be even stronger in myometrium: the share of diploid EVT cells increased up to 46.0%, a prominent amount of tetraploid (45.1%) and highly polyploid (8c and 16c) cells retained in the EVT cell population (7.4 and 1.1%, respectively). Immunohistochemical staining of Ki-67 protein (MIB1), which labels cells held in the cell cycle, showed a high incidence of MIB1-positive stem cells (93.7%) and the EVT cells of the proximal part of CC (85.5%) characterized by high mitotic activity. A lower MIB1-positivity (43.2%) was found in the distal part of CC, whereas invasive EVT cells showed no MIB1-labeling. The presence of MIB1-positive nuclei in the distal part of CCs in the absence of mitoses, taken together with data on polyploidization of these cells, indicates their switch to the endoreduplication cycle. As a whole, the data obtained evidence that differentiation of EVT cells of the invasive pathway is accompanied by polyploidization. However, in a population of trophoblast cells capable of most profound invasion (up to myometrium), the proportion of diploid cells rose. These results suggest that the human cytotrophoblast invasion into the uterine wall requires an optimum, not the highest, ploidy level, whereas highly polyploid cells may form a subpopulation at the border between the maternal and fetal parts of placenta.

Expression of a cytokeratin 18 neo-epitope is a specific marker for trophoblast apoptosis in human placenta.

In epithelial cells the caspase-mediated cleavage of cytokeratin 18 during apoptosis leads to the formation of a specific neo-epitope, recognized by the antibody M30. To test whether this antibody can be used as a specific marker for apoptotic trophoblast, we have stained serial sections of villi and junctional zone of first and third trimester human placenta with antibodies against cytokeratins 7 and 18, and against active caspase 3, with M30 and with the TUNEL reaction. Comparison of M30 immunoreactivities with TUNEL positivity and immunoreactivities for cytokeratins 7 and 18 clearly demonstrates that M30 specifically labels late apoptotic trophoblast cells. This finding is supported by the fact that in trophoblast, M30 immunoreactivities largely overlap with those for active caspase 3. As compared to the TUNEL test, the M30 immune reaction appears to be a highly reproducible marker for apoptotic trophoblast. This antibody stains a larger number of cells within the apoptosis cascade as compared to the TUNEL reaction, since cytokeratin 18 cleavage starts earlier than cleavage of DNA and since endonuclease activation can be bypassed in some trophoblast cells. The data suggest that M30 is superior to the TUNEL reaction as a marker for the detection of trophoblast apoptosis since it is easier to handle, more specific for apoptosis and less prone to artifacts.

[Morphofunctional disorders of uterine-placenta blood flow in multiple uterine myomas]. / Morfofunktsional'nye narusheniia matochno-platsentarnogo krovotoka pri mnozhestvennykh miomakh matki.

Results of the right uterine artery dopplerometry at pregnancy 39-39 weeks and those of subsequent histotopographic and immunomorphologic study of the placental bed in the amputated uteri were compared in 50 pregnant women. Morphofunctional equivalent of a considerable reduction of the uterine-placental circulation depending on the myomatous tissue spread and particularly in rare observations of "the placenta on the node" were found. Pathogenesis of these disturbances is determined by deficiency of the second wave of interstitial cytotrophoblast invasion, by phenomenon of the uterine-placental circulation decrease due to arterial supply of myomatous nodes and local hormonal changes produced by alterations of cellular-tissular correlations in the uterine-placental bed.

Increased fetoplacental angiogenesis during first trimester in anaemic women.

BACKGROUND: Epidemiological studies describe an association between relative size of the placenta at delivery and cardiovascular morbidity and mortality during adult life. Some determinants of placental size, such as maternal anaemia, have been acknowledged, but no plausible mechanism has been advanced to explain the initiation of postnatal disease. METHODS: Placental villous vascularisation in anaemic women (Hb<90 g/L) was assessed in the first and third trimesters of pregnancy by immunohistochemical identification of villous capillaries and compared with that of gestational age-matched groups of women with normal (Hb>110 g/L; control group) concentrations of haemoglobin, and an intermediate group (Hb 90-110 g/L). FINDINGS: Anaemia, especially in the first trimester, was associated with increased numbers of capillaries per villous cross section (mean 11.70 [SE 0.35] vs 4.14 [0.27]) located mainly in the outer third of the stroma beneath the trophoblast (94% [1.15] vs 67% [1.82]) and with increased numbers of villous macrophages and of proliferating MIB-1-positive cells compared with the control group. INTERPRETATION: Maternal anaemia in early pregnancy seems to influence the pattern of placental vascularisation. Such changes might alter placental vascular impedance during early fetal life, thereby exerting important effects on cardiovascular development.

[Placental macrophages (Kashchenko-Gofbauer cells) and their role in pathology]. / Platsentarnye makrophagi (kletki Kashchenko-Gofbauera) i ikh rol' v patologii.

The review deals with the origin, development, histo- and ultrastructure of placental macrophages, their link with stromal channels and their role in pathology. The necessity of further ultrastructural and immunohistochemical studies of these cells in the antenatal infection and early placental failure is emphasized.

[Functional morphology of the placental bed of the uterus]. / Funktsional'naia morfologiia platsentarnogo lozha matki.

The review deals with the literature and authors' data on the structure and function of main components of the uterus placenta bed under normal course of pregnancy. The attention is focused on the most important process, namely, the invasion of non-villous or interstitial cytotrophoblast into the walls of spiral arteries of the endo- and myometrium, their gestational restructuration which ensures sequential increase of the uterus-placenta circulation and, consequently, optimal foetal development. This information is necessary for the understanding of the pathological processes in the zone of uterus-placenta contact.

[Comparative study of dopplerometry and morphological study of placenta and uterine arteries in the evaluation of hemodynamic disorders in the "mother-placenta-fetus" system]. / Sravnitel'nyi analiz Dopplerometrii i morfologicheskogo issledovania platsenty i spiral'nykh arterii v otsenke gemodinamicheskikh narushenii v sisteme mat'-platsenta-plod.

The paper gives the comparative results of Doppler examination of blood flow in the mother-placenta-fetus system and morphological examination of the vessels of the funis, placenta, and placental bed. The latter examination allowed all the cases of placentomaternal and placentofetal circulatory diseases to be divided into 3 groups. There was a clear-cut correlation between the Doppler metric and morphological findings.

[Pathomorphology of spiral uterine arteries, umbilical vessels and placenta in disorders of maternal-fetal circulation]. / Patomorfologiia spiral'nykh arterii matki, pupochnykh sosudov i platsenty pri narusheniiakh materinsko-plodnogo krovotoka.

The comparison of the spiral uterine arteries structure, umbilical vessels and placenta is performed in 16 women with normal haemodynamics and in 31 women with disturbances of mother-foetus circulation, and the data obtained were compared with results of dopplerometry made before the delivery. Three clinical groups are distinguished according to the predominant localization of the resistance to the blood flow: the first--with the domination of the spiral arteries obliteration (lack of gestational changes, hyperplastic arteriosclerosis) with a characteristic ultrastructural pathology of the syncytiotrophoblast microvilli, rheological disturbances in the intervillous space and large necroses of villi; the second--with a predominance of early immaturity of the stroma and capillaries or sclerosis and compression of foetal villous capillaries, i. e. with a reduction of a capillary bed and increase of the resistance to the blood flow; the third--with a predominance of the umbilical arteries narrowing and supporting villi and signs of a blood stasis in the foetal veins. Morphologic heterogeneity of the mother--foetus circulation disturbances should be taken into consideration when choosing obstetric tactics.

[Ultrasound examination of the hip joint in infants]. / Ul'trazvukovoe issledovanie tazobedrennogo sustava u detei grudnogo vozrasta.

The authors investigated 236 hip joints in children aged from 10 days to 3 months by ultrasound method. Joints were classified after Graph in 4 types. Angular values of osseous and cartilaginous reference points of hip joints are determined and therapeutic measures are prescribed depending on the joint type. Sonography provides quite objective information about non-cartilaginous elements of a joint and deserves spreading in all areas.