Gestational age-dependent repair kinetics of microsurgical defects in monolayers of human amniocytes.

OBJECTIVES: There is paucity of data on the capacity of fetal membranes to repair surgical defects following trauma. We aimed at developing an in vitro model using monolayers of human amnion epithelial cells to study fetal membrane healing. METHODS: Term (n = 6) and preterm (n = 3) fetal membranes were collected at caesarean section. The amnion was digested twice in a trypsin solution. Amniocytes were seeded (250,000-750,000/ml) and incubated at 37 degrees C and 5% CO(2) and 21 or 5% O(2). A microsurgical injury was made centrally in the monolayers and the cultures were incubated for 48 h. Every 6 h, slides were fixed and immunohistochemical staining was performed to quantify proliferation at the site of the defect and centrally in the monolayer. The closure rate was evaluated by measuring the defect size every 6 h. RESULTS: The closure rate of the defects was higher in preterm versus term cultures. Proliferation was significantly higher in the defect zone versus the peripheral zone, and also higher in the preterm group. CONCLUSION: We describe a new model for the study of fetal membrane healing and observed gestational age-dependent repair capacity of the amnion.

Shifting concepts of the fetal-maternal interface: a historical perspective.

The first microscopic images of the human placenta, obtained in the 1830s, revealed the presence of an epithelial lining separating fetal capillaries from maternal blood, which was in later years successively interpreted as maternal endothelial, decidual and finally as "trophoblastic". With this new term, introduced by Hubrecht in 1889, its embryonic/fetal origin was recognized as well as its role in nutrient uptake from maternal blood. Thomas Huxley considered the presence of a decidua as an important feature for mammalian classification, but still mixed up maternal and trophoblastic tissue. Mathias Duval recognized invasive activities by trophoblast in rodents, but over-interpreted the arterial invasion observed in rats. In the human, unusual endovascular cells were first described by Carl Friedländer, but their trophoblastic nature was only recognized in the early 20th century. Nitabuch's description of a continuous fibrinoid layer underneath the basal plate led to the erroneous concept of a borderline separating the trophoblast-invaded upper decidua from the deeper non-invaded uterine tissue. This concept - based on the study of one pregnant uterus - has been made obsolete by later studies of trophoblast invasion. Many erroneous interpretations of placental histology in the past were logical in the context of then current knowledge. A better understanding depended on improved technology which allowed tracing of histological continuity of structural features in space and time. Although identification of cell types increasingly relies on molecular markers, classical histological principles should still be applied in conjunction with newer techniques in order to arrive at a broad understanding of placental development. Understanding past errors in interpreting placental histology should guard us against overconfidence in so-called breakthrough discoveries.

Endovascular trophoblast invasion, spiral artery remodelling and uteroplacental haemodynamics in a transgenic rat model of pre-eclampsia.

The aim of the present study was to evaluate the depth of endovascular trophoblast invasion and associated remodelling of spiral arteries in a transgenic model of pre-eclampsia in the rat, a species showing a comparable deep invasion during normal pregnancy as the human. Pre-eclamptic (PE) transgenic rats (TGR) (hAngiotensinogen female x hRenin male) and non-PE reversely mated (RM) TGR rats were compared to normal Sprague-Dawley rats (C). Day 18 implantation sites were collected and the presence of endovascular trophoblast, fibrinoid, endothelial and smooth muscle cells were evaluated in spiral arteries in three parallel layers in the mesometrial triangle using an image analysis system (KS-400). In a separate group of animals peak-systolic and end-diastolic velocities were measured by Doppler in uterine and arcuate arteries, and the resistance indices (RI) were calculated. In PE and RM rats, the entire mesometrial triangle contained significantly more endovascular trophoblast and vascular fibrinoid deposits than the C group. No difference was found between the groups in the overall amount of smooth muscle surrounding the lumen, but in the PE and RM groups significantly more muscle was present in parts of the contours covered by trophoblast. There was significantly less CD31-positive endothelium in the total lumen contours of the PE and RM groups than in the C group, but in parts of the contours covered by trophoblast more residual endothelium was present in both TGR groups. Comparison of the three layers indicated deeper invasion in both the PE and RM groups than in the C group. By Doppler analysis of the proximal uterine artery the RI was found to be significantly lower in the PE and the RM group than in the C group. In the arcuate artery, the RI was significantly lower in the PE group as compared to the RM and C groups. We conclude that in this transgenic PE rat model there is deeper endovascular invasion of spiral arteries and decreased RI of uterine arteries at day 18 of pregnancy.

Erasmus Darwin's enlightened views on placental function.

In his major work "Zoonomia", Erasmus Darwin (1731-1802) devoted one chapter to the placenta, in which the new knowledge of the recently discovered element oxygen was applied to the functioning of this organ. He considered the "cavities" or "lacunae" in the placenta as the main areas for oxygenation of the fetal blood, as he thought them to be structurally comparable to the lungs and the gills of fish. He obviously was aware of species differences in the uterine arterial blood supply to the placenta between humans and cows, assuming a higher contractility of the vasculature in the latter species. The new evidence for a primarily respiratory role overshadowed ideas of a possible nutritive function of the placenta. Since Hunter's definitive demonstration of separate maternal and fetal blood circulations, nutritive functions of the placenta needed to be explained by transmembrane transport processes, which were unknown at that time. Instead Erasmus Darwin erroneously considered the amniotic fluid as the main source of nutrients for the fetus. His understanding of placental respiration found expression in his long poem on the history of life on earth.

The role of the placenta in the initiation of spiral artery remodelling in an early pregnant chimpanzee uterus.

INTRODUCTION: In this study we evaluated the full extent of placental bed changes (centre to periphery) in a pregnant chimpanzee uterus, kept at the Museum for Central Africa in Tervuren, Belgium. According to placental size the specimen was equivalent to an 8 weeks pregnant human uterus. METHODS: Histological sections from central to peripheral tissue blocks of the placental bed were stained to reveal the presence of trophoblast, endothelium, vascular smooth muscle and elastic laminae. As an indicator for early arterial remodelling, we evaluated endothelial nuclear rounding and subendothelial vascular changes within the maternal vasculature in decidua and adjacent inner myometrium. RESULTS: While interstitially invading trophoblasts were present, endovascular trophoblast invasion seemed about to start into one spiral artery outlet at the centre of the placental bed, confirming our previous impression of a later onset of endovascular trophoblast invasion as compared to the human. An early sign of spiral artery remodelling was rounding of the endothelial nuclei. This phenomenon was not related to the local presence of interstitial trophoblast. DISCUSSION: Endothelial nuclear rounding turned out to be a feature of the placental bed as a whole, being significantly less prominent in the adjacent non-placental bed part of the uterus, indicating an effect of the presence of the placenta. The different time-course of early spiral artery remodelling in the chimpanzee as compared to the human may have had a significant impact upon our evolution.

Mathias Duval on placental development in mice and rats.

Mathias Duval (1844-1907) was one of the pioneers in elucidating the intricate placental histology of different mammalian groups, notably the rodents. Using a well-dated series of mouse conceptuses, he described in detail the successive steps in placental development, and for confirmation he included observations on a (undated) collection of rat specimens. Not only was he able to identify correctly the different extra-embryonic cell layers, but he was also the first to recognize trophoblast invasion in rodents. Not all his interpretations are still valid, however. Re-reading his extensive and detailed work "Le placenta des rongeurs" (1890-1892) confronts us with still existing gaps in our present understanding of placental development, notably the morphogenesis of the different placental layers and the differentiation of invasive trophoblast. His understanding of uteroplacental blood flow was still limited, and he failed to recognize the complexity of the maternal decidua and its vasculature, which is essential for correctly understanding the pathways and extent of trophoblast invasion. Although Duval was active in promoting Darwin's evolutionary ideas, he refrained from extrapolating too quickly his findings in rodents to other mammalian groups including humans. In his view detailed histological studies on complete series of specimens had to come first, and thus provide a firm basis for a proper understanding of placental function and evolution.

The uterine spiral arteries in human pregnancy: facts and controversies.

Uterine spiral arteries play a vital role in supplying nutrients to the placenta and fetus, and for this purpose they are remodelled into highly dilated vessels by the action of invading trophoblast (physiological change). Knowledge of the mechanisms of these changes is relevant for a better understanding of pre-eclampsia and other pregnancy complications which show incomplete spiral artery remodelling. Controversies still abound concerning different steps in these physiological changes, and several of these disagreements are highlighted in this review, thereby suggesting directions for further research. First, a better definition of the degree of decidua- versus trophoblast-associated remodelling may help to devise a more adequate terminology. Other contestable issues are the vascular plugging and its relation with oxygen, trophoblast invasion from the outside or the inside of the vessels (intravasation versus extravasation), the impact of haemodynamics on endovascular migration, the replacement of arterial components by trophoblast, maternal tissue repair mechanisms and the role of uterine natural killer (NK) cells. Several of these features may be disturbed in complicated pregnancies, including the early decidua-associated vascular remodelling, vascular plugging and haemodynamics. The hyperinflammatory condition of pre-eclampsia may be responsible for vasculopathies such as acute atherosis, although the overall impact of such lesions on placental function is far from clear. Several features of the human placental bed are mirrored by processes in other species with haemochorial placentation, and studying such models may help to illuminate poorly understood aspects of human placentation.

Interstitial trophoblast invasion in the decidua and mesometrial triangle during the last third of pregnancy in the rat.

Like other species with haemochorial placentation, pregnant rats show marked invasion of the uterine wall by trophoblast. While an endovascular pathway of invasion has been recognized for a long time, only recently, by application of cytokeratin immunostaining, the existence of an interstitial pathway of invasion has been established. Interstitial invasion is mainly effected by glycogen cell-like trophoblast arising from glycogen cell islands of the trophospongium opening up into the decidua, and from glycogen cell sheaths surrounding the intraplacental maternal arterial channels which are connected with the spiral arteries in decidua and mesometrial triangle. Quantitative evaluation of interstitial invasion in both maternal compartments was carried out on days 15-21, using PAS staining and cytokeratin and alpha-actin immunostaining for detecting trophoblast and defining maternal tissue compartments. Measurements of compartment size, cytokeratin-positive areas and invasion extent were performed using the KS400 image analysis system. A distinct pattern of interstitial trophoblast invasion emerged, starting from central decidual areas around the maternal arterial channels, and mushrooming into the mesometrial triangle reaching a peak at day 18, followed by gradual regression of the invaded areas. These measurements may serve as a basis for further experiments to evaluate factors which may influence the depth of trophoblast invasion.

The placental bed in pregnancies complicated by primary antiphospholipid syndrome.

UNLABELLED: Pregnancy in women with primary antiphospholipid syndrome (APS) is frequently associated with placental insufficiency leading to intrauterine growth restriction (IUGR)+/-fetal death, pre-eclampsia, placental abruption, premature delivery or thrombosis. The aim of this study was to investigate the placental bed in APS pregnancies for evidence of impaired trophoblast invasion, endothelial cell activation (ECA) and macrophage infiltration. METHODS: Biopsies from the presumed site of the placental bed were obtained from 12 women with treated primary APS and 16 controls. Immunohistochemical methods were employed to investigate expression of cytokeratin (trophoblasts), alpha-actin (smooth muscle), CD68 (macrophages) and VCAM-1 (as marker of ECA). Fibrinoid and elastin distribution and expression were determined by periodic acid/Schiff and orcein stain, respectively. RESULTS: Three APS pregnancies developed IUGR, one with concurrent pre-eclampsia. Eight of 12 APS biopsies were confirmed to be from the placental bed; one patient failed to meet APS criteria and was excluded from analysis; six included spiral arteries in the biopsy; 11 of 16 controls' biopsies were from the placental bed. APS biopsies had a higher concentration of inflammatory cells (p=0.0001), particularly macrophages (p=0.014). Three APS biopsies showed necrosis with hyperplastic vessels; one demonstrated arterial thromboses, but none had profound vasculopathy/atherosis or ECA. CONCLUSION: Inflammatory mechanisms in the placental bed may contribute to APS pregnancy complications.

Emil Selenka on the embryonic membranes of the mouse and placentation in gibbons and orangutans.

BACKGROUND: Emil Selenka made important contributions to embryology in marsupials, rodents and primates that deserve wider recognition. Here we review his work on early development of the mouse and placentation in the great apes. FINDINGS: Selenka was intrigued by germ layer theory, which led him to study inversion of the germ layers in the mouse and other rodents. He found it was growth of the ectoplacental cone that caused a downward shift in the position of the underlying ectoderm and endoderm, leading to an inside-outside inversion of these layers. In primates he made the important discovery that the embryos of gibbons and orangutans develop under a decidua capsularis. Thus all great apes, including humans, exhibit interstitial implantation; this is in contrast to other primates where implantation is superficial. CONCLUSIONS: Selenka's work was thorough and brilliantly illustrated. It was an important influence on his contemporaries and was well known to scientists of the following generation. Embryologists continue to advance our knowledge of fetal membranes and placentation in the mouse, but Selenka's work on gibbons is unique and our knowledge of orangutan placentation is restricted to his specimens.

Endovascular trophoblast invasion and associated structural changes in uterine spiral arteries of the pregnant rat.

The involvement of endovascular trophoblast in fibrinoid deposition, replacement of the endothelium and vascular smooth muscle breakdown is studied in spiral arteries of the mesometrial triangle from day 15 to day 21 of rat pregnancy, by examining arterial cross sections after staining for cytokeratin, PAS, CD31 and alpha-actin. From day 15 to day 18 of pregnancy, fibrinoid deposition underneath the endovascular trophoblast increases gradually, whereas the amount of endovascular trophoblast in invaded arteries remains constant. CD31 staining is significantly reduced in sub-ET (= underlying the endovascular trophoblast) as compared to extra-ET (= outside the endovascular trophoblast) and no-ET (= non-invaded arterial sections) at each time-point of pregnancy examined (P < 0.005 and P < 0.0005 at each day of pregnancy), whereas alpha-actin staining is reduced both in sub-ET and in extra-ET as compared to no-ET. During pregnancy, CD31 staining in sub-ET initially declines, but increases significantly on day 21 (P < 0.001 versus d20) suggesting re-endothelialization of the vascular wall. In conclusion, changes in spiral arteries of pregnant rats reveal striking similarities with physiological changes seen in human pregnancy, thus emphasizing the usefulness of this species as an experimental model for studying normal and complicated pregnancies in humans.

Thomas Huxley and the rat placenta in the early debates on evolution.

The 19th century debates on mammalian classification in the light of the new evolutionary thinking led to controversies between Thomas Huxley and Richard Owen concerning the value of the placenta as a representative key organ. As a main point in his argument, Huxley provided a detailed description of a sectioned rat placenta, highlighting the importance of decidualization of the uterus as an argument supporting an evolutionary relationship between rodents, insectivores and primates, an idea hotly contested by Owen. In addition, he illustrated and correctly interpreted the maternal blood supply from uterus to placenta in striking detail. During the succeeding decades the key role of trophoblast in placenta formation was discovered, and the decidua became neglected in later comparative studies. Nevertheless, at the present time trophoblast-decidual interaction is regarded as an extremely important feature of placental development in both primates and rodents, and Huxley can therefore rightfully be considered as an early pioneer in placental research.

Decidual changes in the endometrium and morphological adaptation of the associated supplying arteries in the normal and diabetic pseudopregnant rat.

After an electrically induced pseudopregnancy in rats in which deciduomas were produced by an intraluminal oil injection, the decidual tissue was studied morphologically on days 7, 10 and 13. A constant and dynamic wave of mitotic figures was found, which started on day 7 in the antimesometrial decidua, moving to the mesometrial decidua on day 10 and finally to the mesometrial triangle area on day 13 of pseudopregnancy. This and other morphological changes were compared with those found in pseudopregnant rats with streptozotocin-induced diabetes. On days 7 and 10 the incidence and distribution of mitotic figures were practically identical in both groups and statistically no significant difference was found in the uterine weight between the two groups on these two days. On day 13, two statistically significant differences were observed in the diabetic group: a fall in the uterine weight and a fall in the incidence of mitotic figures in the mesometrial triangle decidual cells. Associated with decidualization, a series of histological changes were studied in the arteries of the mesometrial triangle. Although the difference was not statistically significant, changes seemed to occur to a lesser degree in the diabetic group. It is concluded that the diabetic state has no influence in the early decidualization period, but it does have influence late in pseudopregnancy: a fall in the mitotic activity in the decidual tissue, a fall in the uterine weight and a less marked change in the spiral arteries which during pregnancy would supply the developing placenta with maternal blood.

Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy.

Trophoblastic invasion of the human decidua has been studied in 48 intact uteri with pregnancies ranging from 8 to 18 weeks after the last menstrual period. Some cytotrophoblast invades the distal segments of the spiral arteries to become endovascular while the rest diffusely infiltrates the decidua as an interstitial invader. The interstitial cytotrophoblast reaches the myometrium and gives rise to the characteristic placental bed giant cells. As the placental site enlarges the lateral spiral arteries come to lie obliquely; new openings into the intervillous space are created but this readjustment of the placental blood supply may cause focal superficial decidual necrosis. The physiological changes converting the spiral to the uteroplacental arteries are effected in the upper decidua by the action of endovascular and perivascular cytotrophoblast, whereas in the deeper decidua endovascular trophoblast is principally involved. Endometrial granulocytes aggregate in the region of maternal tissue degeneration with the heaviest trophoblast invasion but the role played by these cells in placentation is unknown.

Interaction of interstitial trophoblast with placental bed capillaries and venules of normotensive and pre-eclamptic pregnancies.

While endovascular trophoblast invasion of the human placental bed spiral arteries has been studied extensively, no information is available on the interaction between interstitially invading trophoblast and uterine capillaries and venules. Placental bed biopsies of eight normotensive and 15 pre-eclamptic patients were double-immunostained for cytokeratin and the endothelial marker CD31, providing satisfactory staining results in six and 10 biopsies, respectively. Interstitial trophoblast tissue density did not differ between the two series of biopsies, implying that this pathway of invasion is not impaired in pre-eclampsia. Both groups showed a similar incidence of approach of non-arterial vascular structures by perivascular trophoblast. Differences in CD31 staining intensity were noticed in different vascular cross-sections. Lower staining intensity was related to the presence of perivascular trophoblast. Because of the identity of CD31 with the platelet-endothelial cell adhesion molecule (PECAM)-1, the trophoblast-dependent downregulation of CD31 may play a role in the control of leukocytic traffic within the placental bed. The phenomena described in this paper did not show any difference between the normotensive and pre-eclamptic patients, implying that interaction of interstitial trophoblast with venous and capillary structures is not related to the pathogenesis of pre-eclampsia.

Histological study of decidual spiral arteries and the presence of maternal erythrocytes in the intervillous space during the first trimester of normal human pregnancy.

During the first trimester in normal human pregnancy, endovascular trophoblast migrate along the decidual spiral arteries and invade their walls to produce physiological change. There is controversy as to whether invading trophoblast plug the arteries and prevent blood flow into the intervillous space. Using light microscopy, placental bed sections from 25 first trimester gravid hysterectomy specimens were examined. From each specimen, one section was divided into equal central and peripheral compartments. Maternal red blood cells were present in the intervillous space in all specimens, in both central and peripheral areas. In total, 232 decidual spiral arteries were found, each of those represented by several cross sections, 136 in the central area and 96 in the periphery. Seventy-nine per cent had undergone physiological change (significantly more in the centre than in the periphery), 63 per cent contained scattered endovascular trophoblast, 20 per cent had plugs of trophoblast partially occluding the vessel and 17 per cent had plugs totally filling the vessel lumen. These data confirm that in the first trimester of normal pregnancy, maternal blood enters the intervillous space, total plugging of the arterial system by trophoblast is not common, and more spiral arteries undergo physiological change in the centre than in the periphery.

Review article: trophoblast invasion and the establishment of haemochorial placentation in man and laboratory animals.

Trophoblast invasion is an essential component of haemochorial placentation and has to be considered to relation to reactive changes in the maternal uterine tissues. Some comparative aspects of human and laboratory rodents are discussed and, although there is an obvious phylogenetic gap between the two, many characteristics of placental development are found to be analogous. Trophoblast growth into the uterus is different in different species: localized trophoblast growth forming a bulky tissue (mouse, rat, hamster) contrast with a dispersion of independent trophoblastic elements, forming an interstitial invasion (guinea pig, man). In the rat, mouse, hamster and man retrograde intra-arterial trophoblast migration occurs in maternal vessels supplying blood to the developing placenta. Early changes in maternal tissue might influence trophoblast behaviour. Decidualization probably is a key phenomenon, and the relation of decidual necrosis to trophoblast invasion is considered. Some kind of controlled immune response by the mother also may be involved. These considerations apply to stromal or interstitial invasion as well as to intravascular trophoblast migration but, for the latter, haemodynamic factors probably influence tissue reactions.

Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy.

Morphometric and statistical techniques were used to assess the relation of myometrial interstitial trophoblast to the uteroplacental vasculature in 27 intact hysterectomy specimens ranging from 8 to 18 weeks' gestation. It was found that the volume density of cytotrophoblast in the myometrium and in particular the proximity of such trophoblast to the placental bed spiral arteries correlated significantly with morphological alterations in these vessels. The changes included swelling of endothelium, hypertrophy of individual medial smooth muscle cells, and oedema and disruption of the architecture of the vessel wall as a time-related continuum. Some of the changes, such as swollen endothelium and basophilia of medial smooth muscle cells were noted also in spiral arteries in the non-placental bed endometrium but to a considerably less extent than in the placental bed. Intimal vacuolation was common to placental bed and non-placental bed arteries, increased with gestational age and can be considered as a non-specific feature. The migration of endovascular trophoblast into the myometrial spiral arteries in the second trimester occurred only when these arteries had been considerably altered in their morphology. These findings indicate that migratory interstitial cytotrophoblast probably has a role to play in the preparation of the myometrial segments of the uteroplacental arteries for the second wave of endovascular trophoblast migration that occurs in the second trimester of human pregnancy.

Alpha-2 macroglobulin controls trophoblast positioning in mouse implantation sites.

In humans, functional deficiency of alpha-2M is not known, implying alpha 2M is essential for gestational success. Mice, deficient in two members of the alpha-2 Macroglobulin (alpha 2M) family, i.e. alpha-2 macroglobulin (MAM) and murinoglobulin-1 (MUG-1) are viable, fertile and phenotypically normal, unless stressed (Am J Pathol, 155 (1999), 983). Here, we analysed implantation sites in MAM(-/-)/MUG-1(-/-)mice during pregnancy, a strong physiological stressor. Despite some post-implantation fetal loss, mean litter size was comparable to congenic C57Bl/6J (B6) mice, but MAM(-/-)/MUG-1(-/-)pups were significantly lighter and the sex ratio was skewed towards males. Implantation sites appeared histologically normal up to gestational day (gd) 8. By gd 10, extensive over-development of trophoblasts was evident, accompanied by relative deficits in decidua, in the mural mesometrial lymphoid aggregates of pregnancy and in uterine Natural Killer cells. At gd 10-12, decidual spiral arteries were dilated but abnormally cuffed by trophoblasts that extended anomalously, for midgestation, to the myometrial circular smooth muscle. Ultrastructurally, trophoblasts in the mesometrial decidua made intimate contact with endothelial cells that were shedding membrane fragments. These findings demonstrate that alpha 2M, and thereby proteinases and/or cytokines whose bio-availability is regulated by alpha 2M, exert significant decidual regulation on trophoblast invasion.

Identification of 'renin'-containing cells in the choriodecidua.

Chorionic trophoblast, decidual cells, and macrophages have all been named as the site of renin in the placental membranes. To establish more clearly the nature of the renin-containing cells in the placental membranes, double immunostaining techniques were used to stain renin and specific cell markers in the same tissue sections. Cytokeratin was selected as an ectodermal cell marker and CD68 as a cytoplasmic macrophage marker. Cross-binding between antibodies was prevented by blocking species-related binding sites between the first and second sequence of the double-immunostaining procedures and by using highly selective immunostaining techniques in the second sequence. The results clearly show renin immunostaining in CD68-positive macrophages and not in cytokeratin-positive trophoblast. The anti-renal renin monoclonal antibody showed high affinity cross-reactivity with cathepsin D, another aspartic proteinase that can release angiotensin I from angiotensinogen. This should be seen in the context of earlier findings that only two of four anti-renal renin monoclonal antibodies showed staining in uterine and placental tissues and both cross-reacted with cathepsin D. The results indicate that differentiation between renin and cathepsin D and, possibly, other substances with shared properties and epitope homology deserves more attention than it has received thus far.