Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
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Abstract |
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Key words: decidualization/immunohistology/spiral arteries/trophoblast
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Introduction |
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EVT cells arise during early development as cytotrophoblast cell columns when cells move away from the anchoring villi which border the decidua and fuse to form the cytotrophoblast shell. From this shell, the trophoblast invades into the decidual tissue, with the rounded cohesive cells changing to an isolated, elongated, pleomorphic morphology as the trophoblast infiltrates between the stromal cells (Boyd and Hamilton, 1970). These interstitial trophoblast cells appear to preferentially home towards the uterine spiral arteries and encircle them (Pijnenborg et al., 1980
, 1983
). Cells from the cytotrophoblast shell also give rise to endovascular trophoblast. Where the shell lies over the distal opening of the uterine spiral arteries, endovascular trophoblast cells migrate along the lumen in a retrograde manner. There is associated fibrinoid necrosis of the media and loss of endothelium. Brosens named these collective spiral arterial transformations as `physiological change' (Brosens et al., 1967
). Interestingly, the veins are never transformed in this way.
The dramatic structural alterations of muscular spiral arteries into dilated sac-like vessels, unresponsive to vasoconstrictive agents and capable of high conductance, are essential to accommodate the huge increase in the blood flow required to the intervillous space (Brosens et al., 1967). The central importance of this process to normal fetal growth is demonstrated when the vessels are not adequately converted. In these pregnancies, poor fetal growth and even stillbirth may occur, with pre-eclampsia arising as a secondary systemic complication in susceptible women (Robertson et al., 1967
; De Wolf et al., 1980
, Khong et al., 1986
).
Controversy has surrounded the role of trophoblast in physiological change. Some believe the transformation occurs due to the presence of the trophoblast (Brosens et al., 1967; De Wolf et al., 1973
; Pijnenborg, 1996
), while others have argued that some features of vascular remodelling occur as a consequence of decidualization with the trophoblast being unnecessary (Craven et al., 1998
). Analysis of the spiral arteries in decidua where the trophoblast is absent would allow distinction between the arterial changes which arise as a result of trophoblast invasion and those which are associated with decidualization. Tubal ectopic pregnancies provide one source of such tissue, as there is decidualization of the uterine mucosa but no trophoblast is present. In this study, we have compared spiral arteries in the decidua of normal first trimester pregnant hysterectomy specimens with the uterine decidua from patients with ectopic Fallopian tubal pregnancies of similar gestational age. Non-pregnant endometrium was also studied to examine any changes in the arteries during the normal menstrual cycle as decidualization begins during the luteal phase (de Feo, 1967
). Immunohistology was performed with a panel of antibodies to delineate the cellular and structural components of the blood vessel walls.
While the conversion of myometrial spiral arteries has been well described (Pijnenborg et al., 1983), the earlier physiological change in decidual spiral arteries is less well documented. Hence, in addition, this paper seeks to describe more fully the changes in decidual spiral arteries at the implantation site of pregnancies in early gestation with particular emphasis on the relative contribution of interstitial and endovascular trophoblast to the medial changes.
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Materials and methods |
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Sections from 10 cases of non-pregnant hysterectomies were also examined, five cases of which were of endometrium in proliferative phase (early n = 2, mid n = 1, late n = 2) and five were secretory endometrium (early n = 1, mid n = 2, late n = 2). Again these hysterectomies were performed for conditions unrelated to the endometrium (e.g. cervical neoplasia or uterine prolapse). These samples, which were from normal cycling women who were not taking oral contraceptives or had an intrauterine device (IUD), were histologically dated according to methods previously described (Noyes, 1950; King et al., 1989
). Uterine decidua from seven cases of ectopic pregnancies were used as examples of uterine mucosa from the pregnant state, but where trophoblast was absent. Ectopic pregnancy was verified by the presence of trophoblast in the Fallopian tube. The gestational age was clinically estimated to be 68 weeks.
Haematoxylineosin (H&E) blocks from all the cases used were reviewed. Several blocks of tissue (n = 24) from each case were cut into sections 7 µm thick and stained with periodic acidSchiff (PAS) and silver (PAAg) using standard techniques (Prophet, 1994), as well as with immunohistochemistry.
A panel of five mouse monoclonal antibodies (Table I) was used to immunostain serial sections from all the samples. The slides were immersed in histoclear to remove the wax and rehydrated through a gradient of ethanol (100%, 90%, 70%, 50%) before washing in phosphate-buffered saline (PBS). The slides were boiled in tri-sodium citrate buffer at pH 6 to reveal the antigens and washed in PBS. Serum blocking was performed by incubating for 15 min with normal horse serum (NHS) (SigmaAldrich Co. Ltd, Poole, Dorset, UK) diluted to 1/50 with PBS. The sections were incubated with the primary antibody (see Table I
) at the optimal dilution, determined by previous titration, for 30 min. The slides underwent washing in PBS. The secondary antibody, biotinylated horse anti-mouse IgG (Vector, Peterborough, Cambs, UK) made up in 10% human serum (Sigma) at 1/200 dilution was prepared and left at room temperature for 30 min. This was then microfuged at 9000 g for 5 min to remove secondary antibodyhuman antigen complexes from the solution. The sections were incubated with the secondary antibody for 30 min and then the slides underwent washing in PBS. The avidinperoxidase complex (ABC reagent; Vector) was prepared by adding one drop of Reagent A and one drop of Reagent B to 2.5 ml of PBS, and left at room temperature for 30 min. Incubation with the ABC reagent was for 30 min after which the slides underwent a PBS washing. Peroxidase activity was demonstrated using diaminobenzidine tetrahydrochloride (DAB; Sigma), made according to manufacturer's instructions. This was then applied to the sections for 46 min before washing in PBS. The slides were counterstained in Carazzi's haematoxylin for 6 min and washed in tap water. Dehydration was carried out in 100% ethanol and after immersion in Histoclear (manufactured by National Diagnostics; supplied by Flowgen, Lichfield, Staffs, UK), the slides mounted with Histomount (Flowgen). All incubations in the above steps were carried out in a humidified chamber and the PBS washing consisted of two 5 min immersions in PBS at room temperature.
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Results |
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In non-pregnant endometrium, the CD31+ endothelial cells of the spiral arteries were found to be plump in contrast to the flat venular endothelial cells. Actin staining was used to delineate the smooth muscle cells of the media. A semiquantitative method was used to measure the numbers of layers of medial cells. In non-pregnant endometrium the measurement was estimated at the same depth from the surface epithelium (Table II). The media was found to be more prominent, with more layers of smooth muscle cells, in the secretory phase than in the proliferative phase. These actin layers decreased as the uterine artery reached the surface (not shown).
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Decidua basalis
The decidual spiral arteries in the decidua basalis of the three cases of intrauterine pregnancy hysterectomy specimens were similarly examined (Figure 3). The invasive trophoblast cells were easily identified using the anti-CK7 mAb which yielded staining that was restricted to trophoblast and glandular cells. The presence of interstitial trophoblast was always denser around the spiral arteries than around any other structure in the decidua. Indeed, the arteries could easily be located by scanning the sections at low power for the areas with abundant interstitial trophoblast.
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In these samples where the gestational ages were between 7 and 9 weeks, no transformation of the myometrial spiral arteries was seen. Trophoblast was seen around some veins; there was no modification of the walls of these vessels.
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Discussion |
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In humans, unlike other species, the process of decidualization starts in the mid-luteal phase of the menstrual cycle with enlargement of stromal cells forming a cuff around the spiral arteries and an increase in numbers of NK cells (de Feo, 1967; Bell, 1983
; Finn, 1994
). Decidualization involves all elements of the mucosa, stromal cells, leukocytes, glands and the extracellular matrix (Aplin, 1989
). From this present study it appears that spiral arteries should be considered to be involved in the decidualization process as they show increased endothelial swelling and an increase in the loosely arranged actin-positive medial cells. Like other features of decidualization, the arterial changes become more marked in true gestational decidua. The functional implications of these changes are likely to be related to the increased blood flow in pregnancy and be an example of physiological vascular remodelling (Gibbons and Dzau, 1994
).
A recent report has described similar changes in the media and endothelial cells in decidua and interpreted them as early features of physiological change of the spiral arteries of pregnancy (Craven et al., 1998). However, the term physiological change was originally used to describe the `disappearance of the normal muscular and elastic structures of arteries and their replacement by fibrinoid material in which trophoblast cells are embedded', (Brosens et al., 1967
). We believe that physiological change should be restricted to this definition and not confused with the arterial changes seen as a result of decidualization alone.
It would obviously be important to define the relative contributions that interstitial and/or endovascular trophoblast make to specific destruction of the medial smooth muscle cells. Several publications have implied that it is the incorporation of endovascular trophoblast into the vessel wall which causes this destruction (Zhou et al., 1997; Damsky and Fisher, 1998
). By using immunostains for CK7 and CD56, we were able to distinguish between the two types of trophoblast. Although both interstitial and endovascular trophoblast stain for cytokeratin, which is an intracellular intermediate filament characteristic of epithelial cells (O'Guin, 1990
), only the endovascular trophoblast cells express the adhesion molecule CD56 (NCAM), which is thought to function in the formation of the endovascular plugs (Burrows et al., 1994
). Our findings support the possibility that the medial destruction and fibrinoid necrosis results from interstitial trophoblast. PAS-positive fibrinoid necrosis and loss of actin reactivity was only seen in vessels surrounded by interstitial trophoblast. Furthermore, the deeper portions of such arteries had no trophoblast in the lumen and were still lined with endothelial cells. It was only in the superficial portions of the decidual spiral arteries that endovascular trophoblast was identified in continuity with the cytotrophoblast shell. In these sections of the arteries, endothelial cells had been replaced by the endovascular trophoblast cells. Replacement of the endothelial cells by the endovascular trophoblast appears to be initially focal, occurring only where the endovascular plug is in direct contact with the arterial wall. Thus the likely sequence of events seems to be that the interstitial trophoblast homes to the spiral arteries and destroys the vessels' media as a priming process. The subsequent migration of endovascular trophoblast down the arterial lumen is accompanied by the destruction of the endothelial cells. This course of events in the conversion of the decidual spiral arteries is similar to that seen in the myometrial portions of spiral arteries (Pijnenborg et al., 1983
).
The term extravillous trophoblast, therefore, does encompass several subpopulations which have different phenotypes and functions. Although interstitial trophoblast and endovascular trophoblast both arise from the cytotrophoblast shell, they differ by invading through tissue or into arterial channels. Interstitial trophoblast cells are interesting as they appear to move through decidual tissue with minimal tissue destruction until reaching the arterial media. They then appear to initiate the fibrinoid necrosis so characteristic of physiological change. Future work should focus on the mechanisms interstitial trophoblast uses to exert this medial-specific destruction. This mechanism is central to our understanding of pathological pregnancies including miscarriage, pre-eclampsia, fetal growth retardation and stillbirth where arterial invasion transformation by trophoblast is abnormal.
In summary, decidualization per se is associated with certain changes in the spiral arteries such as swelling of endothelial cells and increase in medial thickness. However, the true physiological change, which involves medial necrosis and replacement with fibrinoid material, only occurs in the presence of interstitial trophoblast. Finally, it is another subpopulation of trophoblast, endovascular trophoblast, which appears to be responsible for replacing the endothelial cells in these transformed arteries
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Acknowledgments |
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Notes |
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References |
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Aplin, J. (1991) Implantation, trophoblast differentiation and haemochorial placentation: mechanistic evidence in vivo and in vitro. J. Cell Sci., 99, 681692.[ISI][Medline]
Bell, S.C. (1983) Decidualization: regional differentiation and associated function. In Finn, C.A. (ed.), Oxford Reviews of Reproductive Biology. Clarendon Press, Oxford, Vol. 5, pp. 220271.
Boyd, J.D. and Hamilton, W.J. (1970) The Human Placenta. W.Heffer, Cambridge.
Brosens, I., Robertson, W.B. and Dixon, H.G. (1967) The physiological response of the vessels of the placental bed to normal pregnancy. J. Path. Bact., 93, 569579.[ISI][Medline]
Burrows, T.D., King, A. and Loke, Y.W. (1994) Expression of adhesion molecules by endovascular trophoblast and decidual endothelial cells: implications for vascular invasion during implantation. Placenta., 15, 2133.[ISI][Medline]
Craven, C., Morgan, T. and Ward, K. (1998) Decidual spiral artery remodelling begins before cellular interaction with cytotrophoblasts. Placenta, 19, 241252.[ISI][Medline]
Damsky, C.H. and Fisher, S.J. (1998) Trophoblast pseudo-vasculogenesis: faking it with endothelial adhesion receptors. Curr. Opin. Cell. Biol. 10, 660666.[ISI][Medline]
De Feo, V.J. (1967) Decidualization. In Wynn, R.M. (ed.), Cellular Biology of the Uterus. North-Holland, Amsterdam, pp. 191290.
De Wolf, F., De Wolf-Peeters, C. and Brosens, I., (1973) Ultrastructure of the spiral arteries in the human placental bed at the end of normal pregnancy. Am. J. Obstet. Gynecol., 117, 177191.[ISI][Medline]
De Wolf, F., Brosens, I. and Ranaer, M. (1980) Fetal growth retardation and the maternal arterial supply of the human placenta in the absence of sustained hypertension. Br. J. Obstet Gynaecol., 87, 678685.[ISI][Medline]
Finn, C.A. (1994) Implantation. In Lamming, G.E. (ed.), Marshall's Physiology of Reproduction. Chapman and Hall, London, pp. 157231.
Gibbons, G.H. and Dzau, V.J. (1994) The emerging concept of vascular remodelling. New Eng. J. Med., 330, 14311438.
Khong, T.Y., de Wolf, F., Robertson, W.B. and Brosens, I. (1986) Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants. Br. J. Obstet. Gynaecol., 93, 10491059.[ISI][Medline]
King, A., Wellings, V., Gardner, L. and Loke, Y.W. (1989) Immunohistochemical characterisation of the unusual large granular lymphocytes in human endometrium throughout the menstrual cycle. Hum. Immunol., 24, 195205.[ISI][Medline]
Loke, Y.W. and King, A. (1995) Human Implantation. Cambridge University Press, Cambridge.
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 325.[ISI][Medline]
O'Guin, W.M. (1990) Differentiation-specific expression of keratin pairs. In Goldman, R.D. and Steinert, P.M. (eds), Cellular and Molecular Biology of Intermediate Filaments. Plenum Press, New York, pp. 301334.
Pijnenborg, R. (1994) Trophoblast invasion. Reprod. Med. Rev., 3, 5373.
Pijnenborg, R. (1996) The placental bed. review article. Hypertens. Pregn., 15, 723.[ISI]
Pijnenborg, R. et al. (1980) Trophoblastic invasion of the human decidua from 8 to 18 weeks of pregnancy. Placenta, 1, 319.[ISI][Medline]
Pijnenborg, R. et al. (1983) Uteroplacental arterial changes related interstitial trophoblast migration in early human pregnancy. Placenta, 4, 397414.[ISI][Medline]
Prophet, E.B. (1994) Laboratory Methods in Histotechnology. American Registry of Pathology, Washington.
Robertson, W.B., Brosens, I. and Dixon, H.G. (1967) The pathological response of the vessels of the placental bed to hypertensive pregnancy. J. Path. Bact., 93, 581592.[ISI][Medline]
Zhou, Y., Damsky, C.H. and Fisher, S.J. (1997) Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. J. Clin. Invest., 99, 21522164.
Submitted on February 5, 1999; accepted on April 23, 1999.